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Clinical Microbiology Reviews, October 2000, p. 615-650, Vol. 13, No. 4
INSERM U 479, Faculté Xavier Bichat,
75018 Paris, France
0893-8512/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Interference of Antibacterial Agents with Phagocyte
Functions: Immunomodulation or "Immuno-Fairy Tales"?
SUMMARY
INTRODUCTION
BRIEF HISTORY OF IMMUNOMODULATION
Hopes and Enthusiasm in the Preantibiotic Era:
Phagocytes and Bacteria
Midcentury: "Miracle Drugs" and the End of
Infectious Diseases?
The "Immunologic Burst": Expanding Complexity of the
Immune System
Hopes and Wisdom at the Dawn of the New Millennium
PHAGOCYTES: DEFENDERS OR OFFENDERS?
Phagocyte Lineages: from Amoebae to Diversity
Phagocyte Life and Functions: the Old and the New
Origin and fate of phagocytes.
Phagocyte functions.
(i) Classical view: PMNs and macrophages as warriors
cooperating in the battle against foreign invaders.
(ii) New aspects of phagocyte functions.
Phagocyte-Speak: Cell-Cell Communication and
Intracellular Messages
Phagocytes and the Host: "Trick or Treat"
Adventureland: How To Explore Phagocyte Functions
ANTIBACTERIAL AGENTS AND PHAGOCYTES
Complex Game for Two or More Players with High Stakes
Clinically relevant effects.
(i) Antibiotic-induced
toxic and immunotoxic effects.
(ii) Intracellular bioactivity.
Effects with a potential clinical impact.
(i)
Modulation of bacterial virulence.
(ii) Modulation of antibiotic activity by phagocytes.
(iii) Modulation of phagocyte antibacterial activity by
antibiotics.
Miscellaneous effects.
(i) Modulation of the
specific immune response.
(ii) Impact on the host microflora.
Lexicon of Immunomodulatory Antibacterial Agents
Aminoglycosides.
Ansamycins.
Benzylpyrimidines (trimethoprim and analogs).
-Lactams.
Chloramphenicol.
Cyclines.
Fosfomycin.
Fusidic acid.
Gyrase B inhibitors.
Isoniazid.
Lincosamides.
Macrolides.
Peptides.
Quinolones.
Riminophenazines.
Sulfones and sulfonamides.
Other antibacterial agents.
NONANTIBIOTIC EFFECT OF ANTIBACTERIAL AGENTS:
POTENTIAL THERAPEUTIC RELEVANCE?
Immunostimulation or Restoration?
Immune response modifiers and immunocompromised patients:
the example of cefodizime.
Fluoroquinolones: a future prospect?
Immunodepression and Anti-Inflammatory Activity of
Antibacterial Agents
Classical use of antibacterial agents in inflammatory
diseases.
Cyclines and ansamycins.
Macrolides.
Prospects: fosfomycin and fusidic acid?
DO WE NEED IMMUNOMODULATING ANTIBACTERIAL AGENTS?
Immunocompromised Individuals
Sepsis
CONCLUSIONS: IMMUNOMODULATING EFFECTS OF ANTIBACTERIAL
AGENTS
"NEVER SAY NEVER"
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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Professional phagocytes (polymorphonuclear neutrophils and monocytes/macrophages) are a main component of the immune system. These cells are involved in both host defenses and various pathological settings characterized by excessive inflammation. Accordingly, they are key targets for immunomodulatory drugs, among which antibacterial agents are promising candidates. The basic and historical concepts of immunomodulation will first be briefly reviewed. Phagocyte complexity will then be unravelled (at least in terms of what we know about the origin, subsets, ambivalent roles, functional capacities, and transductional pathways of this cell and how to explore them). The core subject of this review will be the many possible interactions between antibacterial agents and phagocytes, classified according to demonstrated or potential clinical relevance (e.g., neutropenia, intracellular accumulation, and modulation of bacterial virulence). A detailed review of direct in vitro effects will be provided for the various antibacterial drug families, followed by a discussion of the clinical relevance of these effects in two particular settings: immune deficiency and inflammatory diseases. The prophylactic and therapeutic use of immunomodulatory antibiotics will be considered before conclusions are drawn about the emerging (optimistic) vision of future therapeutic prospects to deal with largely unknown new diseases and new pathogens by using new agents, new techniques, and a better understanding of the phagocyte in particular and the immune system in general.
INTRODUCTION
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When preparing this overview of a field in which I have been working for 15 years, I thought it would be easy to summarize the main data on the immunomodulatory potential of antibacterial agents on phagocytes. Since the understanding of the possible interferences of these bacterium-targeting agents with host cells (and their clinical impact) requires some knowledge of the main actor in the play, namely, the phagocyte, and the way in which the potential therapeutic value of immunomodulation has come to the forefront, I intended to present a brief overlook of a century's research on immunology and infection and then discuss the phagocyte itself. However, when I started to address the question of the complexity of this cell at the functional, transductional, and regulatory levels, I soon realized that, despite a substantial amount of published material in this field, we have so far only seen the tip of an iceberg. Consequently, the following two sections, which address the therapeutic relevance of the observed effects and future research prospects, will certainly raise more questions than answers.
Immunomodulation, a therapeutic need for the third millennium, is still in its infancy, and antibiotic therapy itself is only now approaching maturity. Many current antibacterial agents have not revealed all their facets, and new antimicrobial agents are forthcoming. The microbial world, the phagocyte, and the host still have tricks up their sleeves, holding the promise of a new and exciting research enterprise in years to come. I hope this review will provide a basic framework for those interested in this field.
BRIEF HISTORY OF IMMUNOMODULATION
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The main data reported in this section have been taken from a number of excellent books and papers (10, 76, 79, 105, 111, 145, 146, 190, 234, 252, 293, 294, 415).
It is generally agreed that the concept of immunomodulation emerged in 1796 when Jenner undertook the first "vaccination". Since then, many attempts have been made to help the immune system face external (bacteria, viruses, etc.) or internal (cancer and autoimmunity) attacks. These new therapeutic strategies ("prohost" treatment) have been made possible by about a century of fundamental discoveries and the recognition of immunology and microbiology as distinct scientific disciplines.
Hopes and Enthusiasm in the Preantibiotic Era: Phagocytes and Bacteria
The roots of immunology and microbiology date back to the last decades of the nineteenth century. In 1879, Pasteur discovered, largely by accident, that an attenuated culture of chicken cholera bacteria could immunize against subsequent challenge. The Pasteur Institute was opened in the fall of 1888. After years of patient observation, the first concept of a true host defense mechanism was forwarded by Metchnikoff in December 1882. Space is lacking here to list the names of all these passionate pioneers who, in the short period from the 1850s to the 1880s, amidst great excitement and confusion, established the theoretical and methodological bases for the new science of microbiology. Some notables include Koch, the founder of laboratory bacteriology; Behring, Kitasato, and Ehrlich (1890 and 1891), who developed the theory of humoral immunity; Wright (Sir Almroth "Almost" Wright, 1902), who reconciled the humoral and cellular aspects of immune defense with the concept of opsonins (humoral components aimed at preparing for and activating phagocytosis), and Ivanovski and Beijerinck (1892 and 1899), who found the first filterable agent (virus). These are but some of the many microbe hunters who lent their names to almost all bacterial genera. By the end of the nineteenth century, microbiology was a well-established discipline which had split into several specialized branches. Textbooks, journals, institutes, and courses on microbiology sprang up almost as quickly as newly discovered bacteria. In 1879, Pasteur's associate E. Duclaux established a course in microbiology at the Sorbonne. In 1884, Koch introduced a comprehensive course in medical microbiology at the University of Berlin. Microbiology techniques were sufficiently advanced for scientists to name many diseases caused by a specific bacterium or protozoan. The definition of viruses (as we know them) would take almost 50 years, from 1892 up to the first portrait of a tobacco mosaic virus obtained by an electron microscope in 1939. In the same short period (1884 to 1895), the three great discoveries relating to host defense mechanisms (phagocytes, antibodies, and complement) provided the foundations for host resistance and immunology. Immunomodulation was then thought of as an induction of immunity to pathogens. Several methods were devised to counteract infectious agents: vaccination with laboratory-modified pathogens (to create specific protection, although the immune participants were largely unknown); induction of active and passive immunity by transfer of humoral factors (serum therapy, replacement therapy), which seemed to deal the final blow to infectious diseases; and, by the followers of Metchikoff's cellular theory of immunity, some somewhat adventurous therapies to create beneficial inflammation. "Stimulation of the phagocyte" and the concept of "stimulins" were a great hope in the early nineteenth century (415). However, enthusiasm soon vanished when potentially immune-mediated diseases started to emerge; for example, tuberculin not only failed to cure tuberculosis but even worsened it (although it proved a wonderful diagnosic tool); the experiments by Richet and Portier in the 1900s demonstrated life-threatening hypersensitivity and anaphylactic reactions; Metchnikoff himself, after first refuting the principles of noxious inflammation of Conhein and Helmholz, turned his interest to a possible role of phagocytes in senility and the way in which bacterial toxins could transform the friendly phagocyte into a fearsome foe, and he suggested disinfection of the digestive tract to increase life expectancy; and P. Ehrlich recognized the limitations of serum therapy. Within 10 years, immunologic euphoria was replaced by profound frustration and a period that has been called the Dark Ages of Immunology (234).
Scientists then shifted from immunologic stimulation to the creation of chemotherapeutic "magic bullets," which culminated with Preparation 606 (Salvarsan) by Ehrlich in Hoechst's laboratory in 1912. After the interest in antibiosis in the late nineteenth century and the discovery by Twort (1915) of bacteriophages (the possible "microbes of immunity"), the scientific community was ready to acknowledge the birth of chemotherapy.
Midcentury: "Miracle Drugs" and the End of Infectious Diseases?
In the latter decades of the nineteenth century, many observations on microbial antagonism and attempts to apply this phenomenon to treating diseases created a favorable climate for the advent of antibiotics. The birth of chemotherapy is officially assigned to 1928, with the discovery by Fleming of the potent lytic effect of a mold contaminant, Penicillium notatum, on a staphylococcal culture. It took almost 10 years before the therapeutic activity of penicillin G was demonstrated, thanks to Florey and Chain, among others (294), and a few more years to elucidate its chemical structure and produce it industrially by fermentation. The isolation of tyrothricin in 1939 and the demonstration of its powerful therapeutic effect greatly stimulated the development of antibiotic research. The golden age of antibiotics was starting, stimulated by the needs of World War II. Antibiotic screening and the search for "miracle molds" all around the world resulted in the discovery of almost all the main classes of these therapeutic agents within about 10 years. The term "antibiotics" was coined by Waksman and defined as "compounds produced by microorganisms that can inhibit the growth of other microorganisms or even destroy them." Streptomycin was found in 1944, and gramicidin S was found in 1942. Chloramphenicol, erythromycin, neomycin, cephalosporins, and many others were found in the 1940s and 1950s. The natural backbones were chemically modified to improve stability, efficacy, pharmacokinetics, or toxicity from the 1960s up to the present, and chemical research gave birth to the modern fluoroquinolones. However, the use of these miracle drugs failed to take account of the fact that microorganisms have an extreme capacity to evolve resistance strategies and that creating new antibacterial weapons is an endless effort. Could the sentence of Sir Almroth Wright, "The physician of the future will be the immunisator," (60) be a premonition?
The "Immunologic Burst": Expanding Complexity of the Immune System
By the 1920s, the only thing which was perfectly clear in immunology was that "immunity, whether innate or acquired, is extremely complex in character" (363). Later, as more scientists have become involved, the evolution of immunology has been so dynamic that it has become a fundamental discipline of medicine and biology. Parallel technical advances have made it possible to identify and explore the various interconnected cellular and humoral components of the immune system. The first breakthrough came in the 1960s with the clonal selection theory (44) and the elucidation of the primary structure of the antigen receptor (82). The 1970s and 1980s saw remarkable theoretical and practical contributions to our understanding of the immune network, its cellular subsets and mediators (cytokines), and its involvement in cancer, autoimmunity, and organ transplantation. The development of new technologies (hybridomas and monoclonal antibodies [186] and PCR [293]), the birth of molecular biology, and the identification of intracellular messengers (cyclic AMP to heterotrimeric G proteins, the low-molecular-weight G proteins, and other intracellular biochemical cascades) illustrate the burst of immunology that has now "phagocytized" almost all fundamental disciplines from histology to chemistry, genetics, and even mathematics. New generations of genetically engineered drugs ("poison arrows") are being proposed, targeted not only to microbes but also to chronic diseases, and will open a new era centered on recombinant DNA technology to design proteins with specific desirable functions that act on specific receptors or specific enzyme isoforms.
Hopes and Wisdom at the Dawn of the New Millennium
Technicians and theoreticians have given us a tremendous potential armamentarium against pathogenic microorganisms. However, lessons from the past have shown that announcements of medical miracles have been highly exaggerated. In 1976, it was widely believed that infectious diseases had been conquered; diseases like tuberculosis, cholera, and smallpox were of little concern to people in wealthy industrialized nations. Only the threat of AIDS from 1983, the discovery of infectious proteins (prions) in 1982, and the possibility of microbial terrorism, not to mention the potential nonterrestrial (space-borne) pathogens of tomorrow, have tempered our enthusiasm. The gospel of specific etiology from Koch's postulate, "a bacteria, a disease, a treatment," has furnished the guidelines for medical research for about a century. The development of immunology has resulted in further complexity by combining external (environment and pathogens) and internal (neuro-endocrine-immune system) factors in the pathophysiological scenario of infectious diseases. With the advent of powerful new techniques and drugs, the challenge is to learn how to modulate the immune response to external conditions. Immunopharmacology is still a young science, and the immune system has not yet unveiled its molecular complexity. Nevertheless, along with Metchnikoff, we can say, "we therefore have the right to hope that in the future, medicine will find more than one way to bring phagocytes into play for the benefit of health." Let us now approach this multifaceted cell.
PHAGOCYTES: DEFENDERS OR OFFENDERS?
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Phagocytes, etymologically "devouring cells," are characterized by the process of engulfing relatively large particles (phagocytosis) into vacuoles by a clathrin-independent process that generally requires actin polymerization (reviewed in reference 86a). This property is essential for their role in host defenses and is conserved throughout the evolutionary tree.
Phagocyte Lineages: from Amoebae to Diversity
"Phagocytes are merely the remnants of the digestive system of primitive beings" (252). Amoebae can be considered as a model of the primitive phagocyte and indeed possess all the functional characteristics and transductional systems of the "civilized" phagocytes present in metazoans (8, 9, 258, 334). A general overview of the defense system from primitive invertebrates to mammals reveals a constant role of hemocytes/phagocytes, which are derived from cells of the mesoderm when they were freed from nutritional duties in advanced multicellular invertebrates. Production of reactive oxygen intermediates and possibly of cytokine/cytokine-like molecules (maybe the remnants of a pheromone system in single-celled protozoa) is observed in hemocytes of invertebrates (5, 27). However, at the upper end of the evolutionary scale (mammals), phagocytes have evolved to an extreme diversity. Not only can phagocytes from different species possess peculiar antigenic markers, functional molecules, and activities, but also different phagocytic cell lineages and subsets can be identified in a given species. Roughly speaking, two main lineages exist: polymorphonuclear cells (polymorphonuclear neutrophils [PMNs] and polymorphonuclear eosinophils [PMEs]) and mononucleated cells, referred to as professional phagocytes (the subject of this review). Other cells (such as fibroblasts and epithelial cells) can occasionally phagocytose a more limited range of particles, but in general they do not possess bactericidal mechanisms (oxidants and antibiotics) or opsonin-binding receptors. The professional phagocytic lineages also show an extreme diversity. For instance, PMNs from healthy adults have a heterogenous response to the chemotaxin formyl-methionyl-leucyl-phenylalanine (fMLP) that correlates with the oxidative responsiveness of the cells; stable intersubject differences can also be detected (94). Technologic advances in flow cytometry that allowed the rapid evaluation of PMN membrane responses have shown intrinsic antigenic heterogeneity among PMNs (349). Functional heterogeneity has also been demonstrated between the various PMN pools, i.e., the bone marrow reserve (released by corticosteroids), the circulating granulocyte pool (the most commonly studied compartment), the marginated pool (cells adherent to the endothelium, released by epinephrine), and the tissue pool (245, 365). The second phagocytic lineage in mammals, the monocyte/macrophage system, has an even greater functional and morphological heterogeneity (124). The monocyte/macrophage system consists of bone marrow precursor cells, blood monocytes, and both mobile and fixed tissue macrophages (420). In the late 1960s, the term "mononucleated cell system" replaced the earlier term, reticuloendothelial system" (17), which also encompassed vascular endothelial cells, reticular cells, and dentritic cells of lymphoid germinal centers. Tissue macrophages are derived from blood monocytes which differentiate into specialized cell types according to their location (for example, the Kuppfer cells in the liver and synovial macrophages in the joint capsule). Some macrophages may pass through epithelia and become, for instance, alveolar macrophages or milk macrophages. Each subset of specialized macrophages possesses specific functional and morphological characteristics, but monocyte/macrophage heterogeneity is further amplified by the possibility of other subsets arising under specific pathological conditions (infection or inflammation), such as the "elicited" monocyte-derived macrophage or the epithelioid multinucleated giant cell derived from monocytes under the inflammatory conditions present in granulomas. Like PMNs, macrophages have interspecies and interindividual functional heterogeneity (4, 405).
The initial hypothesis that phagocytic activity was the hallmark of all myelomonocytic offspring rapidly turned into a dogma but is now increasingly rejected. An extended definition of the phagocytic system to some nonphagocytosing cells could open new horizons in the future, since recent work has shown that macrophages can be converted into potent dendritic cells devoid of classical phagocytic activities. The concept of developmental plasticity may have large implications for immune defenses (300).
Phagocyte Life and Functions: the Old and the New
Origin and fate of phagocytes.
Phylogenetically and
ontogenically, hematopoiesis does not occur in the bone marrow;
however, at birth, hemopoietic activity is distributed throughout the
skeleton in humans, and in adult life it is found almost exclusively in
the bone marrow of the sternum and pelvis. The gradual maturation and
differentiation of myelomonocytic cells is an incredibly complex
process which involves many regulatory factors produced locally or
systemically, as well as cell-cell and matrix-cell adhesion mechanisms
(Fig. 1). All mature blood cell lineages
are derived from a totipotent stem cell which gives rise to the
multipotent stem cell and further to the phagocytic cell precursor
CFU-GM (colony-forming unit for granulocytes and monocytes). Under
specific influences, this cell gives rise to the two specialized
phagocyte precursors (CFU-G-myeloblasts and CFU-M-monoblasts). The
production of PMNs in bone marrow takes approximately 2 weeks and
involves a first compartment of proliferating and differentiating cells
(myeloblasts, promyelocytes, and myelocytes) and a second compartment
of maturing, nondividing cells (metamyelocytes, band cells, and mature
cells [the bone marrow reserve]). PMNs are released into the blood,
where their half-life is about 6 to 20 h, and subsequently migrate
into tissues, where they live for 1 to 2 days before becoming apoptotic
and phagocytosed by resident macrophages. The overall production of
PMNs is about 109 cells/kg/day. The number of circulating
leukocytes (for example, PMNs) can be markedly increased by
administration of foreign protein, in particular bacterial products, a
phenomenon already observed by Lowit in 1892 and Metchnikoff and
associates from 1905 to 1914. In 1949, Menkin expanded their
observations on the striking leukocytosis that occurred during
infection and inflammation. The source of these new circulating
leukocytes was unknown. In 1955, Menkin proposed endogenous regulation
by factors released from sites of inflammation. The bone marrow was
recognized as the primary source of PMNs by Perry et al. in 1957 (299). The homeostatic and pathological mechanisms which
control leukocytosis have been reviewed recently (162, 287).
Details on neutrophil production and differentiation can be found in
references 115, 123, and 347.
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Phagocyte functions. Since the historical experiment by Metchnikoff, phagocytes ("microphages" [PMNs] and macrophages) have been assigned a central role in immediate, nonspecific defenses against external aggression (mainly pathogens and their products). In 1908, in his talk after receiving the Nobel prize (252), Metchnikoff presented a visionary approach of the extreme complexity of these microscopic organisms as well as of their pleiotropic role. He not only pointed out the direct microbicidal mechanism of these cells but also suggested other possible functions which were recognized later, such as the secretion of substances "the complement in the humour originates in the white corpuscles," "endolysins of Petterson and leukins of Schneider do exist," the transfer of immunity by white corpuscles, their destruction of microbial toxins, the existence of "certain elements in the organism that promote phagocytosis, the secretins" (cytokines?), the resistance of microorganisms to phagocytes via "agressins," the role of amboreceptors in increasing phagocytosis, etc.
A dichotomic presentation of phagocyte functions between PMNs and monocytes/macrophages has prevailed until recently. PMNs, which are short-lived but extremely abundant cells, were recognized as playing the fundamental role of destroying extracellular pathogens and some of their toxins, whereas monocyte-derived macrophages, long-lived cells, were thought to have (in addition to their phagocyte microbicidal potency) other important functions such as limiting the growth of obligate intracellular pathogens, producing many bioactive molecules important in regulating other cellular functions (complement components, prostaglandins, cytokines, etc.), controlling neoplasia, removing damaged and senescent cells, controlling wound repair, and processing antigens and transmitting the information to lymphocytes, thus directing and targeting the humoral and cellular specific immune responses. This simplistic scheme has been substantially modified by a revisited approach to PMN capabilities and function (68, 320).(i) Classical view: PMNs and macrophages as warriors
cooperating in the battle against foreign invaders.
The two
phagocytic lineages possess similar means of controlling external
aggression, by a sequential multistep process including oriented
motility (chemotaxis), recognition of foreign particles by membrane
lectins and receptors, engulfment into a vacuole (phagosome), degranulation of intracellular secretory pools (granules) and release
of natural antibiotics and enzymes into the phagosome (now a
phagolysosome), production of reactive oxygen species by a complex
enzymatic system (NADPH oxidase) located on the phagocyte membrane
and/or reactive nitrogen species by an inducible nitric oxide synthase,
and killing and digestion of engulfed material in the complex
phagolysosomal medium. Owing to their abundance, rapidity, and more
destructive bactericidal equipment, PMNs are the first line of defense
(Fig. 2). As soon as a microbial pathogen enters the host, a localized, beneficial inflammatory response is
generated by local resident macrophages, necrotized cells and tissues,
plasma factors, and microbial products. The locally produced factors of
inflammation (cytokines, activated complement protein, kinins, etc.)
and microbial factors generate chemotactic gradients, modify
endothelial cell membrane receptors, and promote a slowing of the blood
flow. PMNs that are rolling along the endothelial surface (weak
adhesion mediated by lectin-like molecules, the selectins) respond to
the chemotactic and cell-mediated signals and are first activated to
firmly adhere to the endothelium via their membrane integrins; the
second step is transendothelial migration, referred to as diapedesis,
followed by oriented migration (chemotaxis) toward the inflammatory
site, a phenomenon which involves recognition of chemoattractants
(complement factor C5a, IL-8, bacterial chemotaxins, platelet
activating factor, leukotriene B4, etc.) by specialized
receptors (serpentins [seven-transmembrane-domain G-protein-linked
receptors]) followed by integrin-mediated attachment to the
extracellular matrix and changes in cell shape by rearrangement of the
actin cytoskeleton. This step can be observed within minutes after an
inflammatory signal is generated. During this migratory phase, PMNs
continue to receive information which will further modify their state
of responsiveness (a phenomenon known as priming). Once they have
arrived at the inflammatory site, PMNs can recognize pathogens via
their membrane receptors for opsonins (e.g., complement factors C3b and
iC3b, and the Fc component of immunoglobulins) which are present on
the microbial surface or via microbial and phagocyte lectins
(opsonin-independent phagocytosis). Lectin- or receptor-mediated
activation of PMNs triggers phagocytosis, classically by a zipper
mechanism of sequential recognition of the pathogen by phagocyte
extensions (pseudopods) which finally engulf the microbe in a vacuole.
Coiling phagocytosis is the most frequent unusual uptake: unilateral
pseudopods wrap around the microorganism in multiple turns, giving rise
to largely self-apposed pseudopodial surfaces. This phenomenon
has been observed with Legionella pneumophila,
Trypanosoma spp., Leishmania promastigotes, and
occasionally with Staphylococcus aureus, Haemophilus
influenzae, and Escherichia coli (323). In
parallel, two phagocyte functions are activated: the release of granule
contents into the phagosome and the oxidative burst. The oxidative
burst was first described in 1933 by Baldridge and Gerard and consists
of explosive oxygen consumption (50- to 100-fold increase) that is
unrelated to mitochondrial respiration and reflects the activity of the
NADPH oxidase system. This enzymatic complex is made up of cytosolic
and membrane constituents, which are separated in resting PMN and are
reassembled upon PMN activation. The primary constituents are a
membrane flavocytochrome b (cytochrome b 558),
which comprises two subunits (gp-91phox [phox
for "phagocyte oxidase"], a glycoprotein of 91 kDa, and p22phox), and three cytosolic components,
p47phox, p67phox, and
p40phox. Other cofactors of this complex are the
two 22-kDa low-molecular-mass GTP-binding proteins, Rac-2
(located in the cytosol) and Rap-1A (present in the plasma membrane and
specific granule membranes). Recent insights into the various
components of this system, their assembly, and molecular interactions
are presented in various overviews (20, 67).
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) by the one-electron reduction
of oxygen using NADPH as the electron donor: 2O2 + NADPH
2O2·
+ NADP+ + H+·. Superoxide anion is further
dismutated into hydrogen peroxide (H2O2),
which, in the presence of myeloperoxidase (MPO) released from PMN
azurophilic (primary) granules and a halide, generates very potent
oxidizing agents such as hypochlorous acid (HOCl) and chloramines.
Other oxidative species such as singlet oxygen, the hydroxyl radical,
and reactive nitrogen species can theoretically be produced by
activated PMNs. Their relevance to bacterial killing inside the
phagosome has recently been examined (133). The other way in
which PMNs kill bacteria, known as the oxygen-independent system, is
dependent on protein and peptide antibiotics, which are among the most
phylogenetically conserved bactericidal molecules (103, 104, 147,
239). Most of these proteins (bactericidal permeability-increasing protein, cationic antimicrobial protein 37, and
defensins) are stored in peroxidase-positive (azurophilic, primary)
granules, where they colocalize with active proteases such as elastase,
cathepsin G, and proteinase 3 (408).
Bactericidal-permeability-increasing protein (BPI) is selectively
active against gram-negative bacteria and has LPS-binding properties.
Defensins are small peptides (3 to 4 kDa) that are active against
gram-positive and gram-negative bacteria, fungi, some viruses, and
tumor cells; there are three major human defensins: human neutrophil
peptides 1 to 3 (H-NP 1 to H-NP 3) and a minor one (H-NP 4). (Some
epithelial cells also contain defensins, but human monocytes do not.)
Peroxidase-negative granules (specific granules) are noted for their
membrane, which contains cytochrome b 558, and also for a
variety of receptors for adhesion and phagocytosis; various
metalloproteinases that are stored as zymogens; and a family of
endotoxin-binding proteins (the cathelicidins) that was recently
identified, one member of which (h-CAP 18) has been demonstrated in
humans (360). The synergistic interaction of
oxygen-dependent and -independent microbicidal PMN systems generally
results in pathogen killing (98). However, pathogens have
developed ways of avoiding PMN phagocytosis or even deactivating or
destroying these cells. A few pathogens, such as some
Ehrlichia spp., can multiply within PMNs. Since the first
description in 1994, 400 cases of human ehrlichiosis (a tick-borne
zoonosis) have been reported. Some other microorganisms can survive and
persist within PMNs. For instance, PMNs have been suggested as a
possible reservoir of intracellular S. aureus in recurrent
human infections and chronic staphylococcal mastitis in dairy cows
(416). By contrast, although macrophages potentially display
bactericidal mechanisms, they represent safe harbors for many
intracellular pathogens (129, 170, 263). Differences in the
bactericidal systems which may account for this decreased potency of
macrophages compared to PMNs are a less potent oxidative burst, the
absence of MPO in differentiated macrophages, which prevents the
terminal phases of oxidant-generating systems, and the absence of
numerous antibacterial peptides and proteins. It has also been
suggested that macrophages are unable to produce oxidants inside the
phagosome because of the lack of the granule pool of NADPH oxidase
(164). This defective bactericidal function can be boosted
by cytokine stimulation. In particular, proinflammatory cytokines,
interferon (IFN), bacteria, and their products synergistically induce
NO synthase, which could be the major pathway of macrophage bactericidal activity. Among the major functions attributed to macrophages in host defenses is the triggering of a specific, antigen-driven immune response, both through the synthesis and release
of various cytokines regulating T-lymphocyte functions and through
the antigen-processing mechanisms which take place in late endosomal or
phagosomal structures (370). Also, macrophages orchestrate
the complex processes of cell proliferation and functional tissue
regeneration within wounds through the generation of bioactive substances (72). Among the macrophage factors involved in
this function are chemoattractants, which recruit and activate
additional phagocytes; growth factors, which promote angiogenesis, cell
proliferation, and protein synthesis; proteases and extracellular
matrix protein; and factors that restrain tissue growth once repair is completed.
Complementary information on the classical role of PMNs and
monocytes/macrophages in host defense can be obtained in references 98, 102, 143, and 314.
Eosinophils and neutrophils have similar life cycles, morphology, many
lysosomal enzymes, and most chemotactic, phagocytic, and oxidative
responses to membrane stimuli. Their role, however, is directed mainly
at controlling metazoan parasite infections. Like neutrophils,
eosinophils may be both beneficial and detrimental for the host. Their
transduction pathways involve various phospholipases, kinases, and
second messengers. Eosinophils will not be reviewed here, but details
concerning their functions can be found in reference 102.
(ii) New aspects of phagocyte functions. Whereas the perception of macrophages as primitive cells involved in host defense has shifted rapidly to the recognition of their role in regulating homeostasis and participating in multiple stages of the complex immune response, PMNs have long been considered important (see the consequences of neutropenia) but simple first-line defenders against infection. New insights were obtained in the late 1980s (320), including a complex metabolism, perhaps a longer half-life (particularly in inflammation and infection), interaction with other cells, a key role in many pathological processes, and the presence of receptors able to respond to immunomodulation. In particular, PMNs produce and synthesize a variety of proteins involved in self-regulation and regulation of other cells, such as cytokines (TNF, IL-1, IL-3, IL-6, IL-8, G-CSF, and GM-CSF), complement protein and receptors, major histocompatibility complex (MHC) class I, heat shock protein, and antiproteases, despite minimal protein synthesis equipment. An expression profile of active genes in granulocytes has recently been published (158). PMNs can regulate gene expression constitutively and inducibly by transcriptional and posttransductional events. Their role in limiting the infectious process of various intracellular pathogens (Listeria, Legionella pneumophila, Shigella, Chlamydia, and even mycobacteria), viruses, and some parasites (Entamoeba histolytica and Plasmodium falciparum) and tumor cells has been demonstrated or is strongly suspected. Novel ways in which PMNs phagocytose peculiar pathogens such as Borrelia burgdorferi have been observed (366). By contrast, a negative role of phagocytes in the dissemination of intracellular Listeria monocytogenes and phagocytosis-facilitated invasion has been suggested in central nervous system infection in vivo (74).
Other possible new functions of PMNs are related to their role in the specific immune response. In particular, PMNs can cooperate with professional antigen-presenting cells, enhancing the uptake and proteolysis of antigens; they can be induced to express MHC class II molecules and can present antigens to virus-specific cytolytic memory T lymphocytes. These properties seem to rely on the potent MPO-H2O2-Cl
pathway and
chlorination activity of PMNs, whose products, acting as
immunomodulators, provide a further link between innate and adaptive
immunity (238). An immunoregulatory function has also been
assigned to lactoferrin, an iron-binding protein present in specific
granules which possesses antimicrobial properties (41).
Other regulatory aspects of PMNs include the production (release) of
various factors which modulate lymphocyte, monocyte, and eosinophil
functions, thereby giving this cell a central role in host homeostasis.
The presence of the proenkephalin system in PMNs also indicates a
possible role in local analgesia.
Other advances in our understanding of phagocyte functions concern the
recognition of their active role in hemostasis and thrombosis (reviewed
in reference 84), their detrimental activities in
many pathological settings (see below), and new insights into the
complexity of cell-cell interactions and intracellular messages (see
below). This knowledge is linked to the newer approaches and
characterization of various cell constituents such as the many
lectin-like macrophage receptors (362) which target highly specific interactions with the environment, other membrane receptors which mediate adhesion (361), intricate and redundant
intracellular kinases, phosphatase and phospholipase activities, and
the way in which extracellular signals (cytokines, microbial and
inflammatory products, neuro and endocrine mediators) may regulate the
functional properties of macrophages.
Phagocyte-Speak: Cell-Cell Communication and Intracellular Messages
Phagocytes respond to the variable conditions in the environment
through selective recognition of other cell surface antigens or humoral
mediators via a myriad of membrane receptors. The nomenclature of these
receptors is often confusing to nonimmunologists, since various names
are used interchangeably. An international committee meets periodically
to reach a consensus on official names and CD (cluster differentiation)
numbers. Here, a more practical designation is used, linking receptors
to a specialized function or ligand and/or to a specific signaling
mechanism, although there is some crossover between these categories.
Roughly speaking, phagocyte membrane receptors include adhesion
molecules (three families, i.e., integrins, selectins, and molecules
belonging to the immunoglobulin superfamily), chemoattractant receptors
(the serpentins), the opsonin protein (for the Fc of immunoglobulins
and C3b/inactivated C3b complement protein), multiple receptors for
other humoral mediators (including cytokine receptors, separated into
five families, i.e., hematopoietic receptors, IFN receptors, TNF
receptor, G-protein-coupled receptors, and those belonging to the
immunoglobulin superfamily), LPS-binding receptors, adenosine
receptors, neuromediator receptors, and lectin-like receptors
(mannose-R, mannose-6-P-R, advanced glycosylation end-product R, etc.).
None of these receptors is engaged in only one strictly defined
function, and cross talk with synergistic or antagonistic effects
occurs between the different molecules. For instance, adhesion
receptors will not only trigger rolling, adhesion, and diapedesis of
phagocytes but will also participate in chemotaxis, phagocytosis, and
activation of the oxidative burst; similarly, chemoattractants can
generally trigger degranulation and the oxidative burst, while
opsonin-engaged receptors also activate these functions. An interesting
phenomenon concerns ligands which do not directly stimulate a
functional response but modulate phagocyte behaviour after a second
stimulus. This is referred to as priming and is observed with some
cytokines, endotoxin, and suboptimal concentrations of directly
activating stimuli (292). Engagement of its ligand by a
receptor molecule triggers a sequence of events known as a biochemical
signaling pathway. The first, proximal event, related to the structure
of the receptor, directs the main signaling pathways. Various receptor subgroups are defined according to the primary signal, including heterotrimeric G-protein-coupled receptors (serpentins and some cytokine receptors), glycosylphosphatidylinositol-anchored proteins (CD14, Fc
-RIII, and urokinase-type plasminogen activator receptor), and tyrosine kinase receptors.
The emerging view of signal transduction is that cell pathways are regulated by the organization in macromolecular assemblies (55) involving not only a cascade of enzymatic activities and their corresponding products but also adapter proteins, which permit close association of effector enzymes and products or their translocation, and molecular switches such as the low-molecular-weight G proteins (Ras, Rho, Rab, Arf, and Ran), whose activity is regulated by their association with guanine nucleotides and by exogenous proteins involved in the GDP/GTP-bound form cycle (35).
Under most normal and pathophysiological conditions, information
received by phagocytes is mediated by one or several receptors. In
vitro (and possibly occasionally in vivo), some phagocyte stimuli may
bypass this step and directly activate an intracellular effector. For
instance, extracellular Ca2+ and the most widely used
phagocyte activator, phorbol myristate acetate (PMA), directly
stimulate some intracellular protein kinase C isoforms (PKCs). It is
beyond the scope of this review to deal in depth with the many cellular
participants in signal transduction, but a schematic approach to the
main groups of enzymes and mediators involved in signaling may help the
reader understand the possible sites of interference of antibacterial
agents with phagocyte function. I will attempt to roughly summarize the
most important pathways. An extremely simplified diagram of neutrophil
activation is given in Fig. 3.
Chemoattractant binding to its receptor activates one (or three?)
associated pertussis toxin-sensitive G-protein which further
dissociates into subunits that interact with three phospholipases, PLC,
PLD, and PLA2. The source of lipids used by these lipases is the phagocyte membrane. The first wave of lipid messengers includes
inositol 3-phosphate (IP3) and diacylglycerol (DAG)
derived from phosphatidylinositol-4,5-diphosphate by activation of
membrane-bound PLC-
, phosphatidic acid (PA) and choline derived from
phosphatidylcholine by PLD activity, and arachidonic acid and
lysophospholipids derived from glycurophospholipids by PLA2
activity. These second messengers act on various cellular targets to
deliver second-wave signals: IP3 releases Ca2+
from intracellular pools (IP3-sensitive calciosomes), and
DAG with Ca2+ activates various PKC isoforms to
phosphorylate important targets such as p47phox;
Ca2+ may also activate Ca2+-dependent PKs to
regulate the actin cytoskeleton. PA can either directly activate
various kinases and phosphatases or be hydrolyzed by PA
phosphohydrolase to give rise to DAG. Arachidonic acid serves mainly as
a substrate for the synthesis of eicosanoids via the cyclooxygenase or
lipoxygenase pathway, but it can also act as a second messenger in
activating several kinases. Chemoattractant signaling also involves
G-protein-coupled activation of various tyrosine kinases of the Src
family, including the tyrosine kinase Lyn, which phosphorylates various
adapter proteins such as Shc; the Shc-P-Lyn complex may serve to
activate phosphatidylinositol 3-kinase (PI3-K), an enzyme which
catalyzes the addition of a phosphate group to the D3 position of
phosphatidylinositol lipids. Its importance in the regulation of
various phagocyte functions is well documented. Another adapter protein
linked to ShC is Grb2, which is generally associated with the Ras
guanine nucleotide exchange regulator Sos, thus mediating the
activation of the monomeric G protein Ras (a possible mechanism
activating the mitogen-activated protein [MAP] kinase cascade). Ras
GTP may also favor the membrane translocation and further activation of
Raf serine/threonine kinase, another possible way of activating the
dual Threo/Tyr-dual function kinase MEK (MAP kinase kinase).
Cross-regulatory pathways involving receptor tyrosine kinases,
receptors linked to soluble tyrosine kinase, and the
low-molecular-weight G proteins (Ras, Rho, Rab, Arf, and Ran) to
amplify or downregulate phospholipase and kinase action are also
involved in phagocyte stimulation, not to mention the negative feedback
involved in turning off the system. Not all the pathways are yet
correctly placed hierarchically. In particular, whether PLC is upstream
of PLD activation or whether both lipases are activated in divergent
pathways is still unresolved. Other classes of receptors may involve
similar pathways. In particular, receptor tyrosine kinases may undergo
autophosphorylation followed by binding to adapter protein and
recruitment of Sos with prolin-rich SH3 domains to the membrane, close
to the small G protein Ras, which binds and activates the protein
kinase Raf-1 and the MAP kinase cascade. Phagocytosis (classical or
coiling phagocytosis) also involves tyrosine phosphorylation and PKC
activation (59). Integrin-mediated signaling through similar
pathways involves many other adapter proteins such as actin-binding
proteins (vinculin, talin, and paxillin) between the cytoskeleton and
signaling effectors. Focal adhesion kinase is central to this pathway:
tyrosine autophosphorylation provides docking sites by SH2 domains for
other kinases such as PI3-K and Src kinase, which amplify the
activating signals (phosphorylation of paxillin, p130Cas,
and focal adhesion kinase initiating the canonical Grb2-Sos-Ras-Raf pathway). The redundancy of signals may explain why Mac-1 (an integrin)
serves as a signaling partner for several other receptors. Lastly, it
is also important to note that these pathways are under the influence
of posttranscriptional events such as prenylation, farnesylation, and
carboxymethylation, which may promote or facilitate the interaction of
Ras-related proteins with specific membrane targets (301).
The multiple isoforms of phospholipases and protein kinases, and the
recognized MAP kinase pathways (at least three) are not presented here,
although they are certainly important for refining the phagocyte
response to external stimuli. Distorsion of intracellular signaling
pathways by various microorganisms has also been recognized recently
(157, 272).
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Detailed insights into the transduction pathways of phagocytes are given in references 143 (a complete approach to the PMN), 348 (G-protein-coupled receptors), 61, 230, and 412 (selectins, integrins, and signal transduction), 35 (chemoattractant signaling), and many other reviews dealing with specific participants: Ca2+ (237, 356), PKC (226, 227), PI3-K (220), PLD (121), and MAP kinases (54, 106).
Phagocytes and the Host: "Trick or Treat"
The beneficial role of phagocytes in host defense is widely
acknowledged (see above). Deficiencies in neutrophil numbers or function are substantial risk factors for developing potentially fatal
bacterial and fungal infections (350). Gram-negative bacilli and S. aureus are the most common pathogens in patients with
neutrophil defects; chronic granulomatous disease and leukocyte
adhesion deficiency are the most frequent (although rare) congenital
forms (235). There are detailed reviews that deal with the
main inherited and acquired defects in neutrophil numbers and function
(37, 91, 223, 235, 246, 350, 375, 409). Deficiencies in
neutrophil function can be accompanied by defects in
monocyte/macrophage function, but no defect strictly targeting the
mononucleated phagocyte system has been identified. However, new immune
deficiencies continue to be described (parallel to the development of
techniques and better-coordinated analysis of rare inherited
defects), such as the recently published defects in the
IL-12-IFN-
-TNF-
"circuit," which is accompanied by severe
atypical mycobacteriosis (350). Other potential beneficial
effects of phagocytes include tissue repair and healing, and it is
obvious that the secretion of many regulatory factors, including
cytokines, is part of their role in the maintenance of host homeostasis.
In contrast to this beneficial role, phagocytes appear to be very
fine-tuned cells which, by uncontrolled use of the same mechanisms as
those used to destroy pathogens (i.e., oxidative species, enzymes, and
mediators), can have detrimental effects on the host. These cells,
which are considered a double-edged sword, play a fundamental role in
the pathogenesis of exaggerated inflammatory responses (19, 359,
413). Neutrophils can defend themselves against the oxidant they
produce through a potent antioxidant system (for example superoxide
dismutase, catalase, glutathione-dependent H2O2-detoxifying system,
-tocopherol, and
ascorbic acid). However, when produced in excess (particularly after
priming by cytokines or endotoxin) in the extracellular medium,
oxidative species can damage host tissue (139). The
imbalance between proteinases and antiproteinases (which may be
inactivated by oxidants), interaction with platelets, phagocyte-induced
thrombosis (by plugging microvessels) and expression of procoagulant
activity by monocytes also contribute to vascular injury
(84). Release of chemotactic mediators (leukotrienes, platelet-activating factor, and IL-8) recruits new, elicited phagocytes that maintain the detrimental inflammatory response (221).
Recently, it was shown that mammalian mitochondria produce
N-formylpeptides (bacterial chemotaxins), raising the
possibility that tissue injury or anoxia leads to the release of such
mitochondrial contents, providing another mechanism for recruiting
PMNs. In addition to causing vascular injury, PMNs can transmigrate and
attack parenchymal cells (161). Disease conditions in which
phagocyte-inflicted tissue damage plays an important role include acute
events such as ischemia-reperfusion injury, shock, acute respiratory
distress syndrome, acute allograft rejection, inflammatory bowel
diseases and the Arthus reaction, and chronic diseases such as
bronchiolitis, bronchiectasis, cystic fibrosis, diffuse
panbronchiolitis, gastric ulceration, rheumatoid arthritis, and asthma.
Dermatopathic and autoimmune diseases are often associated with
neutrophil infiltration (246, 402). Vasculitis
(63) and almost all diseases for which no etiology has been
identified may potentially be related to abnormal phagocyte functions.
Other possible deleterious consequences of phagocyte activities include
a potential effect of MPO-mediated oxidation of anticancer drugs such
as vincristine (343). Material, such as defensin, released
by biomaterial-activated PMNs may contribute to creating an environment
hostile to host defenses at the biomaterial surface by cell
deactivation. Indeed, in most cases, the detrimental effect of
phagocytes seems to originate in the uncontrolled development of
pathogens (either demonstrated or suspected) which subvert phagocytic
functions to their own needs. Extreme activation of inflammatory
responses or deactivation of defence mechanisms will result in acute or
chronic disease. For instance, the role of Mycoplasma or
Chlamydia persistence in triggering chronic diseases such as
asthma and unstable angor is under the spotlight. Intraphagocytic pathogens protected from the milieu (and possibly from therapeutics) are better able to survive. Altered cell signaling and phagocyte deactivation are frequently observed during intracellular infection (318). Some pathogens tip the cytokine balance in their
favor to induce phagocytes to produce anti-inflammatory cytokines that render cells refractory to other activating signals (262).
The concept of phagocytes as crucial for both host defense and
pathogenicity (Fig. 4) supports the new
approach to virulence and pathogenicity, depending on the initial
status of the host (51). This concept forms the fundamental
basis for future therapeutic guidelines in immunomodulation as proposed
by Repine and Beehler (319), i.e., that "every effort be
made to develop highly reversible and targeted drugs that decrease the
harmful while retaining (or even enhancing) the beneficial effects of
neutrophils."
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Adventureland: How To Explore Phagocyte Functions
To analyze drug-mediated immunomodulating properties of phagocytes, one must be familiar with the main techniques used to analyze their functions. It is outside the scope of this review to describe all available techniques, but the main steps in the analysis of drug-phagocyte interactions, with emphasis on the problems encountered in such studies and the overall evaluation of the results, will be presented in this section. A practical and critical description of techniques routinely employed to study neutrophils (and sometimes monocytes and macrophages) is presented in references 153, 165, 251, 346, and 364.
A summary of the advantages and disadvantages of techniques used to
study drug-induced modulation of phagocyte functions is given in Table
1. One of the most widely employed
approaches used to explore drug-induced modulation of phagocyte
functions is the use of animal models of infection or inflammation. In
some cases, the immunomodulatory activity of a compound can also be assessed in humans. These in vivo studies provide a view of the global
efficacy of a drug in a specific clinical setting, and modifications of
immune parameters such as blood leukocyte counts, the number of
infiltrating neutrophils and monocytes/macrophages in infected or
inflammatory sites, phagocyte morphology, and levels of immune
mediators present in serum and other extracellular fluids. These
studies are also the first step toward analyzing the functional properties of phagocytes ex vivo. The problems of extrapolating the
data to the immunomodulatory potential of a drug are related first to
the chosen model: animals differ from humans in many ways, such as
susceptibility to different pathogens, drug metabolism, phagocyte
receptors and functions, and chronobiology. Interspecies differences
exist, and interindividual variability or chronobiological variations
are also well documented in humans and animals (4, 94, 249, 302,
383). All these observations highlight the need for caution when
extrapolating data across species barriers. Evaluation in humans is
also restricted by ethical considerations and by the need for
sufficient healthy individuals and patients (357).
Monitoring of immune therapies is neither simple nor straightforward. It requires "familiarity with principles of immunologic assays, a
great deal of judgment and considerable understanding of biologic, immunologic and therapeutic effects induced by biological response modifiers" (411). Defining the administration schedule
(i.e., dosage, time, and duration of the protocol) and the survey
protocol (e.g., sampling times and parameters assessed) is the most
difficult but also the most important aspect of in vivo and ex vivo
studies. Mention must be made of a rare in vivo method for evaluating
neutrophil function (chemotaxis) by the Rebuck window assay
(neutrophils migrate into a dermal abrasion and adhere to a glass
slide), but this semiquantitative approach (dependent on blood counts)
is poorly standardized and poorly reproducible. Ex vivo analyses can
provide information on how phagocyte functions are modified by
therapeutic concentrations under host conditions (mediators, cytokines,
cell contacts, proteins, enzymes, etc.). In addition to the
above-mentioned problems, problems inherent to these studies concern
the isolation procedures (which, by separating the phagocyte from its
context, may also suppress a drug-induced factor necessary for
phagocyte modulation) and the pools that will be analyzed (405). The different functional capabilities of the various
granulocyte pools and monocyte/macrophage subsets have been mentioned
above. Easily available neutrophils are circulating cells (about 50% of blood PMNs), whereas monocytes are the most readily available mononucleated phagocytes. Monocyte-derived macrophages obtained by in
vitro culture can also be assessed. Depending on the culture conditions, these cells can exhibit different morphologic, phenotypic, or functional characteristics. Alveolar and peritoneal macrophages and
those present in other extracellular fluids are less easily studied (at
least in humans). Ex vivo studies also have many of the problems
inherent to in vitro techniques. In vitro studies analyze a theoretical
question, outside the host context. Various phagocyte functions can be
routinely assessed, such as adhesion, chemotaxis (under agarose or in
Boyden chambers), phagocytosis (by techniques using adherent or
nonadherent cells, radiolabeled bacteria, or staining), bactericidal
activity (CFU counts or bacterial staining), degranulation (release of
various enzymes present in different granule subsets, spontaneously or
following stimulation), and oxidative burst (either in global assays
such as oxygen consumption and luminol-amplified chemiluminescence or
by measuring specific oxygen species, mainly superoxide
anion-superoxide dismutase-inhibitable cytochrome c
reduction and lucigenin-amplified chemiluminescence). Stimulation
of phagocyte functions is generally studied with agents that
mimic bacterial chemotaxins (formylated peptides such as fMLP) or that
directly activate intracellular enzymes (phorbol esters such as PMA) or
increase Ca2+ flux (calcium ionophores such as ionomycin
and A23187). Phagocyte activity can be boosted by priming agents such
as cytokines, before stimulation. Fluorescence-activated cell sorter
analysis is a recent technique which provides information on many
phagocyte functions and membrane antigens and permits rapid evaluation
of individual phagocyte responses (33). Measurement of
cytokine production by various specific immunoassays has also become
routine (69). Lastly, although not directly an
immunomodulatory effect, phagocytic uptake of drugs is currently
measured by using either fluorescence-labeled or radiolabeled drugs to
determine the amount of cell-associated drug or by directly assessing
their cellular bioactivity (in the case of antibiotics, for instance).
At the frontier between routine clinical studies and research is the study of bone marrow progenitors and in vitro differentiation by
culture in semisolid agar medium. The main problems encountered in
vitro are due to nonstandardization of techniques in different laboratories and sometimes artifacts introduced by the technique itself
(407), separation of phagocytes from their context (a phenomenon already stressed by Metchnikoff: "this method [glass test
tubes] cannot account satisfactorily for events that take place in
living organisms"), and, as mentioned above, intra- and interspecies
differences and chronobiology that often generate an unsubstantiated
extrapolation of results. Various tools, mostly used in the research
setting, are available for in-depth analysis of possible
immunomodulatory effects and are becoming more refined as our knowledge
of phagocyte functions progresses. Almost all transductional pathways
can be measured in terms of production of specific messengers, enzyme
activities, or cellular target modifications. Unfortunately, not all
the pathways and their hierarchical organization have been fully
elucidated, clearly hindering analytical studies. Novel pathways cannot
be ruled out, and there are no strictly specific activators or
inhibitors of a given pathway or enzyme. Research techniques include
the detection of transcriptional activity, mRNA isolation, etc.
Technological advances in molecular biology have truly revolutionized
our approach to phagocyte behavior under the influence of various
drugs. It should be noted that while in vivo and ex vivo studies deal
with true phagocyte populations, phagocytic cell lines (HL-60, PLB-985,
J774, and U937) are commonly used in vitro. These standardized cell
lines theoretically avoid the problem of intra- and interspecies
variability and heterogeneity. They are derived from human or animal
cells, and some can be induced to differentiate into more mature forms.
However, although they are convenient, the functional and secretory
properties of these cells differ from those of true phagocytes
(34).
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ANTIBACTERIAL AGENTS AND PHAGOCYTES
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On the basis of Metchnikoff's concept of stimulins, the
possibility of strengthening phagocyte-mediated antibacterial defenses by antibiotic administration was explored very early after their discovery (26, 144, 267). Unfortunately, inconsistent data meant that these analyses become merely laboratory curiosities due to
an insufficient knowledge of immune effectors, inadequate techniques,
and blind faith in the potency of antibacterial drugs. Rapidly,
however, the recognized side effects of some antibacterial agents on
the immune response (particularly neutropenia, anaphylaxis, and
allergy) reactivated the search for immune consequences of antibiotic
use. The explosive interest in the knowledge of the interactions
between these drugs and immunity, which began in the late 1970s, has
come in successive waves, following three unrelated pathophysiological
events: (i) the acknowledged increasing importance of intracellular
pathogens resistant to classical
-lactams and aminoglycosides; (ii)
the growing numbers of new categories of so-called immunocompromised
patients, owing to medical and surgical progress, for whom even the
most effective antibacterial combinations prove ineffective
(exemplified by the AIDS pandemic); and (iii) the emergence of
antibiotic-resistant bacteria, stimulating the search for new
anti-infective approaches.
In addition, observations that various noninfectious diseases were improved by antibiotic therapy administered for concomitant infections and observations of the benefit of various antibiotics on certain inflammatory diseases reinforced interest in the immunomodulatory activity of antimicrobial drugs. A parallel change in our understanding of the functioning of the immune system with improved technology made it easier to conduct such investigations. Although the immunomodulatory profile of any drug encompasses its effect on specific and nonspecific immune mediators, owing to the key role of the phagocyte in innate and adaptive defenses and homeostasis, this cell is a major target for immunomodulation. The relevant literature has been periodically reviewed (16, 25, 113, 140, 197, 199, 202, 203, 205, 208, 236, 330, 386, 417), reflecting a gradual change from basic, fundamental, and sometimes controversial observations (considered epiphenomena of antibacterial activity) to serious, well-founded tests of the effects of some antibacterial agents in noninfectious diseases.
Complex Game for Two or More Players with High Stakes
The steps which may theoretically be modified in the
host-(microbe)-antibiotic interplay are summarized in Fig.
5. There are two main possibilities:
antibacterial agents may directly or indirectly modulate the natural
phagocyte-bacterium interaction (Fig. 5A), or phagocytes may alter the
activity or structure of antibacterial agents with consequences for
drug activity. Direct alteration of phagocyte functions may be a
consequence of interference with myelopoiesis leading to detrimental
effects such as neutropenia (step 1 in Fig. 5A); intracellular uptake
and bioactivity (step 2); modification of a receptor or cellular
effector, leading to altered functional activities (step 3); scavenging
or inhibition of phagocyte products (step 4); indirect alterations of
phagocyte activities due to direct antibacterial activity, with
decreased bacterial load (step 5); alteration of virulence (pathogen
structure and/or metabolism) (step 6) or antigenic structure (step 8);
alteration of serum factors (for example opsonic or chemoattractant
activity (step 7); modification of phagocyte regulatory factors such as the endogenous microflora (step 9), or effector functions of the specific immune system (step 10), or postulated regulatory genes (step
11) and the neuro-endocrino-immune axis (step 12).
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In the second scenario (Fig. 5B), phagocytes may directly or indirectly (through their products such as oxidants and enzymes) alter the structure of antibacterial agents, which may then become either more toxic for phagocytes or their bone marrow precursors (step 1) or trigger the immune system to initiate allergic phenomena (step 3) or have enhanced or decreased antibacterial activity (step 2). Phagocyte-mediated alterations of pathogen metabolism or structure may result in increased or decreased susceptibility of the pathogen to the antibacterial effect of the drug (step 4). Lastly, intracellular antibiotics may use phagocytes as taxis to get to the infected or inflammatory site (the "Battle of the Marne" scenario) (step 5). The problem with this simplistic categorization (often based on in vitro observations) is that it overlooks the fact that the phagocyte-drug interplay is a dynamic process in vivo. Both direct and indirect effects may operate sequentially or simultaneously, and the final outcome is often difficult to link to one or other phenomenon.
Two clinically relevant categories of antibiotic-induced effects are acknowledged: antibacterial drug-induced toxic and immunotoxic effects and intracellular bioactivity. Other effects with a potential clinical impact are phenomena usually observed in vitro, such as modulation of bacterial virulence, leading to antibacterial synergy or a proinflammatory effect; antibiotic activation or inactivation by phagocyte functions; and modulation of phagocyte functions or phagocyte products by antibiotics resulting either in immunodepression or anti-inflammatory activity. Lastly, miscellaneous effects such as modulation of the specific immune response and the impact on the microflora have also been described.
These general aspects will be discussed schematically, leading to a rough classification of antibacterial agents according to their interference with phagocyte functions. Peculiar aspects related to a given antibiotic or class of antibiotics will be dealt with in the following section.
Clinically relevant effects.
(i) Antibiotic-induced
toxic and immunotoxic effects.
The most prominent toxic and
immunotoxic reactions secondary to antibiotic administration (11,
70, 112, 125, 202, 275, 367) are listed in Table
2 (only adverse effects related to the
immune system are envisaged here). Antibacterial agents are leading
causes of neutropenia and agranulocytosis and, to a lesser extent,
other immunotoxic effects. Neutropenia may be secondary to direct
toxicity or immunologic mechanisms. Toxic reactions affect committed
stem cells and/or proliferating precursor cells. Marrow damage is
usually dose dependent and is more likely to occur in patients
receiving high doses for long periods. The effect of drugs on
granulopoiesis can be studied by in vitro marrow culture techniques.
Chloramphenicol and
-lactams are examples of neutropenia-inducing drugs. Immunologic mechanisms of neutropenia usually take 1 to 2 weeks
to be expressed and are not dose dependent. In susceptible patients,
onset may occur within 24 to 48 h of starting the therapy. Immunologic toxicity is diagnosed by adding the patient's serum and
the drug to bone marrow cultures. Penicillins, cephalosporins, and
sulfonamides are frequently involved in such reactions.
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-lactams, particularly penicillin G, are the antibiotics most
frequently involved in these deleterious events. A substantial proportion (3 to 10%) of the population is at risk of anaphylactic reactions to penicillin. Up to 10% of these allergic reactions are
life-threatening, and 2% are fatal. Cross-reactions may occur with
other semisynthetic penicillins and, to a lesser extent, cephalosporins. Other possible drug-mediated allergies include skin
eruptions, febrile mucocutaneous syndrome, fever, and pulmonary, renal,
or hepatic hypersensitivity. Penicillin, sulfonamides, nitrofurantoin,
isoniazid, and erythromycin have all been implicated (11).
Particular stress must be placed on the capacity of phagocytes to
metabolize xenobiotics, including antibacterial agents (125, 202). Myeloperoxidase, prostaglandin synthase, and various
cytochrome P450 isoenzymes, along with reactive oxidative species, can
all be involved in the generation of haptens (125).
Macrophages and PMNs appear to serve as a relay between the
preimmunological phase (regional antibiotic bioactivation
sometimes to
form directly toxic compounds
and neoantigen formation) and the
specific immune response (sensitization) to these neoantigens. Genetic
factors determine individual sensitization to a given drug, in terms of both antibody production and synthesis of enzymes which participate in
drug metabolism and the formation of reactive metabolites (70, 125).
(ii) Intracellular bioactivity.
Following the initial
observation by Rous and Jones in 1911 that the intraphagocytic
environment afforded protection from extracellular factors such as
serum, it was rapidly demonstrated that various intracellular pathogens
were protected from the activity of penicillin or streptomycin
(21, 26, 152, 232, 351). It was unclear whether this
protection was derived from a failure of the antibiotic to enter the
cell or from a particular metabolic state of the pathogen. Repeated
experiments using various phagocytized organisms confirmed the
inability of various
-lactams and other non-cell-penetrating drugs
to destroy intracellular pathogens. The identification of
Legion