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.

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.

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.

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?

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.

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.

"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).

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.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Origin and fate of phagocytes: a brief overview of the differentiation pathways of phagocytes and their fate. Details are given in the text. Abbreviations: CFU-GEMM/GM/G/M: colony-forming unit granulocyte-erythrocyte-monocyte-megakaryocyte/granulocyte-monocyte/granulocyte/monocyte; AMP/MP alveolar macrophage/macrophage; Pox+/ gr, peroxidase-positive/negative granules; hl, half-life; BMR, bone marrow reserve; cGp/mGp, circulating/marginated granulocyte pool; , decrease; solid arrowheads, factors involved in cell maturation (for example, CSF and cytokines); open arrowheads, influence of the microenvironment in cell maturation (cell receptors and matrix proteins); dashed curved lines, self-renewal potential of phagocyte precursors.

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.

(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-91^phox [phox for "phagocyte oxidase"], a glycoprotein of 91 kDa, and p22^phox), and three cytosolic components, p47^phox, p67^phox, and p40^phox. 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).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Summary of PMN functions. The localized inflammation following pathogen invasion generates many activating signals for endothelial cells (EC) and circulating PMNs (cP). Concomittant activation of these cells results in strong binding of PMNs to EC (step 1) and later in their transendothelial migration (diapedesis) (step 2). PMNs are attracted to the infected area (oriented migration, chemotaxis) along the chemotactic gradient (step 3). During their voyage, PMNs are primed by various signals (cytokines [CK], LPS, etc.) and are thus prepared to perform their bactericidal function. PMNs recognize pathogens via their membrane receptors for the Fc of immunoglobulins (Ig) or complement proteins (C3b/iC3b) or via lectins. The adherent pathogen is engulfed (phagocytosis) (step 4) in a vacuole. The contents of specific and azurophilic granules are released into the phagosome, which becomes a phagolysosome (degranulation, exocytosis) (step 6), parallel to the activation of the NADPH oxidase system initiating the oxidative burst (step 5). Oxygen-dependent and -independent (natural protein and peptide antibiotics [AB]) bactericidal systems cooperate to destroy the pathogen (step 7). The last step corresponds to the digestion of the bacterial debris by hydrolases and other lytic enzymes released in the phagolysosome (step 8). In the setting of excessive priming, PMNs stop migrating and the activation (degranulation, production of reactive oxygen species [ROS]) takes place in the tissues, which can be injured (step 9).

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(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).

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 p47^phox; 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, p130^Cas, 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).

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Schematic presentation of the main transductional pathways in phagocytes. Due to space limitation, not all routes and regulatory signals are shown. The main abbreviations are given in the text. Additional abbreviations: R1, R2, R', receptors; L1, L2, L', ligands; Ac, adenyl cyclase; cAMP, cyclic AMP; Cs, calciosome; CaM-K/CaM-Pase, calmodulin-dependent kinase/phosphatase; GAP, GTPase-activating protein; GRF, guanine regulatory exchange factor; GP, heterotrimeric G protein; GPL, glycurophospholipids; MLCK, myosin light-chain kinase; NR-PTK/PTK, nonreceptor/receptor protein tyrosine kinase; P, phosphorylation; PC, phosphatidylcholine.

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).

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."

View larger version (26K): [in this window] [in a new window]   FIG. 4.   The phagocyte: a key effector and regulator in host homeostasis, defense, and disease.

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).

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.

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).

View larger version (54K): [in this window] [in a new window]   FIG. 5.   Interactions between phagocytes and antibacterial agents. (A) Effects of antibacterial agents (ABA) on phagocyte activities. Detailed explanations are given in the text. Solid lines indicate the direct effects of ABA on phagocytes, and broken lines indicate indirect effects. Abbreviations: IC, intracellular; CK, cytokines; OX, oxidants; AB, natural antibiotics; EZ, enzymes; S, stimulation; D, deactivation; R, resistance; Ag, antigenic structure; APC, antigen-presenting cells; IS, immune system; HS, hormonal system; CNS, central nervous system. (B) Effects of phagocytes on ABA. Full explanations are given in the text. ABA*, ABA modified by phagocyte products.

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.

(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.

(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