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Interactions between antibacterial agents and polymorphonuclear neutrophils (PMNs) are a major focus of investigation. Owing to the variable drug susceptibility of PMNs from different individuals, in vitro studies require samples from large panels of healthy volunteers to reach statistical significance. Here, we used a phagocytic cell line, PLB-985, which can differentiate into mature PMNs in vitro, for the study of cellular interactions (drug uptake and antioxidant effects) of two macrolides (azithromycin and roxithromycin) and four ketolides [HMR 3004, HMR 3647 (telithromycin), HMR 3562 and HMR 3787]. The oxidative burst of differentiated (D) cells was inhibited by macrolides and ketolides. IC50% values (concentrations impairing the oxidative burst by 50%), determined after 30 min of incubation, were as follows for azithromycin, roxithromycin, HMR 3004, telithromycin, HMR 3562 and HMR 3787, respectively: 40, 39, 15, 23, 26, and 33 mg/l (fMLP stimulation) and 37, 86, 39, 43, 14, and 31 mg/l (PMA stimulation). These values were similar to those obtained with PMNs. Uptake of the two macrolides was significantly lower in non-differentiated (ND) cells than in D cells and PMNs. The cellular/extracellular (C/E) concentration ratios at 60 min for PMNs, D and ND PLB were respectively 67, 25 and 11 (roxithromycin) and 159, 137 and 48 (azithromycin). Ketolide uptake by ND-PLB was also significantly lower than that obtained with PMNs (C/E ratios at 60 min were about 75 versus 265 (HMR 3004), 36 vs 230 (telithromycin), 75 vs 235 (HMR 3562) and 20 vs 130 (HMR 3787). Although the active carrier system seemed to be present in ND cells, its activation pathway was not functional. Thus, the PLB-985 cell line is a good in vitro model for studying drug-PMN interactions. The use of ND and D cells may shed light on the nature and activation pathways of macrolide transport systems present on the PMN membrane.

HMR 3647 (telithromycin), a new ketolide, is active on intracellular pathogens. It was previously demonstrated that it inhibits superoxide anion production in a time- and concentration-dependent manner, at concentrations which inhibit 50% of the control response of about 55 microg/ml (5 min) to 30 microg/ml (30 min); these values are similar to those obtained with roxithromycin, a classical erythromycin A derivative. Here we investigated whether these drugs modified the bactericidal activity of human polymorphonuclear neutrophils (PMN) on four strains of Staphylococcus aureus with different profiles of susceptibility to macrolides and ketolides. We found that the main factor involved in killing was the antibacterial potency of the drugs, although combinations of antibiotics with PMN were slightly more active than each component used alone against two of the four strains. In addition, high concentrations of the drugs, which impaired the PMN oxidative burst, did not impair PMN bactericidal activity. Likewise, some cytokines which enhance PMN oxidative metabolism did not modify PMN bactericidal activity in the presence or absence of macrolides or ketolides. These data suggest that oxygen-independent mechanisms contribute to the bactericidal activity of PMN on these strains of S. aureus. Both live and/or heat-killed bacteria impaired the uptake of telithromycin and roxithromycin (but not that of levofloxacin, a quinolone) in a concentration-dependent manner, owing to a modulation of PMN transductional systems involved in the activation of the macrolide carrier.

We analyzed the cellular accumulation of two new fluoroketolides, HMR 3562 and HMR 3787, by human polymorphonuclear neutrophils (PMN) in vitro. Both compounds were rapidly taken up by PMN, with a cellular-to-extracellular concentration ratio (C/E) of about 141 (HMR 3562) and 117 (HMR 3787) at 5 min, and this was followed by a plateau at 60 to 180 min, with a C/E of >300 at 180 min. Both ketolides were mainly located in PMN granules (about 75%) and egressed slowly from loaded cells (about 40% at 60 min), owing to avid reuptake. Uptake was moderately sensitive to external pH, and activation energy was also moderate (about 70 kJ/mol). As with other macrolides and ketolides, the existence of an active transport system was suggested by (i) the strong interindividual variability in uptake kinetics, suggesting variability in the number or activity of a transport protein; (ii) the saturation kinetics characteristic of a carrier-mediated transport system (V(max), about 2,300 ng/2.5 x 10(6) PMN/5 min; K(m), about 50 microg/ml); (iii) the inhibitory effects of Ni(2+) (a blocker of the Na+-Ca(2+) exchanger), phorbol myristate acetate (a protein kinase C activator), and H89 (a protein kinase A inhibitor). Although these two ketolides are more related to HMR 3647 (telithromycin), it is interesting that the presence of a fluoride gave these molecules a cellular pharmacokinetics more like those of HMR 3004 than those of HMR 3647. The macrolide transport system has not been yet elucidated, but our data confirm that, despite variations in chemical structure, all erythromycin A derivatives share a transmembrane transport system.

Macrolide antibiotics are strongly concentrated within host cells, a property that sustains their activity against intracellular pathogens and is likely responsible for the modulation of cell metabolism and function. There is extensive literature on the subject of macrolide-induced modulation of immune responses. Erythromycin A derivatives seem to display anti-inflammatory activity in vitro, in some animal models and in various clinical settings such as diffuse panbronchiolitis (DPB). The underlying mechanisms are not yet fully understood: inflammatory cytokine and oxidant production by phagocytes is down-regulated by these drugs, but other possible targets include bacterial virulence factors, bronchial and epithelial cells, etc. Also, a link has been suggested between the macrolide transmembrane carrier system and the P-glycoprotein family, which comprises MDR (multiple drug resistance) and CFTR (cystic fibrosis transmembrane conductance regulator), which are respectively involved in the chemotherapeutic resistance of cancer cells and in the genesis of cystic fibrosis.

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.

Cytokines, the hallmarks of infectious and inflammatory diseases, modify phagocyte activities and thus may interfere with the immunomodulating properties of antibacterial agents. We have investigated whether various proinflammatory cytokines (interleukin 1 [IL-1], IL-6, IL-8, gamma interferon, tumor necrosis factor alpha [TNF-alpha], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) modify two macrolide properties, i.e., inhibition of oxidant production by polymorphonuclear neutrophils (PMN) and cellular uptake. Roxithromycin and two ketolides, HMR 3647 and HMR 3004, were chosen as the test agents. TNF-alpha and GM-CSF (but not the other cytokines) decreased the inhibitory effect of HMR 3647 only on oxidant production by PMN. Fifty percent inhibitory concentrations were, however, in the same range in control and cytokine-treated cells (about 60 to 70 microgram/ml), suggesting that HMR 3647 acts downstream of the priming effect of cytokines. In contrast, the impairment of oxidant production by roxithromycin and HMR 3004 was unchanged (or increased) in cytokine-treated cells. This result suggests that HMR 3004 (the strongest inhibitory drug, likely owing to its quinoline side chain) and roxithromycin act on a cellular target upstream of cytokine action. In addition, TNF-alpha and GM-CSF significantly (albeit moderately) impaired (by about 20%) the uptake of the three molecules by PMN. The inhibitory effect of these two cytokines seems to be related to activation of the p38 mitogen-activated protein kinase. Our data also illuminate the mechanism underlying macrolide uptake: protein kinase A- and tyrosine kinase-dependent phosphorylation seems to be necessary for optimal uptake, while protein kinase C activation impairs it. The relevance of our data to the clinical setting requires further investigations, owing to the complexity of the cytokine cascade during infection and inflammation.

HMR 3647, a new ketolide, is active upon intracellular pathogens. We previously demonstrated that HMR 3004 (RU 64004), another ketolide, is highly concentrated by human polymorphonuclear neutrophils (PMNs). This prompted us to evaluate whether the presence of a 3-keto group instead of an L-cladinose, a neutral sugar characteristic of erythromycin A derivatives, confers peculiar pharmacokinetic properties with regard to cellular accumulation and efflux. After incubation with the radiolabelled drug, HMR 3647 uptake was determined by a velocity gradient centrifugation technique. HMR 3647 was avidly concentrated by PMNs, without saturation, over a 3-h incubation period, with cellular-to-extracellular concentration ratios of 31 +/- 4.2 at 5 min and up to 348 +/- 27.1 at 180 min. About 60% of HMR 3647 was located in the granular compartment; less than 6% was associated with the membranes. HMR 3647 gradually egressed from loaded cells placed in drug-free medium. Uptake was dependent on environmental temperature (activation energy, 128 +/- 9. 4 kJ/mol) but not on extracellular pH. HMR 3647 displayed Michaelis-Menten saturation kinetics with a mean Vmax of 2315 ng/2.5 x 10(6) PMNs/5 min and a mean Km of 117 mg/liter (144 microM). As already observed with erythromycin A-derived macrolides, extracellular Ca2+ was necessary for optimal uptake of HMR 3647. Interestingly, verapamil increased the uptake of HMR 3647 at 5 min, but this was followed by gradual inhibition at later incubation times, a phenomenon probably related to stimulation of drug efflux. The impact of intracellular accumulation of HMR 3647 on PMN functions was also investigated. In contrast to other erythromycin A derivatives, HMR 3647 only weakly triggered granule exocytosis, but it inhibited superoxide anion production in a time- and concentration-dependent manner, with concentrations which inhibited 50% of control response of 55 (67 microM) (5 min) and 30 (36 microM) (30 min) mg/liter for formyl-methionyl-leucyl-phenylalanine stimulation and 117 (143 microM) (5 min) and 44 (54 microM) (30 min) mg/liter for phorbol myristate acetate stimulation.

The possibility that antibacterial agents, primarily directed against microorganisms, also modify host functions is widely recognized. While a knowledge of these non-antimicrobial effects of antibiotics, sometimes considered as 'side-effects', is necessary to prevent antibiotic-associated toxicity, the development of drugs derived from antibacterial agents for use in non-infectious diseases (e.g. motilins and antidiabetic drugs) is a new field of therapeutic research. Interactions between antibacterial drugs and the immune system may contribute to therapeutic efficacy in infectious diseases [1,2]. The immune system itself is a complex pyramid of redundant cellular factors/humoral effectors/mediators, whose fine regulation is just beginning to be unraveled. Phagocytes, ubiquitous and multifaceted cells are key components of cellular immunity, being involved both in immediate defences against non-self targets (pathogens, tumour cells, exogenous molecules, etc.) and in the regulation and triggering of specific immune responses. They are thus, prime targets of immune response modifiers. This review reconsiders the widely explored problem of interactions between antibacterial agents and phagocytes, focusing on future prospects in both infectious and non-infectious diseases.

The important role played by macrolides in the chemotherapy of infectious diseases is well established, but there is still much speculation about their anti-inflammatory potential. A review of in-vitro and ex-vivo studies reported in the literature shows that macrolides have potentially relevant immunomodulatory effects. In-vitro data suggest that erythromycin A derivatives have a direct effect on neutrophil function and the production of cytokines involved in the inflammation cascade. The ex-vivo results indicate that short-term administration of macrolides may enhance the immune response while long-term administration results in immunosuppression. Further research is required to improve our understanding of the therapeutic activity of macrolides.

We analyzed the uptake of RU 64004 by human neutrophils (polymorphonuclear leukocytes [PMNs]) relative to those of azithromycin and roxithromycin. RU 64004 was strongly and rapidly accumulated by PMNs, with a cellular concentration/extracellular concentration ratio (C/E) of greater than 200 in the first 5 min, and this was followed by a plateau at 120 to 180 min, with a C/E of 461 +/- 14.8 (10 experiments) at 180 min. RU 64004 uptake was moderately sensitive to external pH, and activation energy was also moderate (63 +/- 3.8 kJ/mol). RU 64004 was mainly located in PMN granules (about 70%) and egressed slowly from loaded cells, owing to avid reuptake. The possibility that PMN uptake of RU 64004 and other macrolides occurs through a carrier-mediated system was suggested by three key results. First, there existed a strong interindividual variability in uptake kinetics, suggesting variability in the numbers or activity of a transport protein. Second, macrolide uptake displayed saturation kinetics characteristic of that of a carrier-mediated transport system: RU 64004 had the highest Vmax value (3,846 ng/2.5 x 10(6) PMNs/5 min) and the lowest Km value (about 28 microM), indicating a high affinity for the transporter. Third, as observed previously with other erythromycin A derivatives, Ni2+ (a blocker of the Na+/Ca2+ exchanger which mediates Ca2+ influx in resting neutrophils) impaired RU 64004 uptake by PMNs, with a 50% inhibitory concentration of about 3.5 mM. In addition, we found that an active process is also involved in macrolide efflux, because verapamil significantly potentiated the release of all three macrolides tested. This effect of verapamil does not seem to be related to an inhibition of Ca2+ influx, because neither EGTA [ethylene glycol-bis (beta-aminoethyl ether)-N,N',N'-tetraacetic acid] nor Ni2+ modified macrolide efflux. The nature and characteristics of the entry- and efflux-mediating carrier systems are under investigation.

Apart from their direct antimicrobial activity, some antimicrobial agents may interfere in the complex host-microorganism interplay by modulating the natural response to invading pathogens. The three main aspects of this non-antibiotic effect (alteration of bacterial virulence, synergism with/impairment of the natural host response, impact on effector cell progenitors) will be briefly reviewed here, along with their potential incidence in the clinical outcome of infectious diseases.

CELL PENETRATION: One of the main features of macrolides is their capacity to penetrate host cells. This property is the basis of their action against intercellular microorganisms and can explain characteristic pharmacokinetics as well as interference with cell metabolism. INTRACELLULAR CONCENTRATION: Erythromycin A derivatives with a single base group (roxithromycin) accumulate rapidly to a saturation point both the cytoplasm and in granulations. Intracellular concentrations of derivatives with two base groups do not reach saturation due to slow exflux. THERAPEUTIC ACTION: Because of their intracellular concentration, macrolides are indicated for the treatment of cell-associated pathogens. Exflux allows possible selective transfer into infected tissues. Macrolides also have an effect on functional activity of the host cells.

Macrolide antibiotics are taken up and concentrated by host cells, particularly phagocytes, and are likely candidates to modify cell functions. In this study, we extended our previous work concerning the effect of three 14-membered-ring macrolides (dirithromycin, erythromycin and erythromycylamine) on human neutrophil exocytosis, and found that three other erythromycin A derivatives (roxithromycin, clarithromycin and the azalide, azithromycin) also triggered neutrophil degranulation in a time- and concentration-dependent manner. After 30 min of incubation, the correlation coefficients for concentration-dependence for roxithromycin were 0.885, 0.739 and 0.750 (P < 0.005) and for clarithromycin were 0.795, 0.599, 0.733 (P < 0.02), respectively, for lysozyme, beta-glucuronidase and lactoferrin release. Although the underlying mechanism was not elucidated, these and previous data suggest that intracellular accumulation is a prerequisite. Furthermore, comparison of the characteristics of macrolide-induced exocytosis with those of exocytosis triggered by the synthetic chemotactic stimulus FMLP suggested that different mechanisms are involved. In keeping with this possibility, we showed that combined treatment (macrolides plus FMLP) resulted in totally additive exocytosis of azurophilic but not specific granules. The clinical relevance of our data remains to be ascertained.

We compared the uptakes and intracellular locations of four 14-membered-ring macrolides (roxithromycin, dirithromycin, erythromycin, and erythromycylamine) in human polymorphonuclear neutrophils (PMNs) in vitro. Intracellular location was assessed by cell fractionation and uptake kinetics in cytoplasts (granule-poor PMNs). Trapping of dirithromycin within PMN granules (up to 80% at 30 min) was significantly more marked than the intracellular trapping of the other drugs (erythromycylamine, 45% +/- 5.1%; erythromycin, 42% +/- 3.7%; roxithromycin, 35% +/- 3.0%). A new finding was that, in the absence of extracellular calcium, the uptakes of all of the macrolides by PMNs and cytoplasts were significantly impaired, by about 50% (PMN) and 90% (cytoplasts). Furthermore, inorganic Ca2+ channel blockers inhibited macrolide uptake in a concentration-dependent manner, with 50% inhibitory concentrations of 1.6 to 2.0 mM and 29 to 35 microM, respectively, for Ni2+ and La3+. The intracellular distributions of the drugs were unchanged in the presence of Ni2+ and La3+ and in Ca(2+)-free medium supplemented with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The organic Ca2+ channel blocker nifedipine had no effect on macrolide uptake, whereas verapamil inhibited it in a time- and concentration-dependent manner. These data show the importance of extracellular Ca2+ in macrolide uptake by phagocytes and suggest a link with Ca2+ channels or a Ca2+ channel-operated mechanism.

The aim of the development of semisynthetic derivatives was to overcome the problem of chemical stability of erythromycin A in acid medium, with less variability in gastro-intestinal absorption and leading to renewed interest in macrolides. The new macrolides have the same antibacterial spectrum as erythromycin A including Gram-positive and Gram-negative cocci, intracellular bacteria, mycoplasma, Campylobacter sp., Helicobacter pylori, mycobacteria spp., Gram-negative bacilli including Haemophilus influenzae, Bordetella pertussis, Pasteurella multocida, Gram-positive bacilli including Corynebacterium diphtheriae and anaerobic species. In vitro activity against Haemophilus influenzae is still a controversial subject. Macrolides are among the best tolerated antibacterial agents. Theoretically, macrolides could be given to a large range of patients even those suffering from underlying diseases. The new macrolides, roxithromycin, azithromycin, clarithromycin, dirithromycin, rokitamycin and miokamycin, are indicated for the treatment of upper respiratory tract infections and lower respiratory tract infections due to intracellular bacteria or Mycoplasma pneumoniae. Macrolides could be used as first line therapy for non-gonococcal urethritis, especially those due to Chlamydia trachomatis or Ureaplasma urealyticum. In pelvic inflammatory infections in which Chlamydia trachomatis is involved macrolides could also be used. Other non-conventional indications under discussion are H. pylori and Lyme's disease. Macrolides in combination with other antibacterials could be an alternative for Mycobacterium avium-intracellulare infections. The antiparasite effect of erythromycin has been known since the 1950s. Extensive experimental work is currently underway to determine the potential use of these drugs in this setting. Research during the 80s in the macrolide field, led to enhanced pharmacokinetic properties. Current research is focused on expanding the antibacterial spectrum and to overcome cross-resistance among 14-membered-ring macrolides.

Since the pioneer work by Metchnikoff, the goal of cooperation between therapeutics and the host defence system (HDS) has been sought after. This area of research received less attention after the introduction of antibiotics. Although, the predictive efficacy of antibacterial agents (ABA) is still evaluated in terms of MICs, MBCs, and pharmacokinetics, much evidence derived from clinical studies underlines the need for synergy between HDS and these drugs to obtain optimal therapeutic efficacy. The analysis of the immunomodifying properties of ABA has come under intense study. The majority of ABA does not substantially affect the functioning of the immune system at least in vivo, despite in-vitro observations of enhancement/inhibition of various immune parameters by some cephalosporins, macrolides, cyclins, aminoglycosides, etc. By contrast, chloramphenicol, sulphonamides and various beta-lactams may be responsible for drug-induced neutropenia whereas macrolides and quinolones, due to their high phagocytic uptake, synergize with phagocytes to destroy intracellular pathogens. Recently, the concept of Biological Response Modifier (BRM)-antibiotics has come under the limelight with the introduction of cefodizime, a new parenteral cephalosporin, which seems to be endowed with immunomodulating properties. This latter aspect has been demonstrated in vitro (potentiation of the phagocyte antimicrobial activity), ex vivo in immunocompromised animals and humans (restoration of various immune parameters) and in vivo (infection models using both sensitive and resistant species). Although the underlying mechanism has not been elucitated, the chemical structure responsible for this BRM activity has been recognized as the thio-thiazolyl moiety at C3 position of the cephem nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphonuclear neutrophils (PMN) play a prominent role in the host response to infectious diseases. One major bactericidal mechanism used by these cells is the production of reactive oxygen species during what is referred to as the oxidative burst. However, excessive oxidant generation can also be involved in cell and tissue damage associated with severe inflammatory reactions. Macrolide antibiotics are able to penetrate and concentrate within phagocytes and have been successfully used to treat infections due to facultative intracellular pathogens. However, intracellular accumulation of macrolides with possible alkalinization of cellular compartments may interfere with normal cell function. In-vitro and ex-vivo data suggest that macrolides affect various phagocytic functions. This paper presents an overview of the published data concerning the modulation of neutrophil function by macrolides. Preliminary data concerning the in-vitro modulation of the neutrophil oxidative burst by dirithromycin and its metabolite, erythromycylamine, are also discussed.

Polymorphonuclear neutrophils play a crucial role in host defences against infectious diseases. New trends in anti-infectious therapy require knowledge of the possible interactions between the drugs and the natural defence system. This overview summarizes some of the in vitro data on the effects of anti-infectious agents on neutrophils. The relevance for the clinical situation is discussed.

In order to counteract an ever increasing bacterial resistance, a new trend in antibiotic therapy is to try and obtain compounds with "immunostimulating" properties. Although the macrolides have been known for more than 30 years, their interaction with the host defence system has been poorly investigated. These drugs display an outstanding ability to penetrate and concentrate in phagocytes. Few depressive effects on phagocyte functions have been reported, while some macrolides have been shown to exert an immunostimulating effect in vitro and ex vivo. The data published on this subject are summarized in this review paper. Further studies would be required for a better understanding of structure-immunomodulating activity relationships.

We have compared the in-vitro interaction of five macrolides (roxithromycin, erythromycin, spiramycin, oleandomycin and josamycin) with human neutrophils (PMN). Only roxithromycin strongly impaired the oxidative burst of PMN assessed by luminol amplified chemiluminescence, superoxide anion generation, and myeloperoxidase-mediated iodination of proteins. This effect was observed only for high concentrations of this drug (100 and 50 mg/l). Furthermore, the sensitivity of PMN to the depressive effect of roxithromycin permitted the definition of two kinds of PMN: in Highly Sensitive (HS)-PMN, the oxidative response was completely abolished while in Moderately Sensitive (MS)-PMN, a decreased, but yet measurable (20-50% of the control), response was obtained. The roxithromycin-induced depression of PMN was time-dependent and partly reversed by washing. Chemotaxis was also impaired by roxithromycin (100 mg/l) but phagocytosis of Klebsiella pneumoniae was unaltered even at high concentrations of the drug. Since roxithromycin displays the highest intracellular uptake, compared with the other macrolides assessed in this study, this could explain the results observed here. The relevance to the clinical situation needs further study. This effect of roxithromycin could be useful to control the inflammatory process associated in certain infectious diseases, in particular if high concentrations of the drug are obtained in tissues.