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Neutrophils and monocytes are the major classes of phagocytes that migrate from the blood stream and accumulate in inflamed tissues in response to various chemoattractants. Because IL-10 is a potent anti-inflammatory cytokine, we analyzed its in vitro effect on chemotaxis using an under agarose method. We found that, as compared to prednisolone, IL-10 alone was a modest inhibitor of C5a, fMLP and IL-8-induced neutrophil chemotaxis, and C5a-induced monocyte chemotaxis. However, GM-CSF pretreatment of the cells potentiated this inhibitory effect. Similarly, the IL-10 induced modulation of the beta2 integrin CD11b/CD18 adhesion molecule expression was only observed on GM-CSF-preactivated neutrophils and monocytes. Taken together, these results suggest that the migration and accumulation of phagocytes at infection sites would not be significantly affected by IL-10 given as an immunomodulatory therapy.

Vitamin E (alpha-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. alpha-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O-2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of alpha-tocopherol on O-2 production by human monocytes. In activated monocytes O-2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, alpha-tocopherol (but not beta-tocopherol) inhibited O-2 production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that alpha-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47(phox) were reduced in monocytes preincubated with alpha-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47(phox) was similar to that of neutrophil p47(phox), indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47(phox) was immunoprecipitated from alpha-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by alpha-tocopherol in a specific manner compared with that of beta-tocopherol or Trolox(R). Membrane translocation of PKC was not affected. These results show that alpha-tocopherol inhibits O-2 production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47(phox) results from a decrease in PKC activity.

Antibiotics do not act alone but in conjunction with the host defence system. In particular, it has been shown that antibiotics can modify cytokine production. The authors reported here the effects of antibiotics which penetrate inside the cells, such as quinolones and macrolides, on the capacity of blood monocytes to produce IL-I alpha, IL-1 beta, TNF alpha and IL-6 in response to endotoxin. Antibiotics can exert a differential effect on cytokine production: in fact, quinolones, in vitro, at concentrations higher than 25 micrograms/ml decreased IL-1 beta, TNF alpha and IL-6, while they do not modify IL-1 alpha. Moreover, ciprofloxacin orally administered (25 mg/kg for 7 days) transitory increased cytokine production. These results are discussed in terms of tissue concentration. Among the same family of antibiotics such as macrolides, differences on cytokine modulation were observed: spiramycin and erythromycin increased IL-6 production while roxithromycin did not exert any significant effect. All these results tend to prove that some antibiotics are immunomodulators; however interactions between antibiotics and immune responses are complex and studies with patients with infections will be necessary to a better understanding of these relationships.

To gain further insight into the pathogenesis of the adult respiratory distress syndrome (ARDS), the authors studied possible relationships among the activation status of circulating polymorphonuclear neutrophils (PMN), cytokine levels, and the severity of lung injury in 31 patients: 15 with ARDS, 9 with severe pneumonia uncomplicated by ARDS, and 7 mechanically ventilated patients with neither ARDS nor pneumonia. Nine healthy subjects served as controls. Using flow cytometry, the authors identified a subpopulation of PMN with an increased capacity to generate hydrogen peroxide after stimulation ex vivo in all three patient groups; significantly higher values were found in those with ARDS. The PMN stimulation index, a reflection of the degree of hyperresponsiveness, correlated with elevated levels of tumor necrosis factor alpha (TNF-alpha) in plasma, and both spontaneous and lipopolysaccharide (LPS)-induced TNF-alpha production by cultured monocytes. These biological expressions of PMN activation and cytokine generation both correlated with indices of the severity of lung injury, but not with the overall clinical severity. In contrast, IL-6 and IL-1 beta showed little or no relationship with either the degree of lung injury or PMN hyperresponsiveness. We conclude that TNF alpha-primed PMN may play a major role in the pathogenesis of ARDS-associated lung injury.

We have tested the hypothesis that cellular activation events occurring in T lymphocytes and monocytes and mediated through translocation of the transcription factor NF-kappa B are dependent upon the constitutive redox status of these cells. We used phenolic, lipid-soluble, chain-breaking antioxidants (butylated hydroxyanisole (BHA), nordihydroquairetic acid, or alpha-tocopherol (vitamin E) to show that peroxyl radical scavenging in unstimulated and PMA- or TNF-stimulated cells blocks the functions depending on NF-kappa B activation. BHA was found to suppress not only PMA- or TNF-induced, but also constitutive, HIV-enhancer activity concomitant to an inhibition of NF-kappa B binding activity in both lymphoblastoid T (J.Jhan) and monocytic (U937) cell lines. This was also true for KBF (p50 homodimer) binding activity in U937 cells. Secretion of TNF, the product of another NF-kappa B-dependent gene, was abolished by BHA in PMA-stimulated U937 cells. The anti-oxidative effect of BHA was accompanied by an increase in thiol, but not glutathione, content in stimulated and unstimulated T cell, whereas TNF stimulation itself barely modified the cellular thiol level. Oxidative stress obtained by the addition of H2O2 to the culture medium of J.Jhan or U937 cells could not by itself induce NF-kappa B activation. These observations suggest that TNF and PMA do not lead to NF-kappa B activation through induction of changes in the cell redox status. Rather, TNF and PMA can exert their effect only if cells are in an appropriate redox status, because prior modification toward reduction with BHA treatment prevents this activation. It appears that a basal redox equilibrium tending toward oxidation is a prerequisite for full activation of transduction pathways regulating the activity of NF-kappa B-dependent genes.

Ciprofloxacin, a new fluoroquinolone antibiotic, inhibits the in vitro production of interleukin 1 beta and tumor necrosis factor alpha by monocytes. We investigated its activity against type II collagen induced arthritis in rats. It exerted a dose dependent preventive effect at 50 and 75 mg/kg/day against clinical and histologic features of collagen induced arthritis without any influence on the production of anticollagen antibodies and alpha 1-acid glycoprotein. This effect was reversible after early removal of the treatment. Ciprofloxacin did not inhibit collagen induced arthritis in adrenalectomized rats but rather caused an exacerbation of the disease. Its effect was not modified by the simultaneous administration of an antiglucocorticoid, RU 40555.

The existence of IL-1 activity on the cell surface of stimulated mononuclear phagocytes is a matter of controversy. In particular, fixation of IL-1-expressing cells for 15 min in 1% paraformaldehyde (PFA) is commonly used to evidence such "membrane-associated" IL-1 activity but other authors have attributed this to passive leakage of IL-1 alpha from the cells and report no activity with longer fixation times. Using specific IL-1 alpha and IL-1 beta assays, we found that after the mild standard PFA fixation procedure, not only IL-1 alpha but also IL-1 beta were released into the supernatants for up to 96 h following fixation; membrane IL-1 activity cannot thus be measured in these conditions. However, using conditions in which neither immunoreactive IL-1 molecules nor IL-1 activity are found in the supernatants (i.e. assay at 144 h, increased fixation time), we were still able to detect IL-1 activity on LPS-stimulated, PFA-fixed monocytes. This activity was independent of the duration of PFA fixation and was inhibited by anti-IL-1 alpha but not anti-IL-1 beta antibodies. Our data thus underline the importance of technical conditions in the study of membrane-associated IL-1 activity.

The effects of penicillin, macrolides (spiramycin and erythromycin), cephalosporins (cefaclor and cefadroxil), cycline (doxycycline) and quinolones (pefloxacin, ciprofloxacin and ofloxacin) on extracellular and cell-associated interleukin-1 activity from human monocytes were investigated in vitro. When cells were treated with 10 micrograms/ml of quinolones, cephalosporins or penicillin, no effect on IL-1 production could be detected. Using 100 micrograms/ml, extracellular IL-1 activity was found to be decreased by quinolones (about 35% of the control without antibiotic) without modification of the cell-associated IL-1 activity. Extra and intracellular IL-1 was only slightly decreased by cephalosporins, while penicillin did not alter the IL-1 activities. Spiramycin and doxycycline using 100 micrograms/ml increased extracellular IL-1 while cell-associated was decreased. A toxic effect may have been exerted by these antimicrobial agents.