Cellular mechanisms of inflammation

The second meeting of the contact group placed under the patronage of the Fonds National de la Recherche Scientique (FNRS, Belgium) and entitled ‘Cellular Mechanisms of In ammation’ held in Yvoir, Belgium, on 11 October 1996. This meeting began with the master lecture by Dr R. Monteiro (INSERM, Paris) on the potential role of the IgA Fc receptors (FcaR) in the catabolism of IgA. This set the stage for communications by Godding et al. and Hoang et al. dealing with the role on interferon gamma on the production of secretory IgA by human bronchial epithelial cells in vitro and the regulation of cytokine production by human mononuclear cells of the lamina propria, respectively. Results from research on the production of cytokines by human chondrocytes (Henrotin et al.) and during human muscular exercise (Croisier et al.) were presented. Courtois et al. described the purication of a NADH-hypothiocyanite-oxidoreductase in Streptococcus s anguis . Mathy-Hartert et al. documented the protective effect of ceftazidime on human endothelial cells damaged by anoxiareoxgygenation in vitro. Various aspects of neutrophil functions were studied in vitro by Mouithys-Mickalad et al. and in trauma patients by Deby-Dupont et al. We are happy to present the results of these experimental studies in the current issue of Mediators o f In amm ation. This meeting has bene ted from the assistance of the Fonds National de la Recherche Scientique (FNRS, Belgium). We are deeply grateful to all the contributors. Secretory component (SC) production by human bronchial epithelial cells is upregulated by interferon gamma (IFN-c)

We are happy to present the results of these experimental studies in the current issue of Medi ators of In amm ation.
This meeting has bene ted from the assistance of the Fonds National de la Recherche Scienti que (FNRS, Belgium). We are deeply grateful to all the contributors.

Secretory component (SC) production by human bronchial epithelial cells is upregulated by interferon gamma (IFN-c)
V. Godding, 1,2 Y. Sibille, 1,2 M. Delos, 1 D. Giffroy, 2 A. Langendries 2 and J. P. Vaerman 2 1 UCL Mont-Godinne, 5530 Yvoir and 2 Experimental Medicine, ICP, UCL, 1200 Brussels, Belgium Secretory IgA (SIgA) is the major immunoglobulin present in human respiratory secretions. Secretory component (SC) production by the epithelial cell regulates the transcytosis of IgA and its release as SIgA at the apical pole of the epithelial cell. We studied SC production and transcytosis by cultured HBEC monolayers. HBEC were isolated from healthy segments of bronchial tubes obtained from thoracic surgery. In primary culture on collagen-coated plates, HBEC production of SC was upregulated by IFN-c 100 U ml at 48 and 72 h (4.5 4.1 ng ml 10 5 cells vs 8.9 7.1 ng ml 10 5 cells at 48 h; 5.7 ng ml 10 5 cells 3.7 ng ml 10 5 cells vs 13. In early post-traumatic phase, the complement fragments released by the activation of the classical and alternative pathways play a pivotal role early in trauma by increasing permeability and activating phagocytic cells. These cells undergo respiratory burst and release a variety of in ammatory mediators and enzymes, capable of producing local cellular and tissue damages, but also of spreading the in ammatory reaction to other organs leading to the development of a systemic in ammatory reaction and multiple organ failure. In 15 patients (12 men, 3 women; mean age: 42 16 years) with severe polytrauma (at least two major injuries to chest or abdomen and limbs) and a mean Injury Severity Score of 45 14, we studied the evolution of the plasma concentrations of three markers of neutrophil activation, myeloperoxidase (MPO) and elastase (two speci c enzymes from azurophilic granules), and lactoferrin (released from speci c granules) from the rst hour until 84 h after trauma. Nine of these patients developed ARDS ( ve within 24 h) and 10 presented septic complications after 2 to 6 days. High mean values of the three enzymes were observed in the rst hours after trauma: 1605 ng ml for MPO after 1 h, 1046 ng ml for elastase after 6 h and 77 ng ml after 12 h for lactoferrin (control values: 320 80, 97 25 ng ml and 16 5 ng ml for MPO, elastase and lactoferrin respectively). MPO returns to normal values 30 h after trauma, while elastase and lactoferrin decrease but remain above normal values until 84 h after trauma. Uptake of MPO by monocytes or macrophages could explain this rapid disappearance from plasma. These observations rmly establish that multiple trauma induces a rapid and intense activation of phagocytic cells, as a consequence of both complement activation and direct stimulation. This early stimulation can be responsible for an explosive chain reaction resulting in subsequent multiple organ failure. Ninety to 95% of intraepithelial lymphocytes (IEL) are T cells (CD3 ) and of these, approximately 80% are of the CD8 phenotype. Although their precise role in vivo remains enigmatic, it has been shown in vitro that under particular conditions human IEL are able to play some regulatory functions. It is believed that they may communicate with the adjacent epithelial cells and lamina propria cells. As an example, we have previously shown that colonic human IEL can inhibit the proliferation of autologous lamina propria mononuclear cells (LPMNC). IEL were also able to suppress IgA synthesis. Inhibitory mechanisms were shown to occur through a soluble factor, possibly a cytokine.
The aim of the study was to test the ability of colonic human IEL to produce interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10). Furthermore, the putative immunoregulatory function of IEL on cytokine secretion by autologous LPMNC was examined.
Mucosal lymphocytes were obtained from colonic resection specimens. To study the immunoregulatory activity of IEL, 10 6 IEL were cultured with 10 6 LPMNC for 72 h with or without phytohaemagglutinin A (PHA). The control cultures consisted of IEL or LPMNC alone with or without PHA. Concentrations of IL-2, IFNgamma and IL-10 in culture supernatants were measured using an ampli ed sensitivity immune assay.
These data show that colonic IEL are able to produce IFN-gamma, IL-2 and IL-10 following stimulation with PHA even if the absolute amounts of cytokines produced were small. Furthermore IEL can enhance the PHA-induced synthesis of IL-2 and interferon-gamma by LPMNC but not that of IL-10. This phenomenon may prove of importance in the modulation of subsequent immunopathological reactions including both activation of in ammatory cells and the expression of class II expression molecules on epithelial cells. To address the question of whether the traininginduced reduction of delayed onset muscle soreness (DOMS) and musle damage caused by maximal eccentric contractions could be explained by a decrease of the in ammatory response to damaging contractions, 10 moderately active male volunteers were randomly assigned to two age-matched groups: a control group (CG; n 5) and a trained group (TG; n 5). All subjects were submitted to two isokinetic exercise sessions in the eccentric mode consisting of three stages of 30 maximal contractions of the knee extensor and exor muscle groups of both legs separated by 1 min rest phases, on a Kin Trex device at 608 s angular velocity. These exercise sessions were separated by a period of 3 weeks during which the subjects of the CG abstained from strenuous exercise while the volunteers assigned to the TG were submitted to ve training sessions consisting of ve stages of 10 submaximal contractions of the knee extensor and exor muscle groups of both legs according to the above protocol, with one to two training sessions per week. The rst isokinetic exercise test was followed by severe muscle pain in previously active muscles, and by signi cant increases in serum creatine kinase activity (SCK) and myoglobin concentration (SMb) in both groups ( p , 0 001); those reached peak values 48 h after the exercise session. While the mean values of DOMS, SCK and SMb remained practically unchanged over time after the second isokinetic test in the CG, training was accompanied by a signi cant decrease of these variables ( p , 0 05). Blood levels of interleukin-6 (IL-6) and C-reactive protein (CRP) did not change signi cantly over time and were not in uenced by training. The hypothetical relationship between muscle damage and the production of IL-6 and CRP was not con rmed by the present results. Nitric oxide (NO) has been shown to be the mediator of the suppressive effect of IL-1b upon proteoglycans synthesis by chondrocytes. Moreover, NO induced chondrocytes apoptosis and inhibited cells proliferation. This study aimed to investigate the role played by NO in the IL-1b-stimulated cytokine productions by human chondrocytes. Chondrocytes were isolated from knee joint cartilage by enzymatic digestions. Chondrocytes were plated at 2 10 5 cells per 24-well plates and cultured for 48 h in the absence or in the presence of rhIL-1b at the concentration of 4 ng ml and with or without 1 mM of L-N G -monomethylarginine (L-NMA). IL-6 and IL-8 were assayed in the culture medium by speci c EASIAs. NO formation was detected by NO 2 accumulation in the culture supernatants by Griess reaction with sodium nitrite as standard. Chondrocytes synthesized large amounts of nitric oxide (NO) following exposure to rhIL-1b. IL-1b also strongly stimulated IL-6 and IL-8 productions. Treatment of chondrocytes with L-NMA, a competitive inhibitor of NO synthase, inhibited both spontaneous and IL-1b-stimulated NO production but did not signi cantly modify cytokine productions.
These ndings suggest that endogenously synthesized NO is not the mediator of the IL-1bstimulating effect on IL-6 and IL-8 productions. We have shown that the cephalosporin antibiotic ceftazidime (CAZ) inhibits lipoperoxidation, desactivates singlet oxygen and protects endothelial cells (EC) against the oxidant stress induced by stimulated leukocytes. 1 By electron spin resonance (ESR) studies, we recently demonstrated the direct trapping by CAZ of free oxygen radicals (superoxide anion and hydroxyl radical) produced by phorbol myristate acetate stimulated neutrophils. We now study the effects of CAZ on ischaemia-reperfusion (IR) syndrome, a situation which is accompanied by the production of free radicalar species. Conuent EC, from human umbilical vein, were submitted to anoxia (100% N 2 ) at 378 C for 210 min and reoxygenated for 30 min (95%air± 5%CO 2 ) in Hanks' balanced salt solution buffer. CAZ was added to the cells before starting anoxia. Cytotoxicity was assessed by the 51 Cr release method and by electronic microscopic observations. CAZ was protective (with variations from one cell batch to another) in a dosedependent manner (log regression, r 2 0 93): for CAZ concentrations of 3.10 5 , 5.10 5 , 10 4 , 3.10 4 and 10 3 M, we observed a protection of respectively 2 8 8 1, 43 1 5 9, 60 1 15 1, 90 8 5 0 and 96 1 3 3%, statistically signicant ( p , 0 01) from control value (without CAZ) except for the 3.10 5 M concentration. Electronic microscopic observations con rmed the protective action of CAZ: main structures of the cells were conserved, swelling of mitochon-dria and lysis areas were reduced compared with control cells. CAZ can thus be considered as a potential protective agent in clinical situations of IR and organ preservation before transplantation. Ten years ago, Lambeth's group reported the inhibitory effect of sphinganine, a biogenic amine, on NADPH oxidase in whole cells, 1 and ascribed it to the inhibition of protein kinase C. The inhibition of NADPH oxidase by physiological amines is of interest in relation to the regulation of O 2 generating activity. Sphingosine and its N-acetyl analogue are present in mammalian cells (especially in lipid of central nervous system) and seem to be involved in NADPH oxidase activity.
In the present work, we examined the effect of sphingosine analogues (N-acetyl and N-hexanoyl-sphingosine) on active oxygen species generated by stimulated human neutrophils (PMN) using both luminol-dependent chemiluminescence (CL) and electron spin resonance (ESR) associated to spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin trap. PMN (6 3 10 6 cells ml for ESR assays and 1 3 10 6 cells ml for CL measurements) were stimulated by phorbol myristate acetate at an initial concentration of 10 6 Min Hank's balanced salt solution pH 7.4. CL and ESR results clearly demonstrate that sphingosine (from 2 to 8 3 10 6 M), C 2 -ceramide (N-acetyl-sphingosine) and C 6 -ceramide (N-hexanoyl-sphingosine) at 2 3 10 5 and 2 3 10 4 M, inhibit in a dose-dependent manner the NADPH oxidase activity by interacting with phosphatidylserine (PS) component of protein kinase C, and may function physiologically as negative effectors of this enzyme. Sphingosine exhibits more pronounced effects than its analogues. According to Bazzi et al. 2  exert its function by decreasing the availability of PS or by inhibiting the substrate phospholipid interaction, resulting in the inhibition of NADPH oxidase activity.