Pulmonary granulocyte influx and impaired alveolar macrophage adenylyl cyclase responsiveness in developing respiratory distress

Alveolar macrophages have recently been postulated as being involved in the aetiology of adult respiratory distress syndrome (ARDS). To evaluate their role, basal cyclic AMP levels and responsiveness of adenylyl cyclase alveolar macrophages were determined at four intermediate stages of developing respiratory distress in piglets using a protocol with repeated lung lavage. Examination of alveolar cells recovered from the subsequent lavages reveals an influx of granulocytes (neutrophils and eosinophils) within 1 h of two intensive lung lavages. During the developing respiratory distress the basal cyclic AMPlevel of alveolar macrophages increases and adenylyl cyclase responsiveness to prostaglandin E2 (PGE2) and isoprelanaline diminishes. The previously observed impairment of macrophage activity can then be explained at a subcellular level.


Introduction
Polymorphonuclear neutrophils (PMN) and monocytes activated by phagocytic or soluble stimuli produce large amounts of microbicidal oxidants that play a key role in host defences against pathogens. [1][2] This metabolic response, known as the 'respiratory burst', involves two enzyme pathways: (i) NADPH oxidase activation leads to the reduction of molecular oxygen to superoxide anion and the subsequent generation of hydrogen peroxide, 3 and (ii) the activity of neutrophil myeloperoxidase (MPO), an enzyme located in the azurophilic granules that are released following degranulation, catalyses the reaction of H202 with chloride, leading to the formation of other oxidants such as hypochlorous acid and chloramines. 4 These latter, known as long-lived oxidants, may be mediators of phagocyte-induced oxidative damage 6 as they are either released or generated via the extracellular MPO in the extracellular environment. Their ability to inactivate protease inhibitors such as 0q-antiproteinase 7 is one way in which chloramines could potentiate deleterious effects in inflammatory processes. The presence of chloramines in biological fluids seems to be highly dependent on the status of antioxidant defences. Natural killer activity, lymphocyte proliferation in response to mitogens, and immunoglobulin and interleukin-2 secretion are apparently affected by H202 and MPO. 9' The aim of this study was to investigate potential interactions between PMN-derived chloramines and lymphocytes. Glutathione, the major nonprotein thiol, is present in virtually all cell types and is involved in numerous biological functions, acting as a co-enzyme, antioxidant, and regulatory molecule in cell cycle initiation and progression 2 and microtubule formation. 13 In addition, glutathione content is important in lymphocyte activation and proliferation. 14 '5 The authors therefore investigated (1) the influence of lymphocytes on chloramine release by PMN, (2) the biochemical mechanism of this action, and (3) the biological consequences of such an interaction on lymphocyte functions.
Cell preparations: Normal PMN were isolated from heparinized (Liquemine Roche, 10 U/ml) venous blood of healthy donors (Centre de Transfusion de l'H6pital Necker). 16 Briefly, PMN were separated from erythrocytes by plasmagel sedimentation, followed by Ficoll-Hypaque density centrifugation. Residual erythrocytes were lysed by treating the cell pellet with a lysis buffer containing ammonium chloride. The purified PMN were then resuspended in HBSS at 106 cells/ml. Mononuclear cells obtained after Ficoll-Hypaque density centrifugation were added to Falcon 3047 tissue culture dishes (Falcon Labware, Becton Dickinson and Co., Oxnard, CA) and incubated for 60 min at 37C in humidified 95% air/5% COg for monocytes to attach. Lymphocytes (non-adherent cells) were then harvested, washed and seeded at the required density.
Chloramine production and PMN supernatant preparation: PMN incubation and taurine-chloramine production were carried out as described previously, iv Briefly, PMN were incubated for 1 h at 37C with constant gentle shaking in the absence (controls) or presence of PMA, (final concentration 1 #g/ml), or opsonized zymosan (OZ) particles (2mg/ml) prepared as described elsewhere. 16 PMN incubation was performed in the absence or presence of autologous lymphocytes. Taurine-chloramine formation was enhanced by adding taurine (15 mM) to the incubation medium. At the end of the incubation period the samples were centrifuged at 4C (1 200 x g for 10 min) the supernatants stored at -20C for testing on the following day. OZ-stimulated PMN supernatants were used as a source of biological chloramines. To avoid cross-reactions with degranulation proteins, supernatants were filtered with 3-kDa cut-off Centricon filters (Amicon, Grace, France). Filtration did not affect the chloramine concentration (data not shown).
Colorimetric chloramine determination in PMN supernatants: Chloramines were determined by colorimetric measurement of the triiodide ion formed by the oxidation of potassium iodide (KI) iv in 96-well microtitre plates (Falcon Labware, Becton Dickinson and Co.; Oxnard, CA). Two hundred /il of sample (supernatant or the chloramine-T standard solution) was placed in each well. Ten/il of 1 (106 cells/ml in HBSS) were exposed either to autologous PMN-derived chloramines (determined by the colorimetric assay) or to synthetic chloramine (chloramine-T) in HBSS (without serum in order to avoid chloramine scavenging or interference in the glutathione assay). After 1 h of incubation at 37C under gentle shaking, lymphocytes were centrifuged and the chloramine concentration was assayed in the supernatant; the cell pellet was resuspended in 5% serum RPMI 1640 medium for cell culture experiments or in HBSS for GSH determination.
Cell viability: After exposure to chloramines, lymphocyte viability was measured in terms of Trypan blue exclusion. Twenty #1 of Trypan blue (0.5%) was added to 80/il of lymphocyte suspension (106 cells/ml).
Glutathione determination: The total GSH concentration in lymphocytes was determined by means of an enzymic recycling assay. TM After pretreatment with chloramines, lymphocytes (106/tube) were centrifuged and resuspended in 200#1 of 5% sulphosalicylic acid and sonicated. After centrifugation (10 min, 20 000 x g), GSH was measured in the supernatant. The assay was performed in microtitre plates as follows: in each well, 140/il of NADPH (0.3 raM), 30/ll of DTNB (5 raM) and the sample (or GSH standard solution) were mixed and incubated for 10 min at 30C. The optical density at 405 nm was measured 1 min after triggering the reaction with 20/il of glutathione reductase (2 U/ml). The oxidized GSH concentration (GSSG) was determined after sample derivatization by adding 2#1 of vinyl pyridine and 8 #1 of triethanolamine to 100/il of sample (or GSSG standard solution). GSSG was then determined in the same manner as GSH. The concentration of reduced GSH was calculated as the difference between total GSH and GSSG.
Each well contained 2 x 10 lymphocytes and the proliferative response was induced by adding either Con-A (1/ig/ml) or PHA (0.0S/ig/ml) in a final volume of 200/il. Plates were incubated for 44 h at 37C in a humidified atmosphere containing 5% COg. Cultures were pulsed with 5/iCi (1 Ci 37 GBq) of [3H]-thymidine (5 Ci/mmol, Chloramine scavenging by lymphocyte glutathione CEA, Saclay, France), harvested 4 h later and counted for beta emission. Each value of proliferation has been determined in triplicate.
Statistical anasis: Results (mean q-S.E.M.) were compared using Student's two-tailed t-test (paired or unpaired, as appropriate). Differences were considered significant when the p value was below 0.05. Correlation coeflCicients were calculated by using simple regression analysis.

Results
Injquence of mphocytes on PMN chloramine release: Chloramine secretion by a purified PMN suspension (106cells/ml) in the absence and presence of autologous lymphocytes (106/ml) was compared first. As shown in Fig. 1, co-incubation of PMN with viable lymphocytes significantly decreased the chloramine concentration in supernatants of PMAand OZ-stimulated PMN. This effect was even more pronounced when lymphocytes were lysed prior to incubation. In the absence of PMN, lymphocytes exerted a significant scavenging effect on both PMN-derived chloramines and chloramine-T (Fig. 2). In contrast, the supernatant of lymphocytes had no effect on PMN-derived chloramines (data not shown). Role of glutathione in mphocyte scavenging of chloramines: To investigate the molecular basis of chloramine scavenging by lymphocytes, the reactivity of glutathione with chloramines in cell-free conditions was tested. Figure 3   Mediators of Inflammation. Vol 2.1993 ;237 had a concentration dependent inhibitory effect on both chloramine-T and PMN derived chloramines. In order to investigate whether the chloramine scavenging effect of lymphocytes involved intracellular GSH, lymphocyte glutathione content before and after 1-h exposure to either chloramine-T or PMN derived chloramines were compared. As shown in Fig. 4, chloramines significantly decreased the lymphocyte glutathione content in a concentration dependent manner. Although this decrease was observed with both total and reduced GSH in the case of PMN derived chloramines (Fig. 4a), it was highly significant only in the case of chloramine-T (Fig. 4b).
Effect of chloramines on mitogen-induced lymphocyte proliferation: To obtain further evidence of an interaction between chloramines and 1}mphocyte functions, lymphocyte proliferative responses to mitogens were studied following exposure to chloramines. In this part of the study, only OZ was used to stimulate PMN chloramine production since PMA may have a direct effect on lymphocyte proliferation. In addition, supernatants of OZ-stimulated PMN were filtered to remove proteins with molecular weights greater than 3 kDa. Table 1 shows that chloramines did not affect lymphocyte viability, at least up to 50 #M. In the absence of mitogen, neither synthetic nor PMN derived chloramines significantly affected lymphocyte [3HI thymidine incorporation. Fig. 5a shows the influence of PMN derived chloramines on lymphocyte proliferation induced by Con-A and PHA.  a decrease in cellular glutathione. 23 In a cell-free system, it was found that only the reduced form of Significant inhibition was observed only when glutathione is able to scavenge both chloramine-T lymphocyte proliferation was induced by Con-A and PMN derived chloramines. Interestingly, the (respectively 28 and 44% for chloramine concentrareaction of chloramines with GSH has recently been tions of 10 and 50/zM). On the other hand, studied in a model of liver injury. 24 The authors chloramine-T (Fig. 5b) induced a significant found that chloramines (monochloramine and concentration dependent inhibition regardless of taurine-chloramine) were eflqciently detoxified in the mitogen used, for chloramine-T concentration the liver by intracellular GSH, inducing GSH greater than 10/M. This is confirmed by the depletion and a subsequent increase in perfusion correlation (r 0.96) found between lymphocyte pressure and a decrease in bile flow. As a result, GSH content and proliferative responses in case of they suggested that chloramines could contribute to stimulation by Con-A (Fig. 6). In contrast, PMN-induced organ injury through a GSH PHA-induced proliferative response at low chlormediated pathway. amine concentration (10/.zM) seems to be less Our results indicate that both the total and sensitive to chloramine and thus less dependent on reduced glutathione content of lymphocytes were glutathione availability, reduced in a concentration dependent manner after chloramine challenge. Reduced glutathione is able Discussion to scavenge chloramines, giving rise to GSSG; GSSG would subsequently be reduced by intra-This study shows that chloramines, described as cellular glutathione reductase and reconverted to long-lived oxidants, may be important immunomo-GSH. Under conditions of continuous exposure to dulatory agents, the eft'ect of which is mediated by chloramines, GSSG may accumulate because the the ubiquitous thiol-containing molecule, glurate of GSSG formation exceeds that of its tathione.ll reduction; 11 GSSG can then form mixed disul-The results suggest a potentially important phides with intracellular or extracellular proteins, scavenging role of lymphocytes for PMN derived resulting in a net loss of total glutathione, chloramines, which are thought to be exported into as seen in this study. The lymphocyte total the surrounding environment, and to potentiate glutathione content observed (mean _ _ _ S.E.M., PMN-induced oxidative damage. 19 The physio-0.7 _-F 0.15 nmol/106 cells) is in the range of logical relevance of this scavenging mechanism concentrations reported in T cells by other groups needs to be determined while the possible using the same GSH assay. 12q4 2O involvement of other plasma antioxidants and Given the multiple roles of glutathione in cell other cell types, like red blood cells, which contain functions, especially in lymphocytes, the conglutathione and which could compete for chlorsequences of direct chloramine exposure for amines might also be considered. However, in lymphocyte proliferation were investigated. Lyminflammation-induced oxidative stress, when plasphocyte proliferation was eflqciently inhibited by ma antioxidant defence may be overwhelmed, chloramine-T, regardless of the mitogen used. In lymphocytes might play an important role in the case of PMN derived chloramines, only oxidant scavenging. Both viable and lysed lympho-Con-A-stimulated lymphocytes showed signifi-cantly inhibited proliferation. These results suggest that the lymphocyte proliferative response to Con-A is more dependent on the glutathione concentration than that induced by PHA. In addition, chloramine-T was much more efficient than PMN derived chloramines in decreasing both the reduced and oxidized glutathione content of lymphocytes. In lymphocyte proliferation experiments, the same difference was observed. Although PMN supernatants were filtered to remove proteins with a molecular weight greater than 3 kDa, low molecular weight antioxidants could interfere with the effect of biological chloramines, this interference being overcome at high chloramine concentrations. Lymphocyte functions show different degrees of susceptibility to oxidative injury by MPO and H202: PHAand Con-A-induced proliferation displayed intermediate degrees of susceptibility between pokeweed induced proliferation and antibody formation, this latter function being the most sensitive to oxidative injury. 9 These latter authors used a GGO as the HiOi-generating system in the presence or absence of MPO. At a rate of production of 60#mol/ml/min of H202, the lymphocytes were 90% viable. This is compatible with our results showing no effect of chloramines on lymphocyte viability when the chloramine concentration was less than 50 pM and there was only a small loss of viability when the concentration reached 100 pM. The experimental conditions in the two studies were very similar.
Although the precise role of GSH in lymphocyte activation remains unclear, GSH synthesis is clearly essential to maintain normal lymphocyte proliferation and cytokine metabolism. As direct relationships exist between the proliferative response and glutathione availability, 15 lymphocyte proliferation can be enhanced by providing excess glutathione, cysteine or 2-mercaptoethanol, while it is strongly inhibited by I-buthionine-(S,R)-sulphoximine (BSO), a specific and irreversible inhibitor of 3-glutamyl cysteine synthetase that limits the quantity of intracellular GSH available during the activation process. This has been demonstrated both with T cells and purified large granular lymphocytes. 25 GSH enhances the effect of IL-2 and IL-4 on replication and [3H]thymidine incorporation of IL-2 and IL-4 dependent ceils, respectively, in vitro. This potentiating effect of GSH is accompanied by an increase in the intracellular GSH level and an enhancement of binding, internalization and degradation of both cytokines. 26'27 This study provides some insights into the molecular mechanisms involved in the effect of oxidants on mitogen-induced lymphocyte proliferation and identifies a possible mechanism for the immunodepression observed following oxidative stress. Indeed, there was a close correlation between lymphocyte proliferation and GSH content after chloramine-T exposure, suggesting that lymphocyte GSH is one of the molecular targets of chloramines. In conclusion, the results emphasize the relationships between PMN and lymphocytes, two major cell types in host defences.