Effects of nitric oxide on neutrophil influx depends on the tissue: role of leukotriene B4 and adhesion molecules.

BACKGROUND AND PURPOSE: We investigated the effect of nitric oxide synthase (NOS) inhibition on polymorphonuclear cell (PMN) influx in zymosan or lipopolysaccharide (LPS)-induced arthritis and peritonitis. EXPERIMENTAL APPROACH: Wistar rats received intra-articular (i.art.) zymosan (30-1000 microg) or LPS (1-10 microg). Swiss C57/Bl6 mice genetically deficient in intercellular adhesion molecule-1 (ICAM-1(-/-)) or in beta(2)-integrin (beta(2)-integrin(-/-)) received zymosan either i.art. or i.p. PMN counts, leukotriene B(4) (LTB(4)), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) levels were measured in joint and peritoneal exudates. Groups received the NOS inhibitors N(G)-nitro-L-arginine methyl ester (LN), nitro-L-arginine, N-[3-(aminomemethyl)benzyl] acetamide or aminoguanidine, prior to zymosan or LPS, given i.p. or s.c. in the arthritis and peritonitis experiments respectively. A group of rats received LN locally (i.art. or i.p.), 30 min prior to 1 mg zymosan i.art. KEY RESULTS: Systemic or local NOS inhibition significantly prevented PMN migration in arthritis while increasing it in peritonitis, regardless of stimuli, concentration of NOS inhibitors and species. NOS inhibition did not alter TNF-alpha and IL-10 but decreased LTB(4) in zymosan-induced arthritis. LN administration significantly inhibited PMN influx into the joints of ICAM-1(-/-) and beta(2)-integrin(-/-) mice with zymosan-arthritis, while not altering PMN influx into the peritoneum of mice with zymosan-peritonitis. CONCLUSIONS AND IMPLICATIONS: Nitric oxide has a dual modulatory role on PMN influx into joint and peritoneal cavities that is stimulus- and species-independent. Differences in local release of LTB(4) and in expression of ICAM-1 and beta(2)-integrin account for this dual role of NO on PMN migration.

Platelet and leukocyte adhesion and activation in unstable angina and post-PTCA.

BACKGROUND: Unstable atherosclerotic plaques activate blood cells which may adhere to the coronary endothelium causing vessel occlusion. However, it is unknown if different clinical syndromes associated with plaque rupture induce similar blood cell activation and adhesion to the endothelium. METHODS: We studied changes in adhesion molecule expression of platelets (GPIIb/IIIa), neutrophils--CD18, CD11b and L-selectin--and monocytes (CD14) after interaction with active lesions of patients with stable angina subjected to PTCA and patients with unstable angina (UA). Generation of superoxide (SO) radicals from PMNs and PMN sequestration in the coronary circulation were also assessed. Blood samples were collected from the aorta (Ao) and coronary sinus (CS) before and 15 min after PTCA (n=13) and within the first 48 h of UA (n=12). RESULTS: PTCA induced a marked up-regulation of CD18, CD11b, CD14 and GPIIb/IIIa with L-selectin shedding and reduced SO formation, whereas only minor L-selectin down-regulation and decreased SO production indicated activation in UA. However, a significant decrease in neutrophil count in the CS compared to the Ao was only observed in UA. CONCLUSIONS: The magnitude of cellular activation depends on the underlying clinical setting and just partially contributes to cell adhesion to the endothelium which might be modulated by different extent of vascular occlusion and shear forces.

Fibrinogen-CD11b/CD18 interaction activates the NF-kappa B pathway and delays apoptosis in human neutrophils.

The regulation of neutrophil half-life by members of the coagulation cascade is critical for the resolution of the inflammatory response. We have demonstrated that soluble fibrinogen (sFbg) delays human neutrophil (PMN) apoptosis through a mechanism that involves CD11b interactions, and phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2). Since NF-kappa B is a key element in the regulation of apoptotic mechanisms in several immune cells, we investigated whether NF-kappa B is involved in the control of PMN survival by sFbg. We show that sFbg triggers inhibitor protein kappa B (I kappa B-alpha) degradation and NF-kappa B activation. Furthermore, pharmacological inhibition of NF-kappa B abrogates sFbg effects on apoptosis. In addition, specific inhibition of MAPK ERK1/2 significantly reduces NF-kappa B translocation by sFbg, suggesting a relationship between ERK1/2 and NF-kappa B activation. Similar results are obtained when granulocytic-differentiated HL-60 cells are treated with sFbg, making this model highly attractive for integrin-induced gene expression studies. It can be concluded that NF-kappa B participates in the prevention of apoptosis induced by sFbg with the participation of MAPK ERK1/2. These results shed light on the molecular mechanisms that control human granulocyte apoptosis, and suggest that NF-kappa B regulation may be of benefit for the resolution of the inflammatory response.

Downregulation of mac-1 expression in monocytes by surface-bound IgG.

Physical and functional association between the beta2-integrin Mac-1 (CD11b/CD18) and receptors of immunoglobulin G (IgG) (FcgammaRs) has been previously reported. In this study, we examined the modulation of Mac-1 expression by IgG in different leucocyte populations. Our data show that human monocytes, but not neutrophils, macrophages, dendritic or natural killer cells, downregulate the expression of Mac-1 after overnight exposure to surface-bound IgG. This effect, which requires at least 6 h of incubation, is not associated with a general downmodulation of membrane antigens, and is selectively induced by immobilized IgG (iIgG), as the stimulation of monocytes with N-formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide, tumour necrosis factor-alpha (TNF-alpha) or soluble IgG did not modify the Mac-1 expression after 18 h in culture. The loss of Mac-1 was completely prevented by blocking antibodies (Abs) directed to FcgammaRII (CD32) or CD18. On the other hand, the serine protease inhibitor, phenyl methyl sulphonyl fluoride, but not inhibitors of cysteine proteases or neutral endopeptidases, partially prevented the downregulation of Mac-1 by iIgG. Monocytes cultured overnight on iIgG exhibited a dramatic decrease in their capacity to ingest zymosan particles that could be attributed to the reduced expression of Mac-1. However, there was no inhibition of TNF-alpha production induced by zymosan, suggesting that Mac-1-dependent responses require different levels of its expression to be fully activated.

beta1 and beta2 integrins activate different signalling pathways in monocytes.

Integrin-mediated signals play an important but poorly understood role in regulating many leucocyte functions. In monocytes and macrophages, integrins of the beta2 subfamily are involved in cell-cell interactions that are important for migration of the cells through the endothelium and also for phagocytosis. On the other hand, in the same cells, beta1 integrin-mediated adhesion to extracellular matrix proteins results in a strong induction of immediate early genes that are important in inflammation. To investigate the signalling pathways from these two types of integrin in monocytic cells, THP-1 cells were selectively stimulated via beta1 or beta2 integrins by cross-linking each type of receptor with specific monoclonal antibodies or their natural ligands. The involvement of extracellular signal-regulated kinase (ERK), Syk and phosphoinositide 3-kinase (PI-3K) was then analysed. Nuclear factor kappaB (NF-kappaB) activation was also detected in THP-1 cells transiently transfected with an NF-kappaB-driven luciferase reporter gene. We found that binding of both types of integrin to their natural ligands activated ERK in a Syk- and PI-3K-dependent manner. Yet, cross-linking of integrins by anti-beta1 antibodies caused activation of ERK while that by anti-beta2 antibodies did not. Also both types of integrin activated NF-kappaB. However, PI-3K was required for beta1 integrin-, but not beta2 integrin-, mediated NF-kappaB activation. In addition, inhibition of PI-3K with wortmannin and LY294002 blocked beta1 integrin-mediated NF-kappaB activation, but did not affect that mediated by beta2 integrin. These data suggest that distinct integrins activate different signalling pathways in monocytic cells.

Mechanisms of allergen- and LPS-induced bone marrow eosinophil mobilization and eosinophil accumulation into the pleural cavity: a role for CD11b/CD18 complex.

OBJECTIVE: The mechanisms involved in bone marrow eosinophil emigration and recruitment to inflammatory sites are not fully understood. The involvement of CD11b/CD18 in marrow eosinophil release induced by lipopolysaccharide (LPS) or allergen was investigated in mice. METHODS: Eosinophil and neutrophil counts in the pleural cavity, blood and bone marrow were performed at different time intervals after the intrathoracic injection of LPS (250 ng/cavity) or ovalbumin (OVA, 12 microg/cavity; into actively sensitized mice) and compared to anti-CD11b/CD 18 (5C6, 1 mg/mouse) or anti-IL-5 (TRFK-5, 500 microg/kg) treated mice. RESULTS: LPS induced local eosinophil influx, that peaked within 24 h and that was preceded by a decrease in marrow eosinophils at 4 h. Antigenic challenge induced a decrease in marrow eosinophils within 4 h, followed by a long lasting pleural eosinophil accumulation and a persistent increase in marrow eosinophil numbers. Pretreatment with anti-CD11b/CD18 abolished LPS-induced neutrophil and eosinophil accumulation in the pleural cavity at 4 and 24 h, respectively. This pretreatment failed to modify neutrophil emigration from bone marrow, but significantly inhibited marrow eosinophil release at 4 h post-LPS or OVA challenge. Anti-IL-5 pretreatment failed to inhibit LPS-induced pleural eosinophil accumulation and mobilization from bone marrow, but it abolished allergen-induced effects, indicating a role for IL-5 in marrow eosinophil mobilization induced by antigen, but not by LPS challenge. CONCLUSIONS: Our results suggest that eosinophil migration induced by antigen or LPS into the pleural cavity is preceded by bone marrow eosinophil release through a mechanism that depends on CD11b/CD18.

Integrin alpha2beta1 mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar3.

It was previously reported that integrins alpha2beta1, alpha4beta1, and alphaXbeta2 are involved in rotavirus cell infection. In this work we studied the role of integrin subunits alpha2, alpha4, and beta2 on the attachment of rotaviruses RRV and nar3 to MA104 cells. Integrin alpha2beta1 was found to serve as the binding receptor for the neuraminidase-resistant virus nar3, whereas the neuraminidase-sensitive strain RRV interacted with this integrin at a postattachment step. It was shown that nar3 binds alpha2beta1 through the DGE integrin-recognition motif located in the virus surface protein VP5. Integrin subunits alpha4 and beta2 do not seem to be involved in the initial cell binding of either virus.

Secreted glucocorticoids regulate leukocyte-endothelial interactions in inflammation. A direct vital microscopic study.

Leukocytes come into intimate contact with the venular endothelium as they extravasate from blood to the interstitium during inflammation. In exteriorized tissues, the number of leukocytes rolling along the vessel wall was markedly increased in adrenalectomized and metyrapone-treated animals, relative to sham-operated and normal animals. During the development of an acute, local inflammatory response, rollers were numerically decreased and a stronger adhesion of the cells to the endothelium, with a concomitant migration into tissues, was observed. Adhesion and migration were much more marked in adrenalectomized and metyrapone-treated animals than in controls, suggesting that secreted glucocorticoids exert a suppressive effect on leukocyte-endothelial interactions. The increased number of rolling leukocytes in adrenalectomized and metyrapone-treated animals apparently resulted in more cells available to migrate into inflamed tissues. The effect appears to involve receptor occupancy and induction of gene expression because normal animals receiving the steroid antagonist RU 38 486, actinomycin D, or cycloheximide behaved as adrenalectomized or metyrapone-treated animals. Administration to adrenalectomized animals of a monoclonal antibody to the membrane glycoprotein complex CD18 did not affect the number of rolling cells, but dramatically reduced the number of adherent or migrated leukocytes. It is suggested that secreted glucocorticoids, in addition to an effect on rolling behavior of circulating leukocytes, might also modulate the activity of the glycoprotein complex CD18 on white blood cells. The ultimate consequence is a restrictive effect on cell emigration in inflammation.