The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment.

Thrombin, the terminal serine protease in the coagulation cascade, is a proinflammatory molecule in vivo and induces endothelial activation in vitro. The cellular signaling mechanisms involved in this function are unknown. The role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in thrombin-induced chemokine production was studied. Phosphorylation of both p38 MAPK and its substrate, ATF-2, was observed in human umbilical vein endothelial cells (HUVECs) stimulated with thrombin, with a maximum after 5 minutes of stimulation. Using the selective p38 MAPK inhibitor SB203580, there was a significant decrease in thrombin-induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) protein production and messenger RNA steady-state levels. In addition, SB203580 decreased IL-8 and MCP-1 production induced by the thrombin receptor-1 agonist peptide (TRAP), suggesting functional links between the thrombin G protein-coupled receptor and the p38 MAPK pathway. Furthermore, endothelial activation in the presence of SB203580 decreased the chemotactic activity of thrombin-stimulated HUVEC supernatant on neutrophils and monocytic cells. In contrast, the p42/p44 MAPK pathway did not appear to be involved in thrombin- or TRAP-induced endothelial chemokine production, because there was no reduction in the presence of the p42/p44-specific inhibitor PD98059. These results demonstrate that the p38 rather than p42/44 MAPK signaling pathway plays an important role in thrombin-induced endothelial proinflammatory activation and suggest that inhibition of p38 MAPK may be an interesting target for anti-inflammatory strategies in vascular diseases combining thrombosis and inflammation. (Blood. 2001;98:667-673)

Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106).

Thrombin, a central molecule in coagulation, is also involved in inflammation. Notably, thrombin induces endothelial neutrophil adhesion, P- and E-selectin expression, and chemokine production. We show here that thrombin induces expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106) on human umbilical vein endothelial cells (HUVECs) associated with increased adhesion of monocytes. Thrombin increased mRNA steady-state levels and expression of ICAM-1 over 24 hours. Thrombin-induced VCAM-1 expression exhibited unusual kinetics, reaching maximum levels after 6 to 12 hours, but decreasing to near baseline after 24 hours. Thrombin activity on HUVECs was mediated through interaction with its specific receptor, because ICAM-1 and VCAM-1 expression were similarly induced by the 14-amino acid thrombin receptor-activating peptide. Thrombin-induced ICAM-1 and VCAM-1 expression was significantly inhibited by hirudin, but not by interleukin-1 receptor antagonist or anti-tumor necrosis factor alpha monoclonal antibody (MoAb). Thrombin-activated HUVECs significantly increased greater numbers of adhering THP-1 macrophagic cells, peripheral blood mononuclear cells, or purified monocytes than unstimulated HUVECs. This adhesion was inhibited by anti-CD18 and anti-CD49d MoAb, demonstrating that thrombin-induced ICAM-1 and VCAM-1 were functional. These results show that, in addition to selectins, thrombin directly induces a cytokine-independent expression of adhesion molecules of the Ig superfamily on HUVECs that may support firm leukocyte attachment during inflammation.

Thrombin induces endothelial type II activation in vitro: IL-1 and TNF-alpha-independent IL-8 secretion and E-selectin expression.

In addition to its role in coagulation, thrombin is involved in the inflammatory process by inducing vessel neutrophilic infiltration. Thrombin induces endothelial P-selectin expression and platelet activating factor release, which participate to induce early neutrophil adhesion and activation. We employed HUVEC and now show that thrombin induces the production of the chemokine IL-8 in a time- and dose-dependent fashion. Similarly, thrombin induced E-selectin expression on HUVEC. Both IL-8 secretion and E-selectin expression were preceded by an increase in steady state levels of the respective mRNAs. Thrombin action on HUVEC was inhibited by the specific thrombin inhibitor, hirudin. In addition, these effects of thrombin on HUVEC were mimicked by the 14-amino acid thrombin receptor agonist peptide, which triggers the native thrombin receptor in a similar fashion to thrombin itself. Although IL-1 and TNF-alpha also induce IL-8 and E-selectin, the thrombin effects in these experiments were not mediated by those cytokines, since neither IL-1 receptor antagonist nor anti-TNF-alpha Ab inhibited the effects of thrombin. Furthermore, IL-1alpha, IL-1beta, and TNF-alpha were not detected in the supernatants of thrombin-activated HUVEC. Although intracellular IL-1alpha was found in thrombin-activated HUVEC, antisense IL-1alpha had no inhibitory effect on IL-8 secretion. These results demonstrate that in addition to short term endothelial activation, thrombin also functions as a long acting proinflammatory agent by inducing endothelial synthesis of the mediators required for neutrophils activation and extravazation during inflammation.

[Resistance of Leishmania infantum to Glucantime: risk factors and therapeutic management]. / Résistance de Leishmania infantum au Glucantime: circonstances de survenue et prise en charge thérapeutique.

BACKGROUND: Resistance to antimonial drugs is rarely observed in immunocompetent patients. CASE REPORT: A 1-year-old girl was admitted suffering from persistent fever. A diagnosis of visceral leishmaniasis was made. The patient was given two courses of meglumine antimoniate (Glucantime) (60 mg/kg/d for 15 days) and one course of 12 injections of pentamidine (4 mg/kg). She relapsed 8 months later and failed to respond to Glucantime. Immunological tests performed during the relapse showed a suppression of the T cell response to Leishmania antigen and no production of interferon gamma. The patient was then successfully given liposomal amphotericin B (3 mg/kg/d for 10 days). She was asymptomatic 9 months later and had acquired specific cellular immunity against Leishmania. CONCLUSION: Deficient cell-mediated immunity and interferon gamma production are some factors responsible for decreased sensitivity to antimonial drugs. The WHO recommendations treating visceral leishmaniasis with prolonged administration of Glucantime may prevent relapses. Liposomal amphotericin B could be an alternative treatment.

IL-6 and IL-8 production from cultured human endothelial cells stimulated by infection with Rickettsia conorii via a cell-associated IL-1 alpha-dependent pathway.

Mediterranean spotted fever due to infection by Rickettsia conorii, is characterized by a general vasculitis. This vasculitis is thought to be due to a direct injury to endothelial cells induced by R. conorii. However, production and activity of cytokines on endothelial cells is an important pathway in inflammation, and part of the underlying mechanism of vasculitis. In the present studies, human umbilical vein endothelial cells (HUVEC) infected with R. conorii actively secrete high levels of IL-8 and IL-6 (P < 0.002, and P < 0.03, respectively, compared with uninfected cells). IL-1alpha, IL-1beta, or TNFalpha were not detected in the culture supernates. Nevertheless, IL-6 and IL-8 production was due, in a large part, to a cell-associated form of IL-1 alpha expressed on R. conorii-infected HUVEC, since production of these cytokines was suppressed by 80% (P = 0.0001) and 85% (P < 0.04) by the addition of IL-1 receptor antagonist, or anti-IL-1alpha antibodies (60% inhibition, P < 0.01 and 65% inhibition, P < 0.05, respectively) and IL-1alpha was measured after lysis of R. conorii-infected HUVEC but not in uninfected cells (P < 0.01). Rickettsial lipopolysaccharide does not seem to be involved, since polymyxin B did not reduce cytokine secretion. On the contrary, infection by intracellular R. conorii appears to be necessary to induce IL-1alpha and subsequently IL-8, since formalin-fixed R. conorii did not induce cytokine production. These observations demonstrate that R. conorii-infected HUVEC secrete IL-6 and IL-8 via the induction of cell-associated IL-1alpha, providing a possible mechanism for the vasculitis observed in Mediterranean spotted fever.

Interleukin-1 induces interleukin-8 secretion from endothelial cells by a juxtacrine mechanism.

Inflammation is characterized by migration of neutrophils through the endothelium, and the chemokine interleukin-8 (IL-8) appears to be involved. We asked whether adherence of cells bearing a membrane-form of interleukin 1 (IL-1) induces IL-8 secretion from human umbilical vein endothelial cells (HUVEC) and fibroblasts. Human peripheral blood mononuclear cells (PBMC) were stimulated with endotoxin for 12 hours and then fixed for 4 hours with paraformaldehyde. When these cells were added to HUVEC or fibroblasts, IL-8 production was induced. This stimulation by fixed PBMC was attributed to IL-1, because pretreatment of HUVEC or fibroblasts with IL-1 receptor antagonist (IL-1Ra) reduced the induction by 95% and 80%, respectively, P < .005. Using anti-IL-1 alpha monoclonal antibodies, reduction was complete, whereas anti-IL-1 beta had no effect. IL-1 alpha was shown on the surface of monocytes by fluorescence-activated cell sorter (FACS) analysis. Blockade of IL-1 receptors on PBMC did not affect the activity of membrane-associated IL-1 alpha, indicating that IL-1 is not anchored to the membrane through its receptors. However, PBMC treated with D-mannose before fixation resulted in a loss of activity; this loss of activity was associated with release of IL-1 alpha, not IL-1 beta, into the supernatant. Thus, anchoring of IL-1 alpha to the membrane may be via a lectin or mannose receptor-like interaction. Blockade of membrane IL-1 alpha required a 30-fold and fivefold excess of IL-1Ra compared with the amount required to block soluble IL-1 beta and IL-1 alpha, respectively. We conclude that the fixed PBMC IL-8 inducing activity is almost entirely caused by IL-1, that this represents IL-1 alpha bound to a surface lectin or mannose receptor on the monocyte, and that it functions in inflammation via juxtacrine interactions.

A novel role for E- and P-selectins: shape control of endothelial cell monolayers.

The migration of neutrophils from blood vessels to peripheral tissues is a key step of inflammation. This requires the formation of transient gaps between endothelial cells with concomitant leucocyte squeezing through these narrow apertures and immediate restoration of endothelium continuity. It is currently considered that the main role of selectins is to mediate the initial contact between flowing leucocytes and endothelial cells. We show here that the binding of E- or P-selectins by specific antibodies induces a marked 'rounding up' of interleukin-1- or thrombin-activated human endothelial cells, respectively. Also, anti-E-selectin antibodies trigger a transient increase in cytosolic calcium involving intracellular calcium stores. No such effect is observed when von Willebrand factor or intercellular adhesion molecule 1 are similarly bound. Thus, in addition to promoting the initial interaction between activated endothelium and moving leucocytes, selectins might play a role in the induction of subsequent endothelial deformation, which would facilitate leucocyte arrest and transmigration towards peripheral tissues, and enhance the diffusion of soluble molecules between intravascular and peripheral compartments. Our results are consistent with this hypothesis and demonstrate a new property of endothelial selectins.

Pseudo Felty's syndrome. A polyclonal disease with a favorable prognosis. Report of two cases with Southern blot analysis of TCR.

The authors report two patients with large granular lymphocyte (LGL) expansion associated with rheumatoid arthritis corresponding to pseudo Felty's syndrome. These cells have natural killer and T cell surface antigen markers. LGL are a heterogeneous population and expansion of these cells is responsible for leukemia, which is generally a monoclonal proliferation. It has been suggested that Epstein-Barr virus (EBV) is a putative agent in this leukemia. No EBV DNA was found with a polymerase chain reaction analysis in the lymphocyte DNA of our two patients. Some cases of pseudo Felty's syndrome have exhibited a monoclonal pattern on Southern blot analysis of the T cell receptor. On the contrary, our two cases showed a polyclonal pattern with TCR beta chain Southern blot analysis. This fact, associated with the mild course seen in both over more than twenty years, suggest that pseudo Felty's syndrome is a disease with a good prognosis.

Granulocyte-endothelium initial adhesion. Analysis of transient binding events mediated by E-selectin in a laminar shear flow.

The adhesion of moving cells to receptor-bearing surfaces is a key step to many important biological processes. Attachment was subjected to extensive modeling. However, the numerical values of kinetic bonding parameters relevant to realistic models of cell adhesion remain poorly known. In this report, we describe the motion of human granulocytes to interleukin-1-activated endothelial cells in presence of a low hydrodynamic drag (a few piconewtons) estimated to be much weaker than a standard ligand-receptor bond. It was thus expected to visualize the formation and rupture of individual bonds. We observed multiple short-time cell arrests with a median duration of 2.43 s. Stop frequency, not duration, was significantly inhibited by anti-E-selectin antibodies. Binding efficiency exhibited an almost linear relationship with the inverse of cell velocity. The distribution of arrest duration was determined: results were consistent with the view that these arrests reflected the formation/dissociation of single ligand-receptor bonds with a spontaneous dissociation rate of 0.5 s-1. The rate of bond formation was on the order of 0.04 s-1 when cells were freely rolling (mean velocity: 19 microns/s) and it exhibited an approximately 10-fold increase after the formation of a first adhesion.

"NK-like" T cytotoxicity against B lymphocytes in a hypogammaglobulinemic patient.

Physiologically, cells with NK activity appear to exert a negative control on immunoglobulin production. The clinical association of large granular lymphocyte (LGL) proliferation with hypogammaglobulinemia suggests that these functional NK cells could also be involved in pathological situations. We studied in vitro lymphocyte functions in a patient presenting LGL proliferation associated with hypogammaglobulinemia. The CD3+ CD8+ CD57+ CD16- phenotype lymphocytes expressed a high NK type cytotoxicity towards K562 targets, suggesting that they may be considered as "NK-like" T cells. We cultured the patient peripheral blood mononuclear cells (PBMC) with control subject PBMC and with PBMC from two other subjects with B chronic lymphocytic leukemia (B-CLL) of the CD20+ CD21- CD10- phenotype. Patient PBMC exhibited a lytic activity on control PBMC and on the B lymphocytes of one of the two B- CLL but only in the presence of PWM. This activity was not exerted by the culture supernatant and required a cell-to-cell contact. We suggest that the hypogammaglobulinemia observed in this patient may be related to a cytotoxic effect exerted on B lymphocytes by a CD3+ CD8+ CD57+ CD16- LGL proliferation.

[HIV infection in the child after materno-fetal transmission: early treatment with azidothymidine and prevention of secondary infectious complications]. / Infection par le VIH de l'enfant après contamination materno-foetale: prise en charge précoce par un traitement associant azidothymidine et prévention des complications infectieuses secondaires.

Twenty-four perinatally HIV infected children received early treatment as soon as the diagnosis of viral contamination was established. In 13 cases (group 1), this diagnosis was based on a viremia and/or antigenemia during the first 6 months of life. In 11 cases (group 2), children were more than 15 months-old and had a positive HIV antibody test. Therapy included azidothymidine (AZT, 400 mg/m2/d) and the prevention of secondary infectious complications with intravenous immunoglobulin and cotrimoxazole. With a median follow-up of 26 months, we reported no case of severe secondary infection and no case of encephalopathy. Hematological side effects of AZT were rarely observed. Only one patient developed anemia. In all other cases, the only hematological abnormality was macrocytosis of red blood cells. Before treatment, the mean value of T4 cells age-adjusted count was 96, 86 and 91%, respectively, for groups 1, 2 and the entire study group. At the time of analysis, these values were 64, 62 and 63% respectively. This decrease was statistically significant for group 1 and for the entire study group, but did not reach statistical significance for group 2. These data show that AZT is probably insufficient as a long-term therapy for HIV infected children. Other therapeutic approaches need to be developed in the future, notably the combination of anti-retroviral drugs.

Evidence of subjects sensitized to Leishmania infantum on the French Mediterranean coast: differences in gamma interferon production between this population and visceral leishmaniasis patients.

The Marseilles region is an endemic area for visceral mediterranean leishmaniasis, but although the number of dog cases, the parasite's main host, is very high, only a few people develop the disease. We looked for sensitized healthy subjects among 25 healthy individuals living in this area by studying their in vitro lymphoproliferative response to Leishmania infantum antigens and gamma interferon synthesis. We found that 65% of tested subjects were sensitized against L. infantum. We compared their cell mediated immunity to that of 13 active Kala-Azar patients and 13 controls from non-endemic areas. In patients, results showed a specific cellular immuno-deficiency in the lymphocyte response to L. infantum antigens and a global deficiency of gamma interferon production. Interestingly, the healthy individuals from the endemic area who responded to L. infantum antigens were found to produce high gamma interferon levels after L. infantum antigen stimulation. After healing, the cell mediated-immunity of the 3 patients we followed up was similar to that of the sensitized tested healthy subjects, but the former were still producing antibodies at the time of study.

Haemodialysis membranes modulate chronically the production of TNF alpha, IL1 beta and IL6.

As cytokines may play a role in the adverse effects of haemodialysis, TNF alpha, IL1 beta and IL6 were investigated before the haemodialysis session (chronic effect) and after 30 and 60 min (session effect). We found that haemodialysis exerts a chronic effect on cytokines but the type of haemodialysis membrane, Cuprophan or Hemophan, specifically influences each cytokine. Circulating levels of TNF and unstimulated production of TNF and IL1 by monocytes were increased in patients dialysed with Hemophan, whereas a greater LPS-stimulated production of TNF was observed in patients dialysed with Cuprophan. Both types of membrane induced a higher production of IL6 as compared to controls. The alternate use of Cuprophan and Hemophan demonstrated that the production of TNF and IL1 was dependent on the type of haemodialysis membrane. We also found that Cuprophan induced a reversible decrease of spontaneous and LPS-stimulated production of TNF, IL1 and IL6 during the haemodialysis session. Taken together, these results suggest that Hemophan induced a sustained production of cytokines whereas Cuprophan primed monocytes, probably through the activation of the complement pathway.

The soluble receptor of interleukin 2 is not a serum marker of the autoimmune activity in type I diabetes mellitus.

In order to determine if soluble interleukin 2 receptor (IL2R) was useful as a marker in screening for early Type 1 diabetes and in monitoring immunological treatment, we assayed serum IL2R levels in 67 controls, 43 patients with newly diagnosed diabetes and 28 first degree relatives of diabetic patients (5 subjects were islet cell antibody positive). In 23 diabetes, specimens were analysed at 3 and 6 months after diagnosis whether or not cyclosporin A was administered. Seven patients were in a clinical trial using anti IL2R monoclonal antibody and cyclosporin A. Since IL2R level in the normal population is elevated in the first 5 years of life then decreases until adulthood (age:IL2R correlation between 0 and 15 years: r = -0.42, P less than 0.05), subjects were carefully matched in age. In recent onset diabetes, this negative correlation disappeared and IL2R levels tended to decrease particularly in younger subjects. In Type 1 prediabetic subjects presenting persistent islet-cell antibody serum IL2R was not elevated. During immunological treatment of recent onset diabetes, serum IL2R remained stable and was not modified by cyclosporin A. As expected IL2R became undetectable during treatment with anti IL2R MC Ab. But it rebounded when treatment was stopped with no effect on remission. We concluded that IL2R levels in Type 1 diabetic patients is not useful in screening autoimmune activity or in evaluating the effectiveness of immunosuppressors.

[Study of cellular immunity in patients with Mediterranean visceral leishmaniasis]. / Etude de l'immunité cellulaire chez des malades atteints de leishmaniose viscérale méditerranéenne.

Cellular immunity against Leishmania infantum antigens was studied in visceral leishmaniasis patients and healthy subjects living in a endemic area. Only the healthy subjects were TTL positive with production of gamma interferon, whereas the visceral leishmaniasis patients presented a transitory inhibition of their specific cellular response mechanisms.

Changes in serum level and affinity for concanavalin A of human alpha 1-proteinase inhibitor in severe burn patients: relationship to natural killer cell activity.

In serum from five patients with severe burns, alpha 1-proteinase inhibitor (alpha 1-PI) was analyzed and then isolated by immunosorption chromatography. By Con A-Sepharose chromatography alpha 1-PI was separated into two types of fractions: the first containing the Con A-non-reactive isoforms and the second containing the Con A-reactive isoforms. The increase of alpha 1-PI serum level in burn patients is associated on the fifth day after the burn with a significant shift toward species enriched in bi-antennary oligosaccharides (Con A-reactive isoforms). This latter change passed very quickly and ten days after the burn, whereas the alpha 1-PI serum level was still high, the difference in proportions of Con A-reactive and non-reactive isoforms was not statistically significant. With respect to the difference in oligosaccharide structure, it appeared that the glycan moiety was involved in the inhibitory effect on natural killer cell activity. At the same concentration, purified alpha 1-PI and retained alpha 1-PI isoforms had an equal effect, whereas the non-retained alpha 1-PI isoforms were more efficient (P less than or equal to 0.01). Purified alpha 1-PI and its isoforms inhibited the natural killer cell activity in a dose-dependent manner.

[Extreme hypogammaglobulinemia disclosing large granular lymphocytes with hepatosplenorenal infiltration]. / Hypogammaglobulinémie extrême révélant une leucémie à grands lymphocytes granuleux avec infiltration hépato-spléno-rénale.

We report a case of large granular lymphocytosis, or chronic "natural killer" lymphocytosis, a newly described entity. We were able to demonstrate the proliferative character of the disease by the finding of karyotype abnormalities. This case was remarkable for the pre-existence, for at least three years, of severe hypogammaglobulinaemia, for the very slow course of the proliferative process and for the progressive and tumoral infiltration of the spleen and liver, then kidney.