Jean-Paul Dessaint

Human Endothelial-Cell Specific Molecule-1 Binds Directly to the Integrin CD11a/CD18 (LFA-1) and Blocks Binding to Intercellular Adhesion Molecule-1

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca2+, Mg2+, or Mn2+ divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (Kd = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.

Using human CD20-transfected murine lymphomatous B cells to evaluate the efficacy of intravitreal and intracerebral rituximab injections in mice.

PURPOSE The treatment of primary central nervous system lymphoma (PCNSL) and its subset, primary intraocular lymphoma (PIOL), remains of limited efficiency, and salvage therapies are often used without prior testing in adequate animal models. Most PNCSL/PIOL are aggressive B-cell malignancies. Two animal models that closely mimic the human situation were established to evaluate the efficiency of intravitreal and intracerebral anti-CD20 monoclonal antibody (rituximab) injections. METHODS Human CD20-transfected murine B-lymphoma cells (38C13 CD20(+)) were inoculated in the vitreous through the pars plana or in the caudate nucleus with the use of a stereotaxic frame in immunocompetent syngeneic mice. Animals were monitored clinically and by funduscopic and histologic examination. Rituximab was injected intravitreally or intracerebrally. Occurrences of exophthalmia, neurologic disturbance, and weight loss were monitored over 2 months. RESULTS Inoculation of 38C13 CD20(+) cells in the eye or the brain resulted in tumor occurrence after a median of 15 days or 22 days, respectively, with histologic characteristics closely resembling those of PIOL and PCNSL. Local rituximab injections eradicated tumor colonization in more than half the graft recipients and inhibited tumor progression significantly in the others compared with progression in mice that underwent grafting with the control 38C13 cell line (no human CD20 expression) and in mice that underwent grafting with 38C13 CD20(+) cells that received local injections of an irrelevant antibody (trastuzumab). CONCLUSIONS Inoculation of native or human CD20-transfected murine 38C13 cells in the vitreous or the brain of immunocompetent mice provides useful novel models for evaluating the biology and treatment of PIOL and PCNSL. Intravitreal and intracerebral rituximab injections reduced tumor occurrence and growth in each model.

Implication of cyclosporine in up-regulation of Bcl-2 expression and maintenance of CD8 lymphocytosis in cytomegalovirus-infected allograft recipients

T cell homeostasis and CD4/CD8 ratios are normally reestablished by apoptotic clearance of activated T cells after immune stimulation. In allograft recipients with cytomegalovirus infection, CD8 lymphocytosis persists after negativation of viral cultures, contrary to immunocompetent hosts. We investigated the expression of Bcl-2 protein, an intracellular suppressor of apoptosis, and of CD95 (APO-1/Fas), a membrane inducer of apoptosis, in peripheral blood lymphocytes from 45 solid organ recipients. During the viremic phase of CMV infection, we found absence or diminished expression of Bcl-2 protein and increased expression of CD95 antigen in activated CD8+ T cells. Opposite evolution of these molecular regulators of apoptosis was reflected by the presence of 10-25% of apoptotic lymphocytes with fragmented DNA, as shown by both in situ nick translation and electrophoresis. Normalization of Bcl-2 expression was progressive over several months but still lower than in uninfected allograft recipients. These results suggest that the initial evolution of CMV infection in allograft recipients resembles acute viral infection in immunocompetent hosts. Conversely, we showed that overexpression of Bcl-2 protein in lymphocytes from uninfected allograft recipients, and culture of unstimulated normal lymphocytes with 0.5 micrograms/ml cyclosporine led to an increase in the expression of intracellular Bcl-2. This up-regulation of Bcl-2 protein by cyclosporine suggests the acquisition of resistance to apoptosis. Thus, the reversion of balance between T cell death and survival after acute CMV infection might be impeded by cyclosporine. Combination of CMV latent infection and cyclosporine therapy appears therefore critical to shift the homeostatic maintenance of the peripheral lymphocyte compartment toward persistingly high numbers of CD8+ T cells.

Immunomodulatory effect of pentoxifylline during human allograft rejection: involvement of tumor necrosis factor-alpha and adhesion molecules.

BACKGROUND Pentoxifylline (PTX), a methylxanthine phosphodiesterase inhibitor, is poorly active as an immunosuppressant but prevents the synthesis of proinflammatory cytokines. In a randomized double-blind study comparing PTX versus placebo in 140 patients receiving cadaveric kidney grafts under cyclosporine and prednisone, we have shown that PTX weakened the consequences of rejection on graft survival. To assess the mechanism underlying the beneficial effect recorded during this trial, we analyzed the impact of PTX on tumor necrosis factor (TNF-alpha) production and expression of cell adhesion molecules. METHODS Plasma levels of TNF-alpha and its soluble receptors (sTNF-RI, sTNF-RII) and of soluble vascular cell adhesion molecule 1 (sVCAM-1) were monitored over the 6 months postgraft period when PTX or placebo were administered. Expression of VCAM-1 and intercellular cell adhesion molecule 1 was scored by immunohistochemical staining of biopsy specimens from patients who underwent rejection crisis. Lymphocyte subset composition was analyzed longitudinally during cytomegalovirus (CMV) infections. RESULTS Plasma TNF-alpha levels were significantly reduced in the PTX-treated group over the 6 months of administration, and specifically during isolated rejection episodes and during CMV infections. Plasma levels of sTNFR-I, sTNFR-II, and sVCAM-1 did not differ between the two groups of patients, but a decrease in renal tubular VCAM-1 expression was observed in the PTX group. During CMV infections, CD8 lymphocytosis and expansion of CD57+ (CD28-) CD8+ T cells were similar in the two groups. CONCLUSION The data collected during this double-blind study point to an immunomodulatory role of PTX, the beneficial effect on graft survival resulting from a restraining effect of the drug on the inflammatory conditions involved in acute graft rejection.

Enhancing the effect of secreted cyclophilin B on immunosuppressive activity of cyclosporine.

BACKGROUND Cyclophilin B (CyPB) is a cyclosporine (CsA)-binding protein, located within intracellular vesicles and secreted in biological fluids. In previous works, we reported that CyPB specifically interacts with the T-cell membrane and potentiates the ability of CsA to inhibit CD3-induced proliferation of T lymphocytes. METHODS CyPB levels were measured in plasma from healthy donors and transplant patients. The role of extracellular CyPB on the distribution and activity of CsA was investigated first by studies on the uptake of free and CyPB-complexed drug by blood cells, and second by studies on the inhibitory effects of these two compounds on the CD3-induced proliferation of peripheral blood mononuclear cells. RESULTS A significant increase in plasma CyPB level was observed for CsA-treated patients (13+/-6.4 nM, n=42) in comparison with untreated donors (4.3+/-2.1 nM, n=34). In vitro, extracellular CyPB dose dependently modified CsA distribution between plasma, erythrocyte, and lymphocyte contents, by both retaining the complexed drug extracellularly and promoting its specific accumulation within peripheral blood mononuclear cells. Moreover, the enhanced ability of CyPB-complexed CsA to suppress CD3-induced T-cell proliferation was preserved in the presence of other blood cells, implying specific targeting of the drug to sensitive cells. Furthermore, although a large interindividual variability of sensitivity to the drug was confirmed for 18 individuals, we found that CyPB potentiated the activity of CsA in restoring a high sensitivity to the immunosuppressant. CONCLUSION These results suggest that plasma CyPB may contribute to the acceptance and the good maintenance of organ transplantation by enhancing the immunosuppressive activity of CsA through a receptor-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes.

Defect in recruiting effector memory CD8+T-cells in malignant pleural effusions compared to normal pleural fluid

BackgroundMalignant pleural effusions (MPE) are a common and fatal complication in cancers including lung or breast cancers, or malignant pleural mesothelioma (MPM). MPE animal models and immunotherapy trials in MPM patients previously suggested defects of the cellular immunity in MPE. However only few observational studies of the immune response were done in MPM patients, using questionable control groups (transudate…).MethodsWe compared T cell populations evaluated by flow cytometry from blood and pleural effusion of untreated patients with MPM (n = 58), pleural metastasis of adenocarcinoma (n = 30) or with benign pleural lesions associated with asbestos exposure (n = 23). Blood and pleural fluid were also obtained from healthy subjects, providing normal values for T cell populations.ResultsBlood CD4+ or CD8+ T cells percentages were similar in all groups of patients or healthy subjects. Whereas pleural fluid from healthy controls contained mainly CD8+ T cells, benign or malignant pleural effusions included mainly CD4+ T cells. Effector memory T cells were the main T cell subpopulation in pleural fluid from healthy subjects. In contrast, there was a striking and selective recruitment of central memory CD4+ T cells in MPE, but not of effector cells CD8+ T cells or NK cells in the pleural fluid as one would expect in order to obtain an efficient immune response.ConclusionsComparing for the first time MPE to pleural fluid from healthy subjects, we found a local defect in recruiting effector CD8+ T cells, which may be involved in the escape of tumor cells from immune response. Further studies are needed to characterize which subtypes of effector CD8+ T cells are involved, opening prospects for cell therapy in MPE and MPM.

Improvement in the outcome of rejection with pentoxifylline in renal transplantation: a randomized controlled trial.

BACKGROUND Pentoxifylline (PTX), a methylxantine phosphodiesterase inhibitor commonly used to treat peripheral vascular disease, has been shown to decrease the production of proinflammatory cytokines and reactive oxygen species and to reduce the toxic effects of cyclosporine. Thus, administration of PTX to transplant patients, as an adjunct to immunosuppressive therapy, could prevent numerous posttransplantation complications. METHODS One hundred forty consecutive patients receiving cadaveric kidney grafts were registered in a randomized double-blind study comparing PTX at a dose of 800 mg/day, then 1200 mg/day, versus placebo during the first 6 months after transplantation. All patients were followed up for 1 year. RESULTS Rejection episodes were validated as the only independent risk factor for graft loss in this study. We compared graft survival rates in each group according to the presence or absence of acute rejection. Acute rejection reduced graft survival in the control group (graft survival rate at 1 year, 59% vs. 97%, P < 0.001), but this adverse effect was blunted in the PTX group (72% vs. 89%, NS). This improvement was confirmed by multivariate analysis for risk factors, with graft survival rates being described at best as the interaction between rejection and treatment (PTX vs. placebo, P = 0.045). CONCLUSION Although PTX does not modify the incidence of any posttransplant complications, it weakens the consequences of rejection on graft survival.

CD28+ intraepithelial lymphocytes with long telomeres are recruited within the inflamed ileal mucosa in Crohn disease

Crohn disease is a chronic inflammatory bowel disease that involves all the intestine but predominantly alters the ileum. The disease largely depends on T cells, but the biologic role of intestinal intraepithelial lymphocytes (IEL) in transmural inflammation remains poorly characterized. To address this issue, a comparison of IEL and lamina propria lymphocytes (LPL) isolated from the uninvolved and the inflamed ileal mucosa of Crohn disease patients was performed. More CD8+ IEL (26% versus 8%) from the inflamed ileal mucosa expressed the CD28 receptor and the CD11a integrin than IEL from the uninvolved ileal mucosa, which were mostly CD28-. IEL had longer telomeres in the inflamed than in the uninvolved areas and a TCR Vbeta repertoire more similar to circulating T cells, suggesting that the increased proportion of CD28+ TCRalphabeta+ IEL within the inflamed mucosa is more likely due to recruited lymphocytes from the periphery that populate the epithelial layer than to the acquisition of the CD28 molecule by activated resident lymphocytes. In the uninvolved ileal mucosa, IEL from Crohn disease patients had shorter telomeric lengths than IEL from control patients, suggesting that they have been chronically stimulated. Such perturbation of the IEL population within the ileal mucosa could contribute to the inflammation in Crohn disease.