Catherine Farnarier

DNAM-1 and PVR Regulate Monocyte Migration through Endothelial Junctions

DNAX accessory molecule 1 (DNAM-1; CD226) is a transmembrane glycoprotein involved in T cell and natural killer (NK) cell cytotoxicity. We demonstrated recently that DNAM-1 triggers NK cell–mediated killing of tumor cells upon engagement by its two ligands, poliovirus receptor (PVR; CD155) and Nectin-2 (CD112). In the present paper, we show that PVR and Nectin-2 are expressed at cell junctions on primary vascular endothelial cells. Moreover, the specific binding of a soluble DNAM-1–Fc molecule was detected at endothelial junctions. This binding was almost completely abrogated by anti-PVR monoclonal antibodies (mAbs), but not modified by anti–Nectin-2 mAbs, which demonstrates that PVR is the major DNAM-1 ligand on endothelial cells. Because DNAM-1 is highly expressed on leukocytes, we investigated the role of the DNAM-1–PVR interaction during the monocyte transendothelial migration process. In vitro, both anti–DNAM-1 and anti-PVR mAbs strongly blocked the transmigration of monocytes through the endothelium. Moreover, after anti–DNAM-1 or anti-PVR mAb treatment, monocytes were arrested at the apical surface of the endothelium over intercellular junctions, which strongly suggests that the DNAM-1–PVR interaction occurs during the diapedesis step. Altogether, our results demonstrate that DNAM-1 regulates monocyte extravasation via its interaction with PVR expressed at endothelial junctions on normal cells.

Expression of the Cdx1 and Cdx2 Homeotic Genes Leads to Reduced Malignancy in Colon Cancer-derived Cells

We have previously described an inverse relationship between Cdx1 and Cdx2 mRNA levels and the extent of dysplasia and severity of clinical outcome in colorectal carcinoma, suggesting that altered expression of these genes was associated with colorectal carcinogenesis or tumor progression. To investigate further their involvement in the physiopathology of colorectal cancer, HT29 colon carcinoma cells that show very lowCdx expression were transfected with Cdx1and/or Cdx2 cDNA to elicit their overexpression. Growth rate, tumorigenicity, resistance to apoptosis, and migration potential of the corresponding cells were analyzed. Growth rate of cells overexpressing Cdx2 decreased by half, whereas overexpression of Cdx1 had no effect. However, cells overexpressing both Cdxs had a growth rate reduced to 20% of control. In cells overexpressing Cdx1 orCdx2, tumorigenicity and resistance to apoptosis induced by serum starvation, ceramide, or staurosporine were not changed compared with control cells; yet phorbol ester-stimulated cell migration was decreased by 50%. In cells overexpressing both Cdx1 andCdx2, tumorigenicity was decreased by 50%, resistance to apoptosis was significantly lowered, and stimulated cell migration was further decreased to 15% of control compared with cells expressingCdx1 or Cdx2. Finally, cells overexpressing both Cdxs showed strongly decreased Bcl-2 expression, which could account for their increased sensitivity to apoptosis. These findings show that, in HT29 cells, both Cdx1 andCdx2 genes must be expressed to reduce tumorigenic potential, to increase sensitivity to apoptosis, and to reduce cell migration, suggesting that the two genes control the normal phenotype by independent pathways. This may explain why loss of Cdx1or Cdx2 expression is associated with tumor development and invasiveness in colorectal tumors.

Increased soluble vascular cell adhesion molecule 1 concentrations in patients with primary or systemic lupus erythematosus-related antiphospholipid syndrome Correlations with the severity of thrombosis

OBJECTIVEnRecent studies have shown that in vitro endothelial cells are activated by antiphospholipid antibodies and may support leukocyte adhesion. We studied levels of soluble intercellular adhesion molecule 1 (sICAM-1, sCD54), soluble vascular cell adhesion molecule 1 (sVCAM-1, sCD106), and soluble E-selectin (soluble endothelial leukocyte adhesion molecule 1 [sELAM-1, sCD62E]) in sera from patients with primary antiphospholipid syndrome (primary APS), and compared them with those from patients with systemic lupus erythematosus-associated APS (SLE-APS) or pure SLE, as well as with those from 2 control groups composed of healthy volunteers and patients with thrombosis unrelated to autoimmune diseases.nnnMETHODSnSerum samples from 24 patients with primary APS, 15 patients with SLE-APS, 22 patients with pure SLE, 48 control patients with thrombosis, and 18 healthy volunteers were examined using enzyme-linked immunosorbent assays specific for sICAM-1, sVCAM-1, and sELAM-1.nnnRESULTSnSerum levels of sVCAM-1, but not sICAM-1 or sELAM-1, were significantly increased in all patient study groups compared with thrombosis control patients and healthy volunteers, but did not differ between the groups of patients with primary APS, SLE-APS, or pure SLE. Concentrations of sVCAM-1 were significantly higher in primary APS or SLE-APS patients with severe, recurrent thrombosis and were negatively correlated with platelet counts in primary APS patients. In patients with primary APS, sVCAM-1 levels were higher if there was thrombotic kidney involvement and correlated with creatinemia.nnnCONCLUSIONnSerum sVCAM-1 concentrations are increased in patients with primary APS, especially those with repeated thrombotic events or kidney involvement. These findings suggest that endothelial/ monocyte interaction may be important in the pathogenesis of primary APS.

Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1α

Sterile inflammation resulting from cell death is due to the release of cell contents normally inactive and sequestered within the cell; fragments of cell membranes from dying cells also contribute to sterile inflammation. Endothelial cells undergoing stress-induced apoptosis release membrane microparticles, which become vehicles for proinflammatory signals. Here, we show that stress-activated endothelial cells release two distinct populations of particles: One population consists of membrane microparticles (<1 μm, annexin V positive without DNA and no histones) and another larger (1–3 μm) apoptotic body-like particles containing nuclear fragments and histones, representing apoptotic bodies. Contrary to present concepts, endothelial microparticles do not contain IL-1α and do not induce neutrophilic chemokines in vitro. In contrast, the large apoptotic bodies contain the full-length IL-1α precursor and the processed mature form. In vitro, these apoptotic bodies induce monocyte chemotactic protein-1 and IL-8 chemokine secretion in an IL-1α–dependent but IL-1β–independent fashion. Injection of these apoptotic bodies into the peritoneal cavity of mice induces elevated serum neutrophil-inducing chemokines, which was prevented by cotreatment with the IL-1 receptor antagonist. Consistently, injection of these large apoptotic bodies into the peritoneal cavity induced a neutrophilic infiltration that was prevented by IL-1 blockade. Although apoptosis is ordinarily considered noninflammatory, these data demonstrate that nonphagocytosed endothelial apoptotic bodies are inflammatory, providing a vehicle for IL-1α and, therefore, constitute a unique mechanism for sterile inflammation.

Increased Levels of Soluble Adhesion Molecules in the Serum of Patients with Hepatitis C (Correlation with Cytokine Concentrations and Liver Inflammation and Fibrosis)

Lymphocyte adhesion to endothelium,extravasation, and adhesion to hepatocytes are mediatedby adhesion molecules and constitute important steps inthe liver inflammation due to chronic hepatitis C(HCV-CH). We measured soluble intercellular adhesionmolecule (sICAM-1, sCD54), vascular cell adhesionmolecule (sVCAM-1, sCD106), E-selectin (sCD62E), as wellas interleukin (IL)-1β, IL-8, and tumor necrosis factor-α (TNF-α) concentrations inthe serum of 22 patients with HCV-CH in comparison to 20seronegative healthy volunteers. sICAM-1, sVCAM-1,sCD62E, TNF-α, and IL-8 but not IL-1βconcentrations were significantly elevated in patients.sICAM-1 and sCD62E correlated with TNF-α andaspartate amino transferases levels. sICAM-1 correlatedwith liver lobular inflammation whereas sVCAM-1, sCD62E, and IL-8 correlated with liver fibrosis.Measurement of soluble adhesion molecules may be an easyway to follow liver inflammation and fibrosis duringHCV-CH.

A Novel Leukocyte Adhesion Deficiency III Variant: Kindlin-3 Deficiency Results in Integrin- and Nonintegrin-Related Defects in Different Steps of Leukocyte Adhesion

Leukocyte adhesion deficiency type III is a recently described condition involving a Glanzmann-type bleeding syndrome and leukocyte adhesion deficiency. This was ascribed to a defect of the FERMT3 gene resulting in abnormal expression of kindlin-3, a protein expressed in hematopoietic cells with a major role in the regulation of integrin activation. In this article, we describe a patient with a new mutation of FERMT3 and lack of kindlin-3 expression in platelets and leukocytes. We assayed quantitatively the first steps of kindlin-3–defective leukocyte adhesion, namely, initial bond formation, bond strengthening, and early spreading. Initial bond formation was readily stimulated with neutrophils stimulated by fMLF, and neutrophils and lymphocytes stimulated by a phorbol ester or Mn2+. In contrast, attachment strengthening was defective in the patient’s lymphocytes treated with PMA or Mn2+, or fMLF-stimulated neutrophils. However, attachment strengthening was normal in patient’s neutrophils treated with phorbol ester or Mn2+. In addition, the patient’s T lymphocytes displayed defective integrin-mediated spreading and a moderate but significant decrease of spreading on anti-CD3–coated surfaces. Patient’s neutrophils displayed a drastic alteration of integrin-mediated spreading after fMLF or PMA stimulation, whereas signaling-independent Mn2+ allowed significant spreading. In conclusion, the consequences of kindlin-3 deficiency on β2 integrin function depend on both cell type and the stimulus used for integrin activation. Our results suggest looking for a possible kindlin-3 involvement in membrane dynamical event independent of integrin-mediated adhesion.

Type III procollagen is a reliable marker of ARDS-associated lung fibroproliferation

AbstractPurposeA specific biomarker of post-ARDS fibroproliferation could be useful in the identification of patients who could benefit from therapies aiming to modulate fibroproliferation such as corticosteroids.The aim of thisn prospective study was to determine the best threshold of the N-terminal-peptidetype III procollagen (NT-PCP-III) in non-resolving ARDS to validate this threshold according to the outcome.MethodsConcerning the best threshold of NT-PCP-III, all consecutive patients with a non-resolving ARDS were included if all the following criteria were fulfilled: moderate to severe ARDS lasting for at least 5xa0days, lung biopsy performed, serum and alveolar NT-PCP-III obtained within 1xa0week prior to biopsy, and no documented infection contra-indicating the corticosteroids. In the validation cohort part of the study, patients were included at day 7 if they presented a persistent moderate to severe ARDS.ResultsNineteen of 32 patients had fibroproliferatio nonbiopsy. Serum and alveolar NT-PCP-III were higher in patients with fibroproliferation. Using a threshold of 9xa0µg/L, alveolar NT-PCP-III had the highest accuracy for diagnosing fibroproliferation (sensitivityxa0=xa089.5xa0% and specificityxa0=xa092.3xa0%). Regarding the 51 patients included in the validation cohort, the mortality rate at day 60 was increased in patients presenting an alveolar NT-PCP-III level higher than 9xa0µg/L (69 vs. 17xa0%, pxa0<xa00.001). The mean alveolar level of NT-PCP-III on day 7 was 8.1-fold higher in nonsurvivors (pxa0=xa00.03).ConclusionsThe determination of NT-PCP-III on BAL done at day 7 in persistent ARDS is able to identify patients with fibroproliferation who could be included in a trial of corticosteroids or any other treatment that might help resolve lung fibroproliferation.

Insulin-Induced Lipoatrophy in Type I Diabetes: A possible tumor necrosis factor-α-mediated dedifferentiation of adipocytes

OBJECTIVE To test the hypothesis that tumor necrosis factor (TNF)-α may mediate the loss and the dedifferentiation of subcutaneous fat tissue in the insulin-induced lipoatrophies of a diabetic patient who presented extensive lesions. RESEARCH DESIGN AND METHODS An in vitro exploration of cytokine production by peripheral blood mononuclear cells (PBMC) from the reported case was performed and compared with the same explorations of PBMC from three nondiabetic subjects and three diabetic patients without lipoatrophic lesions. A proliferation test and an evaluation of TNF-α and interleukin (IL)-6 production from PBMC in presence of insulin were studied. RESULTS The production of TNF-α and IL-6 by the macrophages of the patient in presence of insulin were dramatically increased in comparison with control subjects. This process needed cooperation with other lymphoid cells and was abrogated by dexamethasone. CONCLUSIONS In our reported case, a local hyperproduction of TNF-α from macrophages that was induced by the injected insulin could explain the dedifferentiation of the adipocytes of the subcutaneous tissue and the reversion that was induced by the local injection of dexamethasone.

T Cell Polarization toward TH2/TFH2 and TH17/TFH17 in Patients with IgG4-Related Disease

IgG4-related disease (IgG4-RD) is a fibro-inflammatory disorder involving virtually every organ with a risk of organ dysfunction. Despite recent studies regarding B cell and T cell compartments, the disease’s pathophysiology remains poorly understood. We examined and characterized subsets of circulating lymphocytes in untreated patients with active IgG4-RD. Twenty-eight consecutive patients with biopsy-proven IgG4-RD were included in a prospective, multicentric study. Lymphocytes’ subsets were analyzed by flow cytometry, with analysis of TH1/TH2/TH17, TFH cells, and cytokine release by peripheral blood mononuclear cells. Results were compared to healthy controls and to patients with primary Sjögren’s syndrome. Patients with IgG4-RD showed an increase of circulating T regulatory, TH2, TH17, and CD4+CXCR5+PD1+ TFH cell subsets. Accordingly, increased levels of IL-10 and IL-4 were measured in IgG-RD patients. TFH increase was characterized by the specific expansion of TFH2 (CCR6−CXCR3−), and to a lesser extent of TFH17 (CCR6+CXCR3−) cells. Interestingly, CD4+CXCR5+PD1+ TFH cells normalized under treatment. IgG4-RD is characterized by a shift of circulating T cells toward a TH2/TFH2 and TH17/TFH17 polarization. This immunological imbalance might be implicated in the disease’s pathophysiology. Treatment regimens targeting such T cells warrant further evaluation.

Low Circulating Natural Killer Cell Counts are Associated With Severe Disease in Patients With Common Variable Immunodeficiency

Natural Killer (NK) cells have been shown to exert antiviral and antitumoural activities. Nevertheless most available data are derived from mouse models and functions of these cells in human remain unclear. To evaluate the impact of low circulating NK cell counts and to provide some clues to the role of NK cells in natural conditions, we studied a large cohort of patients with common variable immunodeficiency (CVID) included in a multicenter cohort of patients with primary hypogammaglobulinaemia. Patients were classified into three groups on the basis of their NK cell counts: severe and mild NK cell lymphopenia (< 50 and 50–99 × 106/L respectively), and normal NK cell counts (> 100 × 106/L). Clinical events were analyzed and compared between these three groups of patients. During study period, 457 CVID patients were included: 99 (21.7%) with severe NK cell lymphopenia, 118 (25.8%) with mild NK cell lymphopenia and 240 (52.5%) with normal NK cell counts. Non-infectious complications (57% vs. 36% and 35%), and, particularly, granulomatous complications (25.3% vs. 13.6% and 8.8%), were more frequent in patients with severe NK cell lymphopenia than in other groups. Invasive infections (68.7% vs. 60.2% and 48.8%), including bacteraemia (22.2% vs. 5.9% and 8.3%) and infectious pneumonia (63.6% vs. 59.3% and 44.2%), were also more frequent in this population. However, no difference was observed for viral infections and neoplasms. Low circulating NK cell counts are associated with more severe phenotypes of CVID, which may indicate a protective role of these immune cells against severe bacterial infections and other complications and non-redundant immune functions when the adaptive immune response is not optimal.