Search

Objective: IL-1β is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1β-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1β secretion. The first objective of our study was to characterize IL-1β-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo . The second objective was to detect circulating IL-1β-positive MVs in JIA patients., Methods: MVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1β, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo , MVs' ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1β-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry., Results: THP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1β and bioactive TF. IL-1β-positive MVs expressed P2X7 receptor and released soluble IL-1β in response to ATP stimulation in vitro . In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1β-positive MVs were detectable in plasma from 10 active JIA patients., Conclusion: MVs shed from activated macrophages contain IL-1β, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cambon, Rebelle, Bachelier, Arnaud, Robert, Lagarde, Muller, Tellier, Kara, Leroyer, Farnarier, Vallier, Chareyre, Retornaz, Jurquet, Tran, Lacroix, Dignat-George and Kaplanski.)

Lymphocytes are essential for microbial immunity, tumor surveillance, and tissue homeostasis. However, the in vivo development and function of helper-like innate lymphoid cells (ILCs) in humans remain much less well understood than those of T, B, and NK cells. We monitored hematopoietic stem cell transplantation (HSCT) to determine the kinetics of ILC development in both children and adults. It was found that, unlike NK cells, helper-like ILCs recovered slowly, mirroring the pattern observed for T cells, with normalization achieved at 1 year. The type of graft and the proportion of CD34 + cells in the graft did not significantly affect ILC reconstitution. As HSCT is often complicated by acute or chronic graft-versus-host disease (GVHD), the potential role of ILC subsets in maintaining tissue integrity in these conditions was also analyzed. It was found that GVHD was associated with lower levels of activated and gut-homing NKp44 + ILCP, consistent with a non-redundant role of this ILC subset in preventing this life-threatening disorder in lymphopenic conditions., (©2021 Society for Leukocyte Biology.)

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition. Primary HLH occurs early in life as a result of monogenic biallelic mutations affecting lymphocyte cytotoxicity. Secondary HLH occurs mostly in adults secondary to infection, lymphoma, or rheumatic disease. In this latter setting, lymphocyte cytotoxicity status is not known. We conducted a systematic evaluation of natural killer (NK) cell cytotoxicity in adult patients with secondary HLH. Adult patients with secondary HLH were prospectively studied ex vivo for total lymphocyte count and subtype, NK cell phenotype, perforin expression and degranulation, and natural or antibody-dependent cell cytotoxicity, in comparison with patients affected by the same underlying disease without HLH (disease controls [DCs]) and with healthy controls (HCs). Screening for variants of cytotoxity genes was systematically performed. 68 patients were included in the HLH group and 34 each in the DC and HC groups. In HLH patients, severe and transient lymphopenia, activated NK cell phenotype (eg, increased CD69, ICAM-1, HLADR, and CCR5 expression), and decreased capacity of interferon γ production were observed; mean perforin expression was normal; and degranulation tests and NK cell cytotoxicity were not different from those in DCs. A monoallelic variant of uncertain significance affecting a lymphocyte cytotoxicity gene or the perforin variant A91V was observed in almost 50% of the patients. We detected no major intrinsic cytotoxicity dysfunction in secondary HLH patients compared with DCs and no predicted pathogenic gene variant. The activated NK phenotype profile associated with decreased interferon γ production seems similar to those of other hyperinflammatory diseases such as sepsis or systemic juvenile idiopathic arthritis., (© 2020 by The American Society of Hematology.)

Background: Septic shock, a major cause of death in critical care, is the clinical translation of a cytokine storm in response to infection. It can be complicated by sepsis-induced immunosuppression, exemplified by blood lymphopenia, an excess of circulating Treg lymphocytes, and decreased HLA-DR expression on circulating monocytes. Such immunosuppression is associated with secondary infections, and higher mortality. The effect of these biological modifications on circulating innate lymphoid cells (ILCs) has been little studied. Methods: We prospectively enrolled patients with septic shock (Sepsis-3 definition) in the intensive care unit (ICU) of Timone CHU Hospital. ICU controls (trauma, cardiac arrest, neurological dysfunction) were recruited at the same time (NCT03297203). We performed immunophenotyping of adaptive lymphocytes (CD3 + T cells, CD19 + B cells, CD4 + CD25 + FoxP3 + Treg lymphocytes), ILCs (CD3 - CD56 + NK cells and helper ILCs - ILC1, ILC2, and ILC3), and monocytes by flow cytometry on fresh blood samples collected between 24 and 72 h after admission. Results: We investigated adaptive and innate circulating lymphoid cells in the peripheral blood of 18 patients in septic shock, 15 ICU controls, and 30 healthy subjects. As expected, the peripheral blood lymphocytes of all ICU patients showed lymphopenia, which was not specific to sepsis, whereas those of the healthy volunteers did not. Circulating CD3 + T cells and CD3 - CD56 + NK cells were mainly concerned. There was a tendency toward fewer Treg lymphocytes and lower HLA-DR expression on monocytes in ICU patients with sepsis. Although the ILC1 count was higher in septic patients than healthy subjects, ILC2, and ILC3 counts were lower in both ICU groups. However, ILC3s within the total ILCs were overrepresented in patients with septic shock. The depression of immune responses has been correlated with the occurrence of secondary infections. We did not find any differences in ILC distribution according to this criterion. Conclusion: All ICU patients exhibit lymphopenia, regardless of the nature (septic or sterile) of the initial medical condition. Specific distribution of circulating ILCs, with an excess of ILC1, and a lack of ILC3, may characterize septic shock during the first 3 days of the disease., (Copyright © 2019 Carvelli, Piperoglou, Bourenne, Farnarier, Banzet, Demerlé, Gainnier and Vély.)

Background: Fibroproliferative repair phase of the acute respiratory distress syndrome (ARDS) is followed by a restitutio ad integrum of lung parenchyma or by an irreversible lung fibrosis and patients' death. Transforming Growth Factor-β1 (TGF-β1) is involved in collagen production and lung repair. We investigated whether alveolar TGF-β1 was associated with the presence of fibroproliferation and the outcome of ARDS patients., Methods: Sixty-two patients were included the first day of moderate-to-severe ARDS. Bronchoalveolar lavage fluid (BALF) was collected at day 3 (and day 7 when the patients were still receiving invasive mechanical ventilation) from the onset of ARDS. Survival was evaluated at day 60. TGF-β1 was measured by immunoassay. The patients were classified as having lung fibroproliferation when the alveolar N-terminal peptide for type III procollagen (NT-PCP-III) measured on day 3 was > 9 μg/L as recently reported. The main objective of this study was to compare the alveolar levels of total TGF-β1 according to the presence or not a lung fibroproliferation at day 3., Results: Forty-three patients (30.6%) presented a fibroproliferation at day 3. BALF levels of total TGF-β1 were not statistically different at day 3 (and at day 7) according to the presence or not lung fibroproliferation. Mortality at day 60 was higher in the group of patients with fibroproliferation as compared with patients with no fibroproliferation (68.4% vs. 18.6% respectively; p < 0.001). Total TGF-β1 measured on BALF at day 3 was not associated with the outcome. Multiple logistic regression showed that the presence of lung fibroproliferation was associated with death. In contrast, TGF-β1 was not independently associated with death., Conclusions: Pulmonary levels of TGF-β1 during the first week of ARDS were not associated nor with the presence of fibroproliferation neither with death. TGF-β1 should not be used as a biomarker to direct anti-fibrotic therapies., Competing Interests: The authors have declared that no competing interests exist.

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

The syndromic diarrhea/trichohepatoenteric syndrome (SD/THE) is a rare and multi-system genetic disorder caused by mutation in SKIV2L or in TTC37 , two genes encoding subunits of the putative human SKI complex involved in RNA degradation. The main features are intractable diarrhea of infancy, hair abnormalities, facial dysmorphism, and intrauterine growth restriction. Immunologically this syndrome is associated with a hypogammaglobulinemia leading to an immunoglobulin supplementation. Our immune evaluation of a large French cohort of SD/THE patient revealed several immunological defects. First, switched memory B lymphocytes count is very low. Second, IFN-γ production by T and NK cells is impaired and associated with a reduced degranulation of NK cells. Third, T cell proliferation was abnormal in 3/6 TTC37 -mutated patients. These three patients present with severe EBV infection and a transient hemophagocytosis which may be related to these immunological defects. Moreover, an immunological screening of patients with clinical features of SD/THE could facilitate both diagnosis and therapeutic management of these patients.

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 T H 1/T H 2/T H 17, T FH 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, T H 2, T H 17, and CD4 + CXCR5 + PD1 + T FH cell subsets. Accordingly, increased levels of IL-10 and IL-4 were measured in IgG-RD patients. T FH increase was characterized by the specific expansion of T FH 2 (CCR6 - CXCR3 - ), and to a lesser extent of T FH 17 (CCR6 + CXCR3 - ) cells. Interestingly, CD4 + CXCR5 + PD1 + T FH cells normalized under treatment. IgG4-RD is characterized by a shift of circulating T cells toward a T H 2/T FH 2 and T H 17/T FH 17 polarization. This immunological imbalance might be implicated in the disease's pathophysiology. Treatment regimens targeting such T cells warrant further evaluation.

Innate lymphoid cells (ILCs) have potent immunological functions in experimental conditions in mice, but their contributions to immunity in natural conditions in humans have remained unclear. We investigated the presence of ILCs in a cohort of patients with severe combined immunodeficiency (SCID). All ILC subsets were absent in patients with SCID who had mutation of the gene encoding the common γ-chain cytokine receptor subunit IL-2Rγ or the gene encoding the tyrosine kinase JAK3. T cell reconstitution was observed in patients with SCID after hematopoietic stem cell transplantation (HSCT), but the patients still had considerably fewer ILCs in the absence of myeloablation than did healthy control subjects, with the exception of rare cases of reconstitution of the ILC1 subset of ILCs. Notably, the ILC deficiencies observed were not associated with any particular susceptibility to disease, with follow-up extending from 7 years to 39 years after HSCT. We thus report here selective ILC deficiency in humans and show that ILCs might be dispensable in natural conditions, if T cells are present and B cell function is preserved., Competing Interests: E.V. is the cofounder of and a shareholder in Innate Pharma. The other authors have no conflicting financial interest to declare.

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 50-99×10(6)/L respectively), and normal NK cell counts (>100×10(6)/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., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The world of lymphocytes has recently expanded. A group of cells, innate lymphoid cells (ILC), has been defined. It includes lymphoid cells that have been known for decades, such as natural killer (NK) cells and lymphoid tissue-inducer (LTi) cells. NK cells recognize a vast array of tumor cells, which they help to eliminate through cytotoxicity and the production of cytokines, such as IFNγ. Advances in our understanding of NK-cell biology have led to a growing interest in the clinical manipulation of these cells in cancer. The other ILCs are found mostly in the mucosae and mucosal-associated lymphoid tissues, where they rapidly initiate immune responses to pathogens without the need for specific sensitization. Here, we outline the basic features of ILCs and review the role of ILCs other than NK cells in cancer. Much of the role of these ILCs in cancer remains unknown, but several findings should lead to further efforts to dissect the contribution of different ILC subsets to the promotion, maintenance, or elimination of tumors at various anatomic sites. This will require the development of standardized reagents and protocols for monitoring the presence and function of ILCs in human blood and tissue samples., (©2015 American Association for Cancer Research.)

Purpose: A 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 this 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., Methods: Concerning 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 5 days, lung biopsy performed, serum and alveolar NT-PCP-III obtained within 1 week 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., Results: Nineteen of 32 patients had fibroproliferatio nonbiopsy. Serum and alveolar NT-PCP-III were higher in patients with fibroproliferation. Using a threshold of 9 µg/L, alveolar NT-PCP-III had the highest accuracy for diagnosing fibroproliferation (sensitivity = 89.5 % and specificity = 92.3 %). 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 9 µg/L (69 vs. 17 %, p < 0.001). The mean alveolar level of NT-PCP-III on day 7 was 8.1-fold higher in nonsurvivors (p = 0.03)., Conclusions: The 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.

The major histocompatibility complex class I related chain (MIC) is a stress-inducible protein modulating the function of immune natural killer (NK) cells, a major leukocyte subset involved in proper trophoblast invasion and spiral artery remodeling. Aim of the study was to evaluate whether upregulation of soluble MIC (sMIC) may reflect immune disorders associated to vascular pregnancy diseases (VPD). sMIC was more frequently detected in the plasma of women with a diagnostic of VPD (32%) than in normal term-matched pregnancies (1.6%, P < 0.0001), with highest prevalence in intrauterine fetal death (IUDF, 44%) and vascular intrauterine growth restriction (IUGR, 39%). sMIC levels were higher in preeclampsia (PE) than in IUFD (P < 0.01) and vascular IUGR (P < 0.05). sMIC detection was associated with bilateral early diastolic uterine notches (P = 0.037), thrombocytopenia (P = 0.03), and high proteinuria (P = 0.03) in PE and with the vascular etiology of IUGR (P = 0.0038). Incubation of sMIC-positive PE plasma resulted in downregulation of NKG2D expression and NK cell-mediated IFN-γ production in vitro. Our work thus suggests that detection of sMIC molecule in maternal plasma may constitute a hallmark of altered maternal immune functions that contributes to vascular disorders that complicate pregnancy, notably by impairing NK-cell mediated production of IFN-γ, an essential cytokine favoring vascular modeling.

B7-H6, a member of the B7 family of immunoreceptors, is as a cell-surface ligand for the NKp30-activating receptor expressed on natural killer cells. B7-H6 is not detected in normal human tissues at steady state but is expressed on tumor cells. However, whether B7-H6 can be expressed in other conditions remains unknown. We analyzed here the pathways that lead to the expression of B7-H6 in nontransformed cells. In vitro, B7-H6 was induced at the surface of CD14(+)CD16(+) proinflammatory monocytes and neutrophils upon stimulation by ligands of Toll-like receptors or proinflammatory cytokines such as interleukin-1β and tumor necrosis factor α. In these conditions, a soluble form of B7-H6 (sB7-H6) was also produced by activated monocytes and neutrophils. In vivo, B7-H6 was expressed on circulating proinflammatory CD14(+)CD16(+) monocytes in a group of patients in sepsis conditions, and was linked to an increased mortality. sB7-H6 was selectively detected in the sera of patients with gram-negative sepsis and was associated with membrane vesicles that co-sedimented with the exosomal fraction. These findings reveal that B7-H6 is not only implicated in tumor immunosurveillance but also participates in the inflammatory response in infectious conditions.

Objective: The mechanisms involved in cytomegalovirus reactivation in critically ill patients who were previously immunocompetent are still unknown. The current study was designed to evaluate the possible role of natural killer cells in the reactivation of cytomegalovirus in these patients., Design: Prospective observational., Setting: : A medical intensive care unit of a university hospital., Patients: Fifty-one subjects, including 15 patients who experienced cytomegalovirus reactivation (cases) during their intensive care unit stay and 15 patients who matched intensive care unit controls, selected from a cohort of consecutive nonimmunocompromised intensive care unit patients, as well as healthy controls., Interventions: Tests included weekly systematic immunomonitoring and routine screening for cytomegalovirus infection until discharge from the intensive care unit or death. The immunophenotype and functions of natural killer cells were performed by flow cytometry, and serum levels of pro- and anti-inflammatory cytokines were determined by enzyme-linked immunosorbent assay., Measurements and Main Results: The overall occurrence of cytomegalovirus reactivation in the cohort was 27%. No differences of natural killer cell effector functions were observed at admission between cases and controls. Instead, before cytomegalovirus reactivation, the ability of natural killer cells to secrete interferon-γ was significantly reduced in cases as compared with controls upon stimulation with antibody-coated target cells (p = .029) and with K562 cell stimulation (p = .029). No phenotypic or quantitative differences were observed between cases and controls. Cases exhibited higher levels of interleukin 10 (p = .031) and interleukin 15 (p = .021) than controls before cytomegalovirus reactivation., Conclusions: Impaired natural killer cell function with reduced interferon-γ secretion precedes the occurrence of cytomegalovirus reactivation among previously immunocompetent critically ill patients.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition due to the association of an infectious agent with lymphocyte cytotoxicity defects, either of congenital genetic origin in children or presumably acquired in adults. In HLH patients, an excess of lymphocyte or macrophage cytokines, such as IFN-γ and TNFα is present in serum. In animal models of the disease, IFN-γ and TNF-α have been shown to play a central pathogenic role. In humans, unusually high concentrations of IL-18, an inducer of IFN-γ, and TNF-α have been reported, and are associated with an imbalance between IL-18 and its natural inhibitor IL-18 binding protein (IL-18BP) resulting in an excess of free IL-18. Here we studied whether IL-18BP could reduce disease severity in an animal model of HLH. Mouse cytomegalovirus infection in perforin-1 knock-out mice induced a lethal condition similar to human HLH characterized by cytopenia with marked inflammatory lesions in the liver and spleen as well as the presence of hemophagocytosis in bone marrow. IL-18BP treatment decreased hemophagocytosis and reversed liver as well as spleen damage. IL-18BP treatment also reduced both IFN-γ and TNF-α production by CD8(+) T and NK cells, as well as Fas ligand expression on NK cell surface. These data suggest that IL-18BP is beneficial in an animal model of HLH and in combination with anti-infectious therapy may be a promising strategy to treat HLH patients.

Rationale: Natural killer cells, as a major source of interferon-γ, contribute to the amplification of the inflammatory response as well as to mortality during severe sepsis in animal models., Objective: We studied the phenotype and functions of circulating NK cells in critically-ill septic patients., Methods: Blood samples were taken <48 hours after admission from 42 ICU patients with severe sepsis (n = 15) or septic shock (n = 14) (Sepsis group), non-septic SIRS (n = 13) (SIRS group), as well as 21 healthy controls. The immuno-phenotype and functions of NK cells were studied by flow cytometry., Results: The absolute number of peripheral blood CD3-CD56(+) NK cells was similarly reduced in all groups of ICU patients, but with a normal percentage of NK cells. When NK cell cytotoxicity was evaluated with degranulation assays (CD107 expression), no difference was observed between Sepsis patients and healthy controls. Under antibody-dependent cell cytotoxicity (ADCC) conditions, SIRS patients exhibited increased CD107 surface expression on NK cells (62.9[61.3-70]%) compared to healthy controls (43.5[32.1-53.1]%) or Sepsis patients (49.2[37.3-62.9]%) (p = 0.002). Compared to healthy (10.2[6.3-13.1]%), reduced interferon-γ production by NK cells (K562 stimulation) was observed in Sepsis group (6.2[2.2-9.9]%, p<0.01), and especially in patients with septic shock. Conversely, SIRS patients exhibited increased interferon-γ production (42.9[30.1-54.7]%) compared to Sepsis patients (18.4[11.7-35.7]%, p<0.01) or healthy controls (26.8[19.3-44.9]%, p = 0.09) in ADCC condition., Conclusions: Extensive monitoring of the NK-cell phenotype and function in critically-ill septic patients revealed early decreased NK-cell function with impaired interferon-γ production. These results may aid future NK-based immuno-interventions., Trial Registration: NTC00699868.

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.

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 Mn(2+). In contrast, attachment strengthening was defective in the patient's lymphocytes treated with PMA or Mn(2+), or fMLF-stimulated neutrophils. However, attachment strengthening was normal in patient's neutrophils treated with phorbol ester or Mn(2+). 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 Mn(2+) allowed significant spreading. In conclusion, the consequences of kindlin-3 deficiency on β(2) 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.

Severe sepsis and septic shock are still deadly conditions urging to develop novel therapies. A better understanding of the complex modifications of the immune system of septic patients is needed for the development of innovative immunointerventions. Natural killer (NK) cells are characterized as CD3(-)NKp46(+)CD56(+) cells that can be cytotoxic and/or produce high amounts of cytokines such as IFN-γ. NK cells are also engaged in crosstalks with other immune cells, such as dendritic cells, macrophages, and neutrophils. During the early stage of septic shock, NK cells may play a key role in the promotion of the systemic inflammation, as suggested in mice models. Alternatively, at a later stage, NK cells-acquired dysfunction could favor nosocomial infections and mortality. Standardized biological tools defining patients' NK cell status during the different stages of sepsis are mandatory to guide potential immuno-interventions. Herein, we review the potential role of NK cells during severe sepsis and septic shock.

Killer Ig-like receptors (KIRs) are MHC class I-specific receptors expressed by Natural Killer (NK) and T cell subsets. KIRs either inhibit (KIR-L) or activate (KIR-S) lymphocyte functions. Inhibitory KIR2DL1 and activating KIR2DS1 share ligand specificity for the HLA-C2 group, consistent with their almost identical extracytoplasmic domain. This homology hampered the distinction between KIR2DL1 and KIR2DS1. We report here the characterization of the KIR2DS1(+) subsets among primary human NK and T cells. Regardless of the host HLA-C genotype, around 10% of circulating NK cells expressed KIR2DS1 in absence of KIR2DL1. In HLA-C2 individuals, KIR2DS1 was not able to induce NK cell education (i.e., the acquisition of NK cell competence) nor to interfere with KIR2DL1-induced NK cell education. KIR2DS1 was also present on rare oligoclonal TCRalphabeta(+)CD8alpha(+) and TCRalphabeta(+)CD4(-)CD8(-) subsets. As KIR2DS1 has been associated with autoimmunity and hematopoietic stem cell transplantation, these results pave the way to dissect the function of KIR2DS1 in these clinical conditions., (Copyright 2009 Elsevier Inc. All rights reserved.)

Hemophagocytic syndrome (HPS) is characterized by an uncontrolled and poorly understood activation of T-helper 1 (Th-1) lymphocytes and macrophages. We studied 20 patients with HPS secondary to infections, autoimmune disease, lymphoma, or cancer and observed that the concentrations of serum interleukin 18 (IL-18), a strong inducer of Th-1 responses, interferon gamma (IFN-gamma) production, and stimulation of macrophages and natural killer (NK) cells were highly increased in HPS but not in control patients. In contrast, concentrations of its natural inhibitor, the IL-18 binding protein (IL-18BP), were only moderately elevated, resulting in a high level of biologically active free IL-18 in HPS (4.6-fold increase compared with controls; P < .001). Free IL-18 but not IL-12 concentrations significantly correlated with clinical status and the biologic markers of HPS such as anemia (P < .001), hypertriglyceridemia, and hyperferritinemia (P < .01) and also with markers of Th-1 lymphocyte or macrophage activation, such as elevated concentrations of IFN-gamma and soluble IL-2 and tumor necrosis factor alpha (TNF-alpha) receptor concentrations. Despite high IL-18 elevation, in vitro NK-cell cytotoxicity was severely impaired in HPS patients, in part due to NK-cell lymphopenia that was observed in a majority of patients but also secondary to an intrinsic NK-cell functional deficiency. We concluded that a severe IL-18/IL-18BP imbalance results in Th-1 lymphocyte and macrophage activation, which escapes control by NK-cell cytotoxicity and may allow for secondary HPS in patients with underlying diseases.

The prevention of atherosclerosis depends on the high-density lipoprotein (HDL) capacity to stimulate the efflux of unesterified cholesterol (UC). We tested here the effects of 2 HDL apolipoproteins, apo A-I and the 7-kd anionic peptide factor (APF), on the UC efflux by human endothelial ECV 304 cells in culture. Apolipoprotein A-I (10 micromol/L) or APF (3.5 micromol/L) in lipid-free forms or small particles (13 nm with apo A-I or 19 nm with APF) were incubated in the presence of [4-14C]UC. The phosphatidylcholines (PCs) were present either at a low level (0.35 mmol/L with apo A-I or 0.20 mmol/L with APF) or at a high level (1 mmol/L with apo A-I). We also tested either large 53-nm bile lipoprotein complex-like particles (3.5 micromol/L APF [13 microg/500 microL]) with a high PC level (0.65 mmol/L) or a 9-residue synthetic peptide (13 microg/500 microL), derived from the NH2-terminal domain of HDL3-APF, in a lipid-free or low-lipidated (0.20 mmol/L PCs) form. A control was developed in absence of the added compounds. A rapid [4-14C]UC efflux mediated by APF added in free form or in 19-nm complexes was 2.2- to 2.3-fold higher than that mediated by apo A-I in free form or in 13-nm particles (P < .05). The level of this high APF-related efflux was comparable with that obtained with the 12-nm native HDLs (10 micromol/L apo A-I) or free PCs (1 mmol/L). The increase in the UC efflux was much more limited (1.4-fold) in the presence of the 53-nm APF/high-PC particles, but it was higher than that mediated by apo A-I. In addition, the efflux mediated by the synthetic peptide, in lipid-free or low-lipidated form, constituted the major part of that related to the full-length APF. Thus, all these particles are very active HDL components, able to act as cholesterol acceptors. Interestingly, we further showed a new anti-atherogenic property of APF as well as its metabolic importance and clinical relevance. By its involvement in the first step of the reverse cholesterol transport, APF could reduce the risk of cardiovascular disease.

Background/aims: To better characterize the molecules involved in leukocyte tissue infiltration during hepatitis C-mixed cryoglobulinemia (HCV-MC)-associated vasculitis., Methods: The involvement of ELAM, ICAM-1 and VCAM-1 was evaluated in 36 patients with HCV-MC vasculitis using three different approaches: concentrations of soluble forms by specific ELISA, tissue expression by immunohistochemistry on patients nerve biopsies, endothelial expression by FACS analysis, on cells activated in vitro by cryoprecipitates purified from HCV-MC patients., Results: Concentrations of sVCAM-1 were significantly elevated in the serum of HCV-MC patients compared to HCV patients without MC, the highest concentrations being found in severe vasculitis. VCAM-1 expression was detected on blood vessels from nerve biopsies performed in patients with severe vasculitis. When added to endothelial cells in vitro, HCV-MC patients cryoprecipitate induced VCAM-1 but also ELAM and ICAM-1 expression possibly through a mechanism due to the C1q complement fraction interaction with endothelial cells, since C1q was consistently present in the cryoprecipitates., Conclusions: VCAM-1 is mainly involved in the pathogenesis of HCV-MC-associated severe vasculitis and may be a potential interesting therapeutic target.

Objective: High-density lipoproteins (HDLs) have significant cardiovascular benefits by retarding the progression of atherosclerosis. One of the mechanisms is the inhibition by HDLs of the vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells. Our objective was to test the effect on VCAM-1 expression by the human umbilical vein endothelial cells (HUVEC) of a minor HDL2 and HDL3 apolipoprotein, the anionic peptide factor (APF). The peptide has previously been found to develop some beneficial effects against atherosclerosis, i.e. by promoting the cholesterol efflux from endothelial cells., Methods: We examined the effects of two HDL apolipoproteins A-I and APF, either in presence or absence of phosphatidylcholines (PCs), or free PCs, on the expression of VCAM-1 by HUVEC. The cells were stimulated with either the tumor necrosis factor-alpha (TNFalpha, 500 pg/ml) or the calcium bound to heparin (10 microg Ca2+/ml, 50 microg heparin/ml)., Results: In the presence of TNFalpha, only the free PCs (0.25 and 1 mM) developed an inhibitory effect (up to 50%). In the absence of TNFalpha and in the presence of calcium bound to heparin, either the lipid-free APF (3.5 microM) or the APF/PC complexes (1:57 molar ratio) or the free PCs (0.25 mM) exhibited a substantial inhibitory effect (72, 71 and 42%, respectively)., Conclusion: Our present findings suggest for the first time that one of the mechanisms of the antiatherogenic action of APF involves the inhibition of VCAM-1 expression by HUVEC. The peptide, through its phospholipid-binding and its calcium antagonist abilities, appears to confer on the HDLs a protective effect against the early cellular event of the inflammatory process., (Copyright (c) 2005 S. Karger AG, Basel.)

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.

The origin of the Toll-like family of receptors pre-dates the evolutionary split between the plant and animal kingdoms. These receptors are remarkably conserved across the taxonomic kingdoms and have fundamental roles in triggering immune responses. How they trigger such responses, and how these mechanisms arose in evolution, is a topic of extensive debate. We postulate a surveillance model: these receptors "keep watch" for both endogenous and exogenous molecules that indicate tissue inquiry, infection and remodeling. Furthermore, we suggest that the first Toll-like family receptors that arose in evolution might have acted in both development and immunity by recognizing the degradation of endogenous macromolecules.

Interleukin (IL)-6 transsignaling plays a pivotal role in the shift from neutrophil to monocyte recruitment at the inflammatory site. Release of neutrophil IL-6 receptor-alpha (IL-6Ralpha, CD126) in its soluble form is a key step of IL-6 transsignaling, however, its physiological inducers are poorly known. Here, we observed that the neutrophil chemoattractants IL-8, C5a complement fraction, platelet activating factor, leukotriene B4 and N-formyl-methionyl-leucyl-phenylalanine rapidly decreased IL-6Ralpha membrane expression and increased soluble IL-6Ralpha concentrations in the neutrophil supernatants, consistent with a shedding process. IL-6Ralpha shedding involved a TNF-alpha-converting enzyme-type metalloproteinase since it was partly decreased in the presence of a specific inhibitor, but not cathepsin G since PMSF or alpha1 antichymotrypsin had no effect. Neutrophil IL-6Ralpha shedding may be a common feature of neutrophilic infiltrates in various inflammatory situations, allowing IL-6 transsignaling, decreasing neutrophil infiltration and in the meantime favoring monocyte recruitment, thus the initiation of an immune response and subsequently the resolution of inflammation.

Background: The early stage of post-transplant obliterative bronchiolitis (OB) is characterized by an influx of inflammatory cells to the lung, among which neutrophils may play a role in key events. The potential for chemokines to induce leukocyte accumulation in the alveolar space was investigated. We assessed whether changes in the chemotactic expression profile could be used as sensitive markers of the onset of OB., Methods: Serial bronchoalveolar lavage (BAL) fluids from 13 stable healthy recipients and 8 patients who developed bronchiolitis obliterans syndrome (BOS) were analyzed longitudinally for concentrations of interleukin-8 (IL-8), chemokines regulated-upon-activation and normal T-cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1), soluble intracellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These were assessed by enzyme-linked immunosorbent assay (ELISA)., Results: Significantly elevated percentages of BAL neutrophils and IL-8 levels were found at the pre-clinical stage of BOS, on average 151 +/- 164 days and 307 +/- 266 days, respectively, before diagnosis of BOS. There was also early upregulation of RANTES and MCP-1 in the BOS group (mean 253 +/- 323 and 152 +/- 80 days, respectively, before diagnosis of BOS). The level of MCP-1 was consistently higher than that of RANTES until airway obliteration. BAL sICAM-1 and sVCAM-1 levels were not statistically different between the groups., Conclusions: These data support the belief that RANTES, IL-8 and MCP-1 play a crucial role in the pathogenesis of OB. The results show that relevant increased levels of such chemokines may predict BOS, and suggest that there is potential for some of these markers to be used as early and sensitive markers of the onset of BOS. Longitudinal monitoring of these chemokine signals may contribute to better management of patients at risk for developing OB, at a stage when remodeling can either be reversed or altered.