Analysis of CD95 and CCR7 expression on circulating CD4+ lymphocytes revealed disparate immunoregulatory potentials in systemic lupus erythematosus

Emerging data have implicated a critical role for CD4 in the pathogenesis of systemic lupus erythematosus (SLE). This study was designed to delineate the contribution of CD4⁺ T cells in the pathogenesis of SLE disease. Forty-four patients (3 male: 41 female) and 20 healthy volunteers (4 male: 16 female) were included in the study. CD4⁺ lymphocytes analysis was done using three-color flow cytometry with antibodies against human-CD95, a prototype cell death receptor, and the chemokine receptor-7 (CCR7) after gating for lymphocytes based on the forward and side scatter. Serum levels of IL-6, IL-12, IL-17, TNF-α and IL-10 cytokines were assayed using ELISA. Disease activity was assessed using the SLE disease activity index (SLEDAI). Based on the expression of CCR7 and CD95, CD4⁺ lymphocytes were subdivided into three particular subsets; CD4⁺CD95⁺CCR7⁺ cells, CD4⁺CD95⁻CCR7⁺ cells and CD4⁺CD95⁺CCR7⁻ cells. Percentage of CD4⁺CD95⁺CCR7⁺ cell subset was significantly higher in patients with SLE with active disease (SLEDAI > 6) and inactive (SLEDAI < 6) as compared with controls (P = 0.005), and it showed a significant positive correlation with ANA titer (P = 0.01), and a negative correlation with WBCs count (P = 0.001). CD4⁺CD95⁺CCR7⁻ cell subset was significantly higher in active SLE patients in comparison to patients with inactive disease and controls (P = 0.05, P = 0.005 respectively), and it correlates positively with SLEDAI, IL-6 and IL-17 levels (P = 0.001, 0.05, 0.01 respectively), and negatively with blood WBCs counts (P = 0.001). The third CD4⁺CD95⁻CCR7⁺cell subset was found significantly lower in SLE patients compared with controls, and it was found negatively correlated with IL-10, IL-6, and IL-17. The results show that CD4⁺CD95⁺subset lacking expression of CCR7 is associated with cell mediated inflammatory response as manifested by its correlation with signs of inflammation, inflammatory cytokines and disease activity index. Whereas, CD4⁺CD95⁺CCR7⁺ correlate more with antibody immune responses as manifested by association with serum ANA. These data suggest disparate roles of these cell subsets in the pathophysiology of SLE. A better understanding of the characteristics of CD4 cell subsets may shed light on the pathogenesis of autoimmune diseases, particularly SLE.     

Regulation of IL-21 signaling by suppressor of cytokine signaling-1 (SOCS1) in CD8(+) T lymphocytes

Mice lacking the gene for suppressor of cytokine signaling 1 (SOCS1) show defective homeostasis of T lymphocytes due to accumulation of CD8⁺ T cells, resulting at least partly from dysregulated IL-15 signaling. IL-15 alone does not stimulate proliferation of naïve CD8 T cells, but can synergize with IL-21 to induce proliferation, suggesting a potential role for IL-21 in the defective homeostasis of CD8⁺ T lymphocytes in SOCS1^−/− mice. Since IL-21 strongly induced SOCS1 mRNA in CD8⁺ T cells, we investigated whether SOCS1 regulates their response to IL-21. CD8⁺ T cells isolated from SOCS1-deficient mice proliferated vigorously in response to IL-21 + IL-15. In CD8⁺ T lymphocytes expressing transgenic TCR, IL-21 + IL-7 provided a stronger stimulus to naïve cells whereas IL-15 + IL-21 potently stimulated memory cells. Compared to truly naïve or memory cells, SOCS1^−/− H-Y TCR⁺ CD8⁺ T cells displayed CD44^loLy6C^hiCD122^intCD127^lo partial memory phenotype and exhibited stronger response to IL-15 + IL-21 than truly naïve cells. In SOCS1^−/− CD8⁺ T cells, IL-21 caused greater reduction in IL-15 threshold for activation in a dose-dependent manner. SOCS1 deficiency did not modulate IL-21Rα expression or sensitivity to IL-21, but delayed the loss of IL-21-induced phospho-STAT3 signal. These results show that SOCS1 is a critical regulator of IL-21 signaling in CD8⁺ T cells, and support the notion that sustained IL-21 signaling might also contribute to the aberrant T cell homeostasis in SOCS1-deficient mice.     

Chemokine receptors expression on peripheral CD4-lymphocytes in rheumatoid arthritis: Coexpression of CCR7 and CD95 is associated with disease activity

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation triggered by infiltrating CD4 lymphocytes. The positioning and activation of lymphocyte in inflamed synovial tissues are dependent on a number of factors including their chemokine receptor expression profile. We aimed to investigate which chemokine receptors pattern correlate with serum cytokine levels and with disease activity. Forty patients with RA (34 female and 6 male) with age range from 21 to 68 years were included. Twenty healthy volunteers (16 female and 4 male) with matched age (range 21–48 years) were served as healthy controls (HCs). Expression of chemokine receptors (CCR5, CX3CR1 and CCR7) together with the apoptosis-related marker (CD95) was analyzed using three-color flow cytometry analysis after gating on CD4⁺ peripheral blood lymphocytes. Plasma levels of IL-6, IL-10, IL-12 and TNF-α cytokines were measured in all participants using ELISA. Disease activity score (DAS28-CRP) system was assessed and active disease was defined as DAS28 ⩾3.2. Twenty-five (62.4%) patients were classified as active RA (ARA) and 15 (37.5%) patients with inactive RA (IRA). Percentages of CD4⁺ lymphocytes expressing CD95 with either of CCR7 or CCR5 were significantly higher in ARA compared to IRA and HCs groups, while the expression of CX3CR1 on T-cells was found significantly lower in both CD95⁻ and CD95⁺ T-cells in RA groups than HC. Percentages of CD4⁺CD95⁺CCR7⁺ cells correlated positively with IL-6 (r = 0.390). Whereas CD4⁺CD95⁺CX3CR1⁺ were negatively correlated with TNF-α (r = −0.261). Correlation of CD4⁺CD95⁺CCR7⁺ T cell subset with disease activity and inflammatory cytokines suggests a role for this cell subset in the pathogenesis of RA. Further investigation will be required to fully characterize this cell subset and its role in disease progression.     

The role of adipose tissue-associated macrophages and T lymphocytes in the pathogenesis of inflammatory bowel disease

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the gastrointestinal tract that affect more than 3 million people worldwide, but the pathological etiology is still unknown. The overall purpose of our investigations was to elucidate the possibility of pathological causes of IBD, and therefore, we determined the difference of inflammatory cytokine profiles in adipose tissue macrophages (ATMs) and T lymphocytes (ATTs) obtained near active lesions of IBD; investigated whether the alteration in ATM activation induces genes involved in collagen formation; and evaluated the effects of fatty acid oxidation inhibitors on factors involved in inflammation and collagen production by ATMs in IBD. Adipose tissues (ATs) were collected near active lesions and also at the margin of resected segments of the bowel from IBD patients with ulcerative colitis (UC) and CD (n = 14/group). Normal appearing ATs from control subjects (n = 14) who had colon resection for adenocarcinoma were collected as far away from the cancer lesion as possible to rule out possible changes. Compared with inactive disease lesions, ATMs and ATTs from active lesions released more IL-6, IL-4 and IL-13. Treatments of cytokine IL-4 and/or IL-13 to ATMs reduced iNOS expression but increased Arg-I expression which were exacerbated when treated with T cell- and adipocyte-conditioned medium. However, fatty acid oxidation inhibitors prevented the effects of cytokines IL-4 and/or IL-13 on iNOS and Arg-I expressions. This study was the first to show the effect of IL-4 and IL-13 on collagen formation, through iNOS and Arg-I expressions, that was exacerbated in a condition that mimics in vivo condition of active lesions. Moreover, our study was the first to provide potential benefits of fatty acid oxidation inhibitors to ATMs on preventing collagen formation; thus, providing therapeutic implications for individuals with intestinal fibrosis and stricture lesions, although future study should be guaranteed to elucidate the underlying mechanisms.     

IL-9, a local growth factor for synovial T cells in inflammatory arthritis

ObjectiveThe regulatory role of the T_h9 cells along with its signature cytokine IL-9 in human immune system and its aberrant activation in autoimmune diseases is currently under investigation. We are reporting the functional significance of IL-9 in the pathogenesis of autoimmune inflammatory arthritis.MethodsCD3⁺ T cells were obtained from peripheral blood (PB) and synovial fluid (SF) of psoriatic arthritis (PsA), rheumatoid arthritis (RA), and osteoarthritis (OA) patients. MTT, FACS based CFSE dilution assay and apoptosis assay (Annexin-V) were performed to determine the pro-growth/survival effect of human recombinant IL-9 on activated CD3⁺ T cells. Immunoblots were performed to determine the signaling proteins responsible for the progrowth/survival effect of IL-9.ResultsSF of PsA and RA was enriched with IL-9 producing CD3⁺ T cells compared to the SF in OA. IL-9 level measured by ELISA was significantly elevated in PsA and RA patients compared to SF in OA (<.001). Activated T cells of PsA and RA had higher levels of IL-9 receptors. IL-9 promoted proliferation and survival of the CD3⁺ T cells of PB and SF of PsA and RA and compared to untreated (media) controls (p < .005, t-test). IL-9 induced proliferation of T cells was dependent on PI3K/Akt/mTOR signaling pathway.ConclusionIL-9 is functionally active, and is a pro-growth/survival factor for the localized pathologic T cells in the synovium of inflammatory arthritis. The pro-growth/survival effect is mediated by the activation of mTOR kinase cascade. To our knowledge, this is the first report of a functional role of IL-9 in human autoimmune arthritis.     

Chemokine and cytokine levels in inflammatory bowel disease patients

Crohn’s disease (CD) and ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), are chronic, relapsing, and tissue destructive lesions that are accompanied by the uncontrolled activation of effector immune cells in the mucosa. Recent estimates indicate that there are 1.3 million annual cases of IBD in the United States, 50% of which consists of CD and 50% of UC. Chemokines and cytokines play a pivotal role in the regulation of mucosal inflammation by promoting leukocyte migration to sites of inflammation ultimately leading to tissue damage and destruction. In recent years, experimental studies in rodents have led to a better understanding of the role played by these inflammatory mediators in the development and progression of colitis. However, the clinical literature on IBD remains limited. Therefore, the aim of this study was to evaluate systemic concentrations of key chemokines and cytokines in forty-two IBD patients with a range of disease activity compared to levels found in ten healthy donors. We found a significant increase in an array of chemokines including macrophage migration factor (MIF), CCL25, CCL23, CXCL5, CXCL13, CXCL10, CXCL11, MCP1, and CCL21 in IBD patients as compared to normal healthy donors (P < 0.05). Further, we also report increases in the inflammatory cytokines IL-16, IFN-γ, IL-1β and TNF-α in IBD patients when compared to healthy donors (P < 0.05). These data clearly indicate an increase in circulating levels of specific chemokines and cytokines that are known to modulate systemic level through immune cells results in affecting local intestinal inflammation and tissue damage in IBD patients. Blockade of these inflammatory mediators should be explored as a mechanism to alleviate or even reverse symptoms of IBD.     

Interleukin (IL)-15 in combination with IL-2, fms-like tyrosine kinase-3 ligand and anti-CD3 significantly enhances umbilical cord blood natural killer (NK) cell and NK-cell subset expansion and NK function

Background aimsInterleukin (IL)-15 and fms-like tyrosine kinase-3 (FLT-3) are crucial factors for the development of human and murine natural killer (NK) cells. Previously, we have demonstrated significant ex vivo expansion and activation of unrelated cord blood (UCB) NK cells with an antibody/cytokine cocktail consisting of anti-CD3 + IL-2 + IL-12 + IL-7 and anti-CD3 + IL-2 + IL-12 + IL-18.MethodsIn the current experiments, we investigated the effects of short-term culture with anti-CD3 + IL-2 + FLT-3 + IL-15 on cord blood (CB) NK cell and NK-cell subset expansion and function. CB mononuclear cells were cultured for 48 h in AIM-V media or AIM-V + IL-2 (5 ng/mL) + anti-CD3 (50 ng/mL) + FLT-3 (50 ng/mL) ± escalating doses of IL-15 (1, 10 or 100 ng/mL). Flow cytometric analysis was performed using various fluorescent-conjugated monoclonal antibodies. In vitro cytotoxicity was determined with a standard europium assay against K562 and Daudi cells.ResultsThere was a 4.8-fold significant increase in NK-cell population (CD3^?/16⁺/56⁺; P < 0.03), 21-fold significant increase in CD3^?/56⁺/158a⁺ (KIR2DL1/S1; P < 0.002), 46-fold significant increase in CD3^?/56⁺/158b⁺ (KIR2DL1/S2; P < 0.002) and 11.5-fold significant increase in CD3^?/56⁺/NKB1⁺ (KIR3DL1; P < 0.01). We also noted a significant increase in both NK and lymphokine-activated killer (LAK) cytotoxicity with IL-2 + anti-CD3 + FLT-3 + IL-15 (100 ng/mL) compared with IL-2 + anti-CD3 + FLT-3 and media alone against K562 (P < 0.01) and Daudi (P < 0.001), respectively.ConclusionsWe have demonstrated a significant increase in UCB NK cells and NK cells expressing a variety of killer immunoglobulin-like receptor (KIR) receptors after short-term culture with anti-CD3, IL-2, FLT-3 and IL-15. Furthermore, there was a significant increase in in vitro NK/LAK cell cytotoxicity.     

Ontogeny and localization of the cells produce IL-2 in healthy animals

IL-2 is a growth factor for activated T cells and is required for maintenance of naturally arising regulatory T cells (nTregs). Mice defective in IL-2/IL-2 receptor signaling pathways have impaired nTregs and suffer from lymphoproliferative disorders, suggesting that IL-2 is present and functional in healthy animals. However, the cellular source of IL-2 is currently unknown. To determine which cells produce IL-2 in healthy animals, we established mice carrying cre gene knock in at the il-2 locus (termed IL-2^cre). When IL-2^cre mice were crossed with EGFP reporter mice, EGFP was exclusively expressed by a fraction of CD4 T cells present in both lymphoid and non-lymphoid tissues. Live imaging of IL-2^cre mice that carry the luciferase reporter showed concentrated localization of luciferase⁺ cells in Peyer’s patches. These cells were not observed in new born mice but appeared within 3 days after birth. Reduction of antigen receptor repertoire by transgene expression reduced their number, indicating that recognition of environmental antigens is necessary for generation of these IL-2 producers in healthy animals. A substantial fraction of EGFP⁺ cells also produce IL-10 and IFN-γ, a characteristic profile of type 1 regulatory T cells (Tr1). The data suggest that a group of Tr1 cells have addition roles in immune homeostasis by producing IL-2 along with other cytokines and help maintaining Tregs.     

Systemic expression of inflammatory mediators in patients with chronic rhinosinusitis and nasal polyps with and without Aspirin Exacerbated Respiratory Disease

BackgroundSystemic reactions are related to the pathogenesis of Aspirin Exacerbated Respiratory Disease (AERD). With this work we wanted to study the changes in the systemic levels of inflammatory mediators in both baseline and after oral aspirin challenge in patients with and without AERD.MethodsPatients with nasal polyposis and asthma with AERD (n = 20) and without (n = 18) were orally challenged with aspirin in a single-blind placebo controlled study. Serum samples and urine were collected before and 6 h after placebo and aspirin oral challenges. Serum levels of inflammatory mediators were assayed by using the Luminex technology and ELISA. The concentrations of 9-alpha, 11-beta prostaglandin F₂, and leukotriene E₄ (uLTE₄) were measured in urine samples by ELISA. The expression of T-cell surface markers was analyzed in peripheral blood mononuclear cells isolated before and after the challenges.ResultsAERD patients showed significantly higher baseline levels of s-IL-5R-alpha, uLTE₄ and percentage of CD4⁺CD25⁺CD127^pos and CD4⁺CD45RA⁻CD45RO⁺ but decreased levels of TGF-β₁ and number of CD4⁺CD25⁺CD127^neg cells. Aspirin challenge induced the release of uLTE₄, IL-6 and increased the number of CD4⁺CD45RA⁻CD45RO⁺ memory T-cells only in AERD patients but failed to reduce the levels of sCD40L as observed in non-AERD subjects. Further, IL-8 and sIL-5R-alpha levels directly correlated with the PD₂₀ASA and the effects of aspirin on IL-6 and number of memory T-cells was more pronounced in subjects showing more strong reaction (bronchial and nasal).ConclusionsAERD patients have a differential baseline inflammatory pattern that supports the role inflammation as underlying mechanism of the disease. Systemic response to oral aspirin challenge was related to an increase in serum IL-6 and the number of circulating memory T-cells in AERD patients.     

Abdominal visceral adiposity influences CD4+ T cell cytokine production in pregnancy

Women with pre-gravid obesity are at risk for pregnancy complications. While the macrophage response of obese pregnant women categorized by body mass index (BMI) has been documented, the relationship between the peripheral CD4⁺ T cell cytokine profile and body fat compartments during pregnancy is unknown. In this study, third trimester peripheral CD4⁺ T cell cytokine profiles were measured in healthy pregnant women [n = 35; pre-pregnancy BMI: 18.5–40]. CD4⁺ T cells were isolated from peripheral blood mononuclear cells and stimulated to examine their capacity to generate cytokines. Between 1 and 3 weeks postpartum, total body fat was determined by dual-energy X-ray absorptiometry and abdominal subcutaneous and visceral fat masses were determined by magnetic resonance imaging. Pearson’s correlation was performed to assess relationships between cytokines and fat mass. Results showed that greater abdominal visceral fat mass was associated with a decrease in stimulated CD4⁺ T cell cytokine expression. IFN-gamma, TNF-alpha, IL-12p70, IL-10 and IL-17A were inversely related to visceral fat mass. Chemokines CCL3 and IL-8 and growth factors G-CSF and FLT-3L were also inversely correlated. Additionally, total body fat mass was inversely correlated with FGF-2 while abdominal subcutaneous fat mass and BMI were unrelated to any CD4⁺ T cell cytokine. In conclusion, lower responsiveness of CD4⁺ T cell cytokines associated with abdominal visceral fat mass is a novel finding late in gestation.     

Interleukin-10-1082A/G polymorphism and inflammatory bowel disease susceptibility: A meta-analysis based on 17,585 subjects

A large number of studies have shown that the interleukin-10 (IL-10)-1082A/G polymorphism is implicated in susceptibility to inflammatory bowel disease (IBD). However, the results are inconsistent. We performed this meta-analysis to estimate the association between -1082A/G polymorphism in the IL-10 gene and IBD susceptibility. A total number of 18 case-control studies including 17,585 subjects were identified. No association was found between -1082A/G polymorphism and ulcerative colitis (UC) susceptibility. However, increased risk of Crohn’s disease (CD) was associated with -1082A/G polymorphism in the dominant genetic model (GG + GA vs. AA: OR = 1.22, 95% CI: 1.02–1.46, P = 0.028) and the heterozygote comparison (GA vs. AA: OR = 1.28, 95% CI: 1.05–1.55, P = 0.015). The results of this meta-analysis provide evidence for the association between IL-10-1082A/G polymorphism and susceptibility of CD. Due to several limitations in the present study, well-designed epidemiological studies with large sample size among different ethnicities should be performed in the future.     

Differential alteration in peripheral T-regulatory and T-effector cells with change in P-glycoprotein expression in Childhood Nephrotic Syndrome: A longitudinal study

IntroductionChildhood Idiopathic Nephrotic Syndrome (INS) responds to glucocorticoid therapy, however, 60–80% of patients relapse and some of them become steroid non responsive. INS may occur because of T cell dysfunction, abnormal cytokines and podocytopathies which reverse on steroid treatment. The reason of relapses could be imbalances in T cells phenotypes and respective cytokines. Herein, we hypothesize that relapses in INS may occur due to imbalance in T-regulatory and T-effector cell with their respective cytokines and overexpression of P-gp on lymphocytes.MethodsThe frequency of peripheral blood CD4⁺CD25⁺FoxP3⁺ Treg, CD4⁺IFN-γ⁺ Th1 and CD4⁺IL-4⁺ Th2 lymphocytes and their respective cytokines and P-gp expression on peripheral blood lymphocytes (PBLs) were analyzed in INS patients at baseline (n = 26), during remission (n = 24) and at relapse (n = 15).ResultsCompared to baseline, the frequency of Tregs was significantly increased at remission and decreased during relapse. In contrast, the frequency of Th1 and Th2 lymphocytes was significantly decreased during remission and increased at the time of relapse. Similarly, expression of P-gp was significantly high at baseline and at the time of relapse as compared to remission. Levels of cytokines IL-10 and TGF-β in the supernatant of stimulated PBMCs was increased during remission and decreased during relapse. In contrast, levels of IFN-γ and IL-4 were decreased during remission and increased at the time of relapse.ConclusionsSteroid therapy in INS induces decreased P-gp expression on PBLs along with increased frequency and cytokine response of T-regulatory cells, and reduced frequency and respective cytokine response of Th1 and Th2 cells during remission. However, reversal in the frequency and respective cytokines of T-regs, Th1 and Th2, and P-gp expression on PBLs occurs during relapses on follow-up.     

Immune mobilization of autologous blood progenitor cells: direct influence on the cellular subsets collected

Background aimsA phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells.MethodsInterleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin's lymphoma n = 2).ResultsToxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 10⁶ CD34⁺ cells/kg (median; range 1.9–6.6 × 10⁶/kg). Immune mobilization increased the number of CD3⁺ CD8⁺ T cells (P = 0.002), CD56⁺ natural killer (NK) cells (P = 0.0001), CD8⁺ CD56⁺ T cells (P = 0.002) and CD4⁺ CD25⁺ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 10⁶ IU/m²/day.ConclusionsImmune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.     

HSP70, a Novel Regulatory Molecule in B Cell-Mediated Suppression of Autoimmune Diseases

B cells have recently emerged as playing regulatory role in autoimmune diseases. We have previously demonstrated that human peripheral blood CD19⁺ CD24^hiCD27⁺ B cells have regulatory function both in healthy donors and in patients with autoimmune disease. However, the mechanism of this regulation is still not fully understood. In this study, microarrays were utilized to compare gene expression of CD19⁺ CD24^hiCD27⁺ B cells (regulatory B cells, Bregs) with CD19⁺ CD24^loCD27⁻ B cells (non-Bregs) in human peripheral blood. We found that heat shock protein 70 (HSP70) expression was significantly upregulated in Bregs. In vitro studies explored that HSP70 inhibition impaired the regulatory function of peripheral blood Bregs. In mouse models of autoimmune disease, using HSP70-deficient mice or HSP70 inhibitors, Bregs suppressed effector cells and rescued disease-associated phenotypes that were dependent on HSP70. Mechanistically, Bregs secreted HSP70, directly suppressing effector cells, such as T effect cells. These findings reveal that HSP70 is a novel factor that modulates Breg function and suggest that enhancing Breg-mediated production of HSP70 could be a viable therapy for autoimmune disease.     

Neutrophil CD64 expression and serum IL-8: Sensitive early markers of severity and outcome in sepsis

The aim of the present study was to investigate which biomarker/s reliably assess severity and mortality early in the sepsis process. In 47 critically-ill patients within the 24 h of septic onset, Interleukins (IL)-8, -1β, -6, -10, and -12p70, tumor necrosis factor-α (TNF-α), procalcitonin (PCT) and C-reactive protein (CRP) were measured in serum. Additionally, CD64 expression was measured in neutrophils. In early sepsis, neutrophil CD64 expression and IL-8 levels are the only biomarkers that increased with sepsis severity, differentiating disease stages: sepsis, severe sepsis and septic shock (p < 0.001). The biomarkers that best evaluate the severity of sepsis (via APACHE II) were CD64, IL-8 and IL-6 (p < 0.01), and the severity of organ failure (via SOFA) were CD64 and IL-8 (p < 0.01). CD64 expression and IL-8 levels were associated with mortality within 28-days (OR = 1.3, p = 0.01 for CD64 and OR = 1.26, p = 0.024 for IL-8 by logistic regression analysis) and ROC curve analysis showed high sensitivity and specificity for predicting sepsis stages and the 28 day mortality. We conclude that there is an early increase of neutrophil CD64 expression and IL-8 levels during sepsis. Based on this single measurement it is possible to reliably assess the stage, detect the severity and predict the 28-day mortality of sepsis.     

Interleukin-21 expanded NKDC in vitro reduces the B16F10 tumor growth in vivo

Innate immunity to tumors is mediated mainly by natural killer cells (NKs) and dendritic cells (DCs). The function of these cells is coordinated by cytokines produced during the inflammatory process. NK cells are highly active against tumors, being an important source of IFN-γ. Natural killer dendritic cells (NKDCs) were recently identified as a group of hybrid cells; some studies claim that they have lytic activity, produce IFN-γ and can also stimulate antigen-specific T cells. Interleukin 21 (IL-21) regulates the proliferation capacity and cytotoxicity of NK and T cells. The main objective of this study was to investigate if IL-21 influences the frequency of NKDCs in vitro as well as IFN-γ production and also to verify if these cells could enhance the antitumor activity against B16F10 tumor model in vivo. Splenocytes from C57BL/6 mice were isolated and the DC were enriched by immunomagnetic beads and cultured for four days with recombinant IL-21 (10, 20, 40 or 100 ng/ml). NKDC population was characterized as CD11c^low/medB220⁺NK1.1⁺. Expanded cells were used to treat B16F10 tumor bearing mice and tumor growth was compared between the doses of IL-21 10 ng/ml and 20 ng/ml. The results indicate that IL-21 increases the expansion of splenic NKDCs in vitro in doses of 10 ng/ml and 20 ng/ml and these cells produce IFN-γ. In vivo, cells expanded with IL-21 and injected directly into the growing tumor efficiently reduced the tumor size. Together, these results showed for the first time that IL-21 influences the biology and the effector activity of NKDCs.     

Effect of transcatheter renal arterial embolization combined with cryoablation on regulatory CD4+CD25+ T lymphocytes in the peripheral blood of patients with advanced renal carcinoma

ObjectiveTo analyze the effect of Argon-Helium cryosurgery (AHCS) combined with transcatheter renal arterial embolization (TRAE) on the differentiation of regulatory CD4⁺ CD25⁺ T cell (Treg) and its implication in patients with renal carcinoma.MethodsSeventy seven patients are included in the study, and divided into two groups: TRAE group (n = 45, receiving TRAE only) and TRAE + cryoablation group (n = 32, receiving cryoablation 2–3 weeks after TRAE). The percentage of Treg cells and T lymphocyte subsets (CD4⁺T, CD8⁺T, and CD4⁺T/CD8⁺T) in the peripheral blood is measured by flow cytometry previous to the therapy and 3 months after therapy. Meanwhile, the extent of tumor necrosis is measured by MRI or CT 1 month after therapy.ResultsThe percentages of Treg cells of patients in TRAE + cryoablation group decrease from (6.65 ± 1.22)% to (3.93 ± 1.16)%, (t = 42.768, P < 0.01), and the percentages of CD4⁺T and CD4⁺T/CD8⁺T increase significantly (P < 0.01). However, the results of patients in TRAE group show that the percentages of Treg, CD4⁺T, CD8⁺T and CD4⁺T/CD8⁺T increase slightly although the differences had no statistical significance (P > 0.05). The tumor necrosis rate of TRAE + cryoablation group is 57.5%, significantly higher than those of TRAE group, which shows 31.6% (t = 6.784, P < 0.01). The median survival duration of the TRAE + cryoablation group is 20 months, significantly longer than that of the TRAE group (χ² = 7.368, P < 0.01). The decreasing extent of Treg cells is correlated with tumor necrosis rates (r = 0.90, P < 0.01) and life time (r = 0.67, P < 0.01).ConclusionThe therapy of TRAE combined with cryoablation contributes to reduce the percentage of Treg cells and improve the immune situation of patients with renal cell carcinoma, which consequently increase tumor necrosis rate and prolong the patients‘ survival duration.     

Aging is associated with circulating cytokine dysregulation

PurposeAging is accompanied by a progressive increase in pro-inflammatory cytokine status. However, little is known about the development of age-dependent modifications in other circulating cytokines. The aim of this study was to investigate in vivo the influence of age on circulating cytokine production in healthy subjects (HC).MethodsCirculating cytokines were measured by CBA and ELISA in 73 HC. Intracellular cytokine production was assessed in CD3+ and CD14+ cells by flow cytometry. Production of cytokines in cell culture supernatants was also studied after polyclonal stimulation.ResultsSubjects were divided into three different groups according to age: 28 young HC (<30 years, 26.2 ± 2.4), 24 middle age HC (30–60 years, 44.7 ± 8.4) and 21 elderly HC (>60 years, 70.6 ± 7.9). Age was positively correlated with the circulating levels of IL-12p70, IL-1β, TNFα, IL-6, and IL-10. Age had a negative correlation with circulating levels of IL-17. Besides, age was positively correlated with spontaneous intracellular expression of proinflammatory cytokines in circulating monocytes. No correlation was found with other intracellular cytokine expression or with the production of cytokines in cell culture supernatants after in vitro stimulation. Gender had a marginal effect on the circulating cytokine profile.ConclusionAging has a significant impact on the production of circulating cytokines in healthy individuals. The circulating cytokine milieu may contribute to the development of age-restricted conditions.     

High salt induces anti-inflammatory MΦ2-like phenotype in peripheral macrophages

Macrophages play a critical role in inflammation and antigen-presentation. Abnormal macrophage function has been attributed in autoimmune diseases and cancer progression. Recent evidence suggests that high salt tissue micro-environment causes changes in macrophage activation. In our current report, we studied the role of extracellular sodium chloride on phenotype changes in peripheral circulating monocyte/macrophages collected from healthy donors. High salt (0.2 M NaCl vs basal 0.1 M NaCl) treatment resulted in a decrease in MΦ1 macrophage phenotype (CD11b⁺CD14^highCD16^low) from 77.4±6.2% (0.1 M) to 29.3±5.7% (0.2 M, p<0.05), while there was an increase in MΦ2 macrophage phenotype (CD11b⁺ CD14^lowCD16^high) from 17.2±5.9% (0.1 M) to 67.4±9.4% (0.2 M, p<0.05). ELISA-based cytokine analysis demonstrated that high salt treatment induced decreased expression of in the MΦ1 phenotype specific pro-inflammatory cytokine, TNFα (3.3 fold), IL-12 (2.3 fold), CCL-10 (2 fold) and CCL-5 (3.8 fold), but conversely induced an enhanced expression MΦ2-like phenotype specific anti-inflammatory cytokine, IL-10, TGFβ, CCL-17 (3.7 fold) and CCR-2 (4.3 fold). Further high salt treatment significantly decreased phagocytic efficiency of macrophages and inducible nitric oxide synthetase expression. Taken together, these data suggest that high salt extracellular environment induces an anti-inflammatory MΦ2-like macrophage phenotype with poor phagocytic and potentially reduced antigen presentation capacity commonly found in tumor microenvironment.     

Inactivation of IL-6 and soluble IL-6 receptor by neutrophil derived serine proteases in cystic fibrosis

The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4 × 10⁶ cells/ml v. 0.35 × 10⁶ cells/ml in controls) (p < 0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62 × 10⁶ cells/ml v. 0.007 × 10⁶ cells/ml in controls) (p < 0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122 pg/ml v. 64 pg/ml in controls, p < 0.01 and MCP-1; median: 692 pg/ml v. 182 pg/ml in controls, p < 0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p < 0.01), was weakly expressed (median: 50 pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung.