Endothelial cell activation leads to neutrophil transmigration as supported by the sequential roles of ICAM-2, JAM-A, and PECAM-1

Leukocyte transmigration is mediated by endothelial cell (EC) junctional molecules, but the associated mechanisms remain unclear. Here we investigate how intercellular adhesion molecule-2 (ICAM-2), junctional adhesion molecule-A (JAM-A), and platelet endothelial cell adhesion molecule (PECAM-1) mediate neutrophil transmigration in a stimulus-dependent manner (eg, as induced by interleukin-1beta [IL-1beta] but not tumor necrosis factor-alpha [TNF-alpha]), and demonstrate their ability to act in sequence. Using a cell-transfer technique, transmigration responses of wild-type and TNF-alpha p55/p75 receptor-deficient leukocytes (TNFR(-/-)) through mouse cremasteric venules were quantified by fluorescence intravital microscopy. Whereas wild-type leukocytes showed a normal transmigration response to TNF-alpha in ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) recipient mice, TNFR(-/-) leukocytes exhibited a reduced transmigration response. Hence, when the ability of TNF-alpha to directly stimulate neutrophils is blocked, TNF-alpha-induced neutrophil transmigration is rendered dependent on ICAM-2, JAM-A, and PECAM-1, suggesting that the stimulus-dependent role of these molecules is governed by the target cell being activated. Furthermore, analysis of the site of arrest of neutrophils in inflamed tissues from ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) mice demonstrated that these molecules act sequentially to mediate transmigration. Collectively, the findings provide novel insights into the mechanisms of action of key molecules implicated in leukocyte transmigration.

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE2 formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β). Treatment of rat and human mesangial cells with S1P led to concentration-dependent enhanced expression of COX-2. Pharmacological and molecular biology approaches revealed that the S1P-dependent increase of COX-2 mRNA and protein expression was mediated via activation of S1P receptor 2 (S1P2). Further, inhibition of Gi and p42/p44 MAPK signaling, both downstream of S1P2, abolished the S1P-induced COX-2 expression. In addition, S1P/S1P2-dependent upregulation of COX-2 led to significantly elevated PGE2 levels, which were further potentiated in the presence of Ang II and IL-1β. A functional consequence downstream of S1P/S1P2 signaling is mesangial cell migration that is stimulated by S1P. Interestingly, inhibition of COX-2 by celecoxib and SC-236 completely abolished the migratory response. Overall, our results demonstrate that extracellular S1P induces COX-2 expression via activation of S1P2 and subsequent Gi and p42/p44 MAPK-dependent signaling in renal mesangial cells leading to enhanced PGE2 formation and cell migration that essentially requires COX-2. Thus, targeting S1P/S1P2 signaling pathways might be a novel strategy to treat renal inflammatory diseases.

Impact of the CTLA-4/CD28 axis on the processes of joint inflammation in rheumatoid arthritis

OBJECTIVE: The importance of the costimulatory molecules CD28 and CTLA-4 in the pathologic mechanism of rheumatoid arthritis (RA) has been demonstrated by genetic associations and the successful clinical application of CTLA-4Ig for the treatment of RA. This study was undertaken to investigate the role of the CTLA-4/CD28 axis in the local application of CTLA-4Ig in the synovial fluid (SF) of RA patients. METHODS: Quantitative polymerase chain reaction was used to analyze the expression of proinflammatory and antiinflammatory cytokines in ex vivo fluorescence-activated cell sorted CTLA-4+ and CTLA-4- T helper cells from the peripheral blood and SF of RA patients. T helper cells were also analyzed for cytokine expression in vitro after the blockade of CTLA-4 by anti-CTLA-4 Fab fragments or of B7 (CD80/CD86) molecules by CTLA-4Ig. RESULTS: CTLA-4+ T helper cells were unambiguously present in the SF of all RA patients examined, and they expressed increased amounts of interferon-γ (IFNγ), interleukin-17 (IL-17), and IL-10 as compared to CTLA-4- T helper cells. The selective blockade of CTLA-4 in T helper cells from the SF in vitro led to increased levels of IFNγ, IL-2, and IL-17. The concomitant blockade of CD28 and CTLA-4 in T helper cells from RA SF by CTLA-4Ig in vitro resulted in reduced levels of the proinflammatory cytokines IFNγ and IL-2 and increased levels of the antiinflammatory cytokines IL-10 and transforming growth factor β. CONCLUSION: Our ex vivo and in vitro results demonstrate that the CTLA-4/CD28 axis constitutes a drug target for not only the systemic, but potentially also the local, application of the costimulation blocking agent CTLA-4Ig for the treatment of RA.

A randomized clinical trial of Behavioral Activation (BA) therapy for improving psychological and physical health in dementia caregivers: results of the Pleasant Events Program (PEP).

Dementia caregiving is associated with elevations in depressive symptoms and increased risk for cardiovascular diseases (CVD). This study evaluated the efficacy of the Pleasant Events Program (PEP), a 6-week Behavioral Activation intervention designed to reduce CVD risk and depressive symptoms in caregivers. One hundred dementia family caregivers were randomized to either the 6-week PEP intervention (N = 49) or a time-equivalent Information-Support (IS) control condition (N = 51). Assessments were completed pre- and post-intervention and at 1-year follow-up. Biological assessments included CVD risk markers Interleukin-6 (IL-6) and D-dimer. Psychosocial outcomes included depressive symptoms, positive affect, and negative affect. Participants receiving the PEP intervention had significantly greater reductions in IL-6 (p = .040), depressive symptoms (p = .039), and negative affect (p = .021) from pre- to post-treatment. For IL-6, clinically significant improvement was observed in 20.0% of PEP participants and 6.5% of IS participants. For depressive symptoms, clinically significant improvement was found for 32.7% of PEP vs 11.8% of IS participants. Group differences in change from baseline to 1-year follow-up were non-significant for all outcomes. The PEP program decreased depression and improved a measure of physiological health in older dementia caregivers. Future research should examine the efficacy of PEP for improving other CVD biomarkers and seek to sustain the intervention's effects.

Human TH9 cells are skin-tropic and have autocrine and paracrine proinflammatory capacity.

T helper type 9 (TH9) cells can mediate tumor immunity and participate in autoimmune and allergic inflammation in mice, but little is known about the TH9 cells that develop in vivo in humans. We isolated T cells from human blood and tissues and found that most memory TH9 cells were skin-tropic or skin-resident. Human TH9 cells coexpressed tumor necrosis factor-α and granzyme B and lacked coproduction of TH1/TH2/TH17 cytokines, and many were specific for Candida albicans. Interleukin-9 (IL-9) production was transient and preceded the up-regulation of other inflammatory cytokines. Blocking studies demonstrated that IL-9 was required for maximal production of interferon-γ, IL-9, IL-13, and IL-17 by skin-tropic T cells. IL-9-producing T cells were increased in the skin lesions of psoriasis, suggesting that these cells may contribute to human inflammatory skin disease. Our results indicate that human TH9 cells are a discrete T cell subset, many are tropic for the skin, and although they may function normally to protect against extracellular pathogens, aberrant activation of these cells may contribute to inflammatory diseases of the skin.

HLA class II antigen expression in colorectal carcinoma tumors as a favorable prognostic marker.

The goal of this study was to determine the frequency of HLA class II antigen expression in colorectal carcinoma (CRC) tumors, its association with the clinical course of the disease, and the underlying mechanism(s). Two tissue microarrays constructed with 220 and 778 CRC tumors were stained with HLA-DR, DQ, and DP antigen-specific monoclonal antibody LGII-612.14, using the immunoperoxidase staining technique. The immunohistochemical staining results were correlated with the clinical course of the disease. The functional role of HLA class II antigens expressed on CRC cells was analyzed by investigating their in vitro interactions with immune cells. HLA class II antigens were expressed in about 25% of the 220 and 21% of the 778 tumors analyzed with an overall frequency of 23%. HLA class II antigens were detected in 19% of colorectal adenomas. Importantly, the percentage of stained cells and the staining intensity were significantly lower than those detected in CRC tumors. However, HLA class II antigen staining was weakly detected only in 5.4% of 37 normal mucosa tissues. HLA class II antigen expression was associated with a favorable clinical course of the disease. In vitro stimulation with interferon gamma (IFNγ) induced HLA class II antigen expression on two of the four CRC cell lines tested. HLA class II antigen expression on CRC cells triggered interleukin-1β (IL-1β) production by resting monocytes. HLA class II antigen expression in CRC tumors is a favorable prognostic marker. This association may reflect stimulation of IL-1β production by monocytes.

Pre-therapy inflammation and coagulation activation and long-term CD4 count responses to the initiation of antiretroviral therapy

OBJECTIVES Pre-antiretroviral therapy (ART) inflammation and coagulation activation predict clinical outcomes in HIV-positive individuals. We assessed whether pre-ART inflammatory marker levels predicted the CD4 count response to ART. METHODS Analyses were based on data from the Strategic Management of Antiretroviral Therapy (SMART) trial, an international trial evaluating continuous vs. interrupted ART, and the Flexible Initial Retrovirus Suppressive Therapies (FIRST) trial, evaluating three first-line ART regimens with at least two drug classes. For this analysis, participants had to be ART-naïve or off ART at randomization and (re)starting ART and have C-reactive protein (CRP), interleukin-6 (IL-6) and D-dimer measured pre-ART. Using random effects linear models, we assessed the association between each of the biomarker levels, categorized as quartiles, and change in CD4 count from ART initiation to 24 months post-ART. Analyses adjusted for CD4 count at ART initiation (baseline), study arm, follow-up time and other known confounders. RESULTS Overall, 1084 individuals [659 from SMART (26% ART naïve) and 425 from FIRST] met the eligibility criteria, providing 8264 CD4 count measurements. Seventy-five per cent of individuals were male with the mean age of 42 years. The median (interquartile range) baseline CD4 counts were 416 (350-530) and 100 (22-300) cells/μL in SMART and FIRST, respectively. All of the biomarkers were inversely associated with baseline CD4 count in FIRST but not in SMART. In adjusted models, there was no clear relationship between changing biomarker levels and mean change in CD4 count post-ART (P for trend: CRP, P = 0.97; IL-6, P = 0.25; and D-dimer, P = 0.29). CONCLUSIONS Pre-ART inflammation and coagulation activation do not predict CD4 count response to ART and appear to influence the risk of clinical outcomes through other mechanisms than blunting long-term CD4 count gain.

EAACI IG Biologicals task force paper on the use of biologic agents in allergic disorders.

Biologic agents (also termed biologicals or biologics) are therapeutics that are synthesized by living organisms and directed against a specific determinant, for example, a cytokine or receptor. In inflammatory and autoimmune diseases, biologicals have revolutionized the treatment of several immune-mediated disorders. Biologicals have also been tested in allergic disorders. These include agents targeting IgE; T helper 2 (Th2)-type and Th2-promoting cytokines, including interleukin-4 (IL-4), IL-5, IL-9, IL-13, IL-31, and thymic stromal lymphopoietin (TSLP); pro-inflammatory cytokines, such as IL-1β, IL-12, IL-17A, IL-17F, IL-23, and tumor necrosis factor (TNF); chemokine receptor CCR4; and lymphocyte surface and adhesion molecules, including CD2, CD11a, CD20, CD25, CD52, and OX40 ligand. In this task force paper of the Interest Group on Biologicals of the European Academy of Allergy and Clinical Immunology, we review biologicals that are currently available or tested for the use in various allergic and urticarial pathologies, by providing an overview on their state of development, area of use, adverse events, and future research directions.

[Polymyalgia rheumatica: What is the current status?]

The diagnosis of polymyalgia rheumatica (PMR) is based on the typical clinical symptoms and elevated inflammatory markers in blood; however, both are unspecific and the differential diagnosis of the disease still represents a challenge for clinicians. The new consensus classification criteria of the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR) established in 2012 have a high sensitivity (92.6 %) and specificity (91.2 %) and therefore contribute to improved diagnostics. Glucocorticoids are still the standard treatment with methotrexate and as an alternative and possibly anti-interleukin (anti-IL) 6 therapy in the future.

Helicobacter pylori Inhibits Dendritic Cell Maturation via Interleukin-10-Mediated Activation of the Signal Transducer and Activator of Transcription 3 Pathway.

Helicobacter pylori infects the human gastric mucosa causing a chronic infection that is the primary risk factor for gastric cancer development. Recent studies demonstrate that H. pylori promotes tolerogenic dendritic cell (DC) development indicating that this bacterium evades the host immune response. However, the signaling pathways involved in modulating DC activation during infection remain unclear. Here, we report that H. pylori infection activated the signal transducer and activator of transcription 3 (STAT3) pathway in murine bone marrow-derived DCs (BMDCs) and splenic DCs isolated ex vivo. Isogenic cagA-, cagE-, vacA- and urease-mutants exhibited levels of phosphoSTAT3 that were comparable to in the wild-type (WT) parent strain. H. pylori-infected BMDCs produced increased immunosuppressive IL-10, which activated STAT3 in an autocrine/paracrine fashion. Neutralization of IL-10 prevented H. pylori-mediated STAT3 activation in both BMDCs and splenic DCs. In addition, anti-IL-10 treatment of infected H. pylori-BMDCs was associated with increased CD86 and MHC II expression and enhanced proinflammatory IL-1β cytokine secretion. Finally, increased CD86 and MHC II expression was detected in H. pylori-infected STAT3 knockout DCs when compared to WT controls. Together, these results demonstrate that H. pylori infection induces IL-10 secretion in DCs, which activates STAT3, thereby modulating DC maturation and reducing IL-1β secretion. These findings identify a host molecular mechanism by which H. pylori can manipulate the innate immune response to potentially favor chronic infection and promote carcinogenesis. © 2014 S. Karger AG, Basel.

Individual IL-3 priming is crucial for consistent in vitro activation of donor basophils in patients with chronic urticaria

The in vivo autologous serum skin test (ASST) is the diagnostic gold standard to detect autoantibodies against FcεRI or IgE itself, as well as other autoreactive serum components, in patients with chronic spontaneous urticaria (CU). Coincubation of patient sera with donor basophils and measuring their degranulation in vitro could be a safe alternative but has shown inconsistent results.

Proviral integration site for Moloney murine leukemia virus 1, but not phosphatidylinositol-3 kinase, is essential in the antiapoptotic signaling cascade initiated by IL-5 in eosinophils

BACKGROUND: Eosinophil differentiation, activation, and survival are largely regulated by IL-5. IL-5-mediated transmembrane signal transduction involves both Lyn-mitogen-activated protein kinases and Janus kinase 2-signal transducer and activator of transcription pathways. OBJECTIVE: We sought to determine whether additional signaling molecules/pathways are critically involved in IL-5-mediated eosinophil survival. METHODS: Eosinophil survival and apoptosis were measured in the presence and absence of IL-5 and defined pharmacologic inhibitors in vitro. The specific role of the serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim) 1 was tested by using HIV-transactivator of transcription fusion proteins containing wild-type Pim-1 or a dominant-negative form of Pim-1. The expression of Pim-1 in eosinophils was analyzed by means of immunoblotting and immunofluorescence. RESULTS: Although pharmacologic inhibition of phosphatidylinositol-3 kinase (PI3K) by LY294002, wortmannin, or the selective PI3K p110delta isoform inhibitor IC87114 was successful in each case, only LY294002 blocked increased IL-5-mediated eosinophil survival. This suggested that LY294002 inhibited another kinase that is critically involved in this process in addition to PI3K. Indeed, Pim-1 was rapidly and strongly expressed in eosinophils after IL-5 stimulation in vitro and readily detected in eosinophils under inflammatory conditions in vivo. Moreover, by using specific protein transfer, we identified Pim-1 as a critical element in IL-5-mediated antiapoptotic signaling in eosinophils. CONCLUSIONS: Pim-1, but not PI3K, plays a major role in IL-5-mediated antiapoptotic signaling in eosinophils.