Podocytes regulate neutrophil recruitment by glomerular endothelial cells via IL-6-mediated crosstalk.

Stromal cells actively modulate the inflammatory process, in part by influencing the ability of neighboring endothelial cells to support the recruitment of circulating leukocytes. We hypothesized that podocytes influence the ability of glomerular endothelial cells (GEnCs) to recruit neutrophils during inflammation. To address this, human podocytes and human GEnCs were cultured on opposite sides of porous inserts and then treated with or without increasing concentrations of TNF-α prior to addition of neutrophils. The presence of podocytes significantly reduced neutrophil recruitment to GEnCs by up to 50% when cultures were treated with high-dose TNF-α (100 U/ml), when compared with GEnC monocultures. Importantly, this phenomenon was dependent on paracrine actions of soluble IL-6, predominantly released by podocytes. A similar response was absent when HUVECs were cocultured with podocytes, indicating a tissue-specific phenomenon. Suppressor of cytokine signaling 3 elicited the immunosuppressive actions of IL-6 in a process that disrupted the presentation of chemokines on GEnCs by altering the expression of the duffy Ag receptor for chemokines. Interestingly, suppressor of cytokine signaling 3 knockdown in GEnCs upregulated duffy Ag receptor for chemokines and CXCL5 expression, thereby restoring the neutrophil recruitment. In summary, these studies reveal that podocytes can negatively regulate neutrophil recruitment to inflamed GEnCs by modulating IL-6 signaling, identifying a potential novel anti-inflammatory role of IL-6 in renal glomeruli.

Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection, galactosylation, and sialylation.

Granulomatosis with polyangiitis (GPA) is associated with circulating immunoglobulin (Ig) G anti-proteinase 3 specific (anti-PR3) anti-neutrophil cytoplasm antibodies (ANCA), which activate cytokine primed neutrophils via Fcgamma receptors. ANCA are class switched IgG antibodies implying T cell help in their production. Glycosylation of IgG Fc, under the control of T cell cytokines, determines the interaction between IgG and its receptors. Previous studies have reported aberrant glycosylation of Ig Fc in GPA patients. We investigated whether aberrant Fc glycosylation was present on anti-PR3 ANCA as well as whole IgG subclass preparations compared to healthy controls and whether this correlated with Birmingham vasculitis activity scores (BVAS), serum cytokines, and time to remission. Here, IgG Fc glycosylation of GPA patients and controls and anti-PR3 ANCA Fc glycosylation were determined by mass spectrometry of glycopeptides. IgG1 and IgG2 subclasses from GPA patients showed reduced galactosylation, sialylation, and bisection compared to healthy controls. Anti-PR3 IgG1 ANCA Fc galactosylation, sialylation, and bisection were reduced compared to total IgG1 in GPA. Galactosylation of anti-PR3 ANCA Fc correlated with inflammatory cytokines and time to remission but not BVAS. Bisection of anti-PR3 ANCA Fc correlated with BVAS. Total IgG1 and anti-PR3 IgG1 Fc galactosylation were weakly correlated, while bisection of IgG1 and anti-PR3 showed no correlation. Our data indicate that aberrant ANCA galactosylation may be driven in an antigen-specific manner.

Recommendations for the use of rituximab in anti-neutrophil cytoplasm antibody-associated vasculitis.

OBJECTIVES: To perform a literature review and develop recommendations for the use of rituximab in ANCA-associated vasculitis. METHODS: A committee of experts (five rheumatologists, five nephrologists and one paediatrician) conducted a modified Delphi exercise to identify five topics for a systematic literature search. The evidence was then reviewed, categorized according to international criteria and assimilated to form five recommendations statements and a research agenda. RESULTS: Forty-three studies met the review criteria. These included two randomized controlled trials and a predominance of small, uncontrolled series. In refractory ANCA-associated vasculitis, remission rates of >80% are obtained with rituximab. In newly diagnosed disease, rituximab is at least as effective as conventional therapy. Fifteen recommendations were made. Their strength was restricted by the low quality of the evidence. Six areas for future research were identified. CONCLUSION: On the basis of the available evidence and expert consensus, recommendations have been made for the use of rituximab as a treatment of ANCA-associated vasculitis. Further questions, in particular regarding long-term outcomes, remain to be explored.

Biology of the renal pericyte.

Pericytes are cells of mesenchymal origin that are intimately involved in the development and stabilization of vascular networks. Novel studies of their role in inflammation have identified that pericytes are not only major contributors to the activated matrix depositing myofibroblast populations seen in progressive renal fibrosis but perhaps even more importantly, the detachment of renal pericytes from the vasculature contributes to the microvasculature rarefaction and subsequent hypoxia associated with chronic kidney disease. In this review, our current understanding of the functioning of renal pericytes will be considered and set in the context of the wider literature that is currently available on this neglected population of cells.

Neutrophil serine proteases mediate inflammatory cell recruitment by glomerular endothelium and progression towards dysfunction.

BACKGROUND: Neutrophil recruitment into glomerular tissues and reduced capillary wall integrity has been implicated in the development of vasculitic glomerulonephritis (VGN). This study investigated the stages and mechanisms through which neutrophil serine proteases (SPs), proteinase 3 (PR3) or elastase contribute to endothelial dysfunction. METHODS: Protease-induced damage to endothelium and adhesion molecule upregulation was measured by viability assays and ELISA. Neutrophil/platelet adhesion to human glomerular and umbilical vein endothelium was assessed using in vitro adhesion assays. RESULTS: PR3 and elastase (1 µg/mL, 2 h) significantly induced neutrophil adhesion to endothelial cells (EnC) whilst PR3 also enhanced platelet-EnC interactions. This neutrophil adhesion was associated with enhanced P-selectin expression and required CXCL8 receptor involvement, and could be inhibited by blocking the P-selectin ligand PSGL-1. SPs induced damage in a time- and dose-dependent fashion, decreasing cell monolayer integrity followed by cell membrane integrity, inducing caspase-3 activation and p21 cleavage. However, SPs caused significant EnC damage with increasing concentrations and prolonged exposures. CONCLUSION: Neutrophil SPs induce a pro-adhesive phenotype in glomerular endothelium primarily by inducing neutrophil and platelet adhesion that transits to dysfunction after high/prolonged exposures. Dysregulated release of these enzymes within glomeruli may contribute to injury during diseases such as VGN.

CD4+CD28- T cell expansion in granulomatosis with polyangiitis (Wegener's) is driven by latent cytomegalovirus infection and is associated with an increased risk of infection and mortality.

OBJECTIVE: Expanded populations of CD4+CD28- T cells with a cytotoxic phenotype have been repeatedly reported in patients with granulomatosis with polyangiitis (Wegener's) (GPA). In healthy individuals expansion of this T cell population follows cytomegalovirus (CMV) infection. We undertook this study to investigate whether CMV infection may be responsible for driving the expansion of CD4+CD28- T cells in GPA patients and how this might relate to clinical features. METHODS: Forty-eight GPA patients and 38 age-matched healthy donors were included in the study. CMV-specific IgG in serum was detected by enzyme-linked immunosorbent assay. Flow cytometric analysis was used to study T cell populations and phenotype. The presence of CMV in renal biopsy tissue from GPA patients was investigated by immunohistochemistry and polymerase chain reaction (PCR). Clinical information was obtained from patient records. RESULTS: Populations of CD4+CD28- T cells were only expanded in CMV-seropositive GPA patients and controls. In CMV-seropositive GPA patients we observed negative correlations between the percentages of CD4+CD28- T cells and both the percentage of naive T cells and the glomerular filtration rate at presentation. There was a significant association between the percentage of CD4+CD28- T cells and risk of infection and mortality. CMV could not be detected in renal tissue by PCR or immunohistochemistry. CMV seropositivity itself was not a risk factor for infection in a cohort of 182 patients with antineutrophil cytoplasmic antibody-associated vasculitis who had been recruited into clinical trials performed by the European Vasculitis Study Group. CONCLUSION: The expansion of CD4+CD28- T cells in GPA patients is associated with CMV infection and leads to a reduction in the number of naive T cells in peripheral blood. Patients with expanded CD4+CD28- T cells have significantly increased mortality and risk of infection.

CD248+ stromal cells are associated with progressive chronic kidney disease.

Stromal fibroblasts are the primary cells of the kidney that produce fibrotic matrix. CD248 is a stromal marker expressed on fibroblasts and pericytes within the human kidney. Here, we tested whether CD248 expression in the kidney colocalizes with fibrosis and if it is associated with known determinants of chronic kidney disease (CKD). CD248 expression was located and quantified in situ by immunohistochemistry in kidney biopsies from 93 patients with IgA nephropathy and compared with 22 archived biopsies encompassing normal kidney tissue as control. In normal kidney tissue, CD248 was expressed by resident pericytes, stromal fibroblasts, and was upregulated in human CKD. The expression was linked to known determinants of renal progression. This relationship was maintained in a multivariate analysis with CD248 expression linked to renal survival. CD248 was expressed by a population of α-smooth muscle actin (SMA)(+) myofibroblasts and α-SMA(-) stromal cells but not expressed on CD45(+) leukocytes. Thus, CD248 defines a subset of stromal cells, including but not limited to some myofibroblasts, linked to albuminuria and tubulointerstitial damage during tissue remodeling in CKD.

A dual role for diacylglycerol kinase generated phosphatidic acid in autoantibody-induced neutrophil exocytosis.

Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.

Addition of infliximab to standard therapy for ANCA-associated vasculitis.

BACKGROUND: Tumour necrosis factor-α (TNF) is implicated in the pathogenesis of anti-neutrophil cytoplasm antibody-associated vasculitis (AAV). Current immunosuppressive therapy is associated with considerable morbidity and mortality. Anti-TNF antibody therapy (infliximab) may help control AAV by providing more targeted immunosuppression and allow reductions in the use of corticosteroids and cyclophosphamide, thereby reducing the burden of immunosuppression with its associated morbidity and mortality. METHODS: 33 patients with active AAV participated in this cohort study. Patients were treated with standard therapy (corticosteroids and cyclophosphamide with additional plasma exchange in the case of life- or organ-threatening disease) or standard therapy + infliximab at weeks 0, 2, 6 and 10. The primary outcome measure was time to remission. Other outcome measures were adverse events, cumulative damage scores and relapse, as well as biomarkers for circulating activated and regulatory T cells. Follow-up was for 12 months. RESULTS: 17 patients received standard therapy alone; 16 patients received additional infliximab. The addition of infliximab to standard therapy did not influence remission rates, adverse events, damage index scores, relapse rates or biomarker levels in this cohort study. CONCLUSION: The addition of infliximab to standard therapy did not confer clinical benefit for patients with active AAV.

Immune profile and Epstein-Barr virus infection in acute interstitial nephritis: an immunohistochemical study in 78 patients.

BACKGROUND: Acute interstitial nephritis (AIN) is a common cause of acute kidney injury and is characterised by a dense interstitial cellular infiltrate, which has not been well defined. Previous studies have demonstrated a correlation between Epstein-Barr virus (EBV) infection and AIN. The purpose of our study was to define the nature of the interstitial immune infiltrate and to investigate the possibility of renal infection with EBV. METHODS: Seventy-eight patients with AIN were identified from renal biopsy reports in a single centre over an 18-year period. Immunohistochemical staining was performed to define the cellular infiltrate. In situ hybridization and immunohistology were used to detect EBV. RESULTS: A positive correlation between CD68 macrophage infiltration and serum creatinine concentration at presentation was identified. IL-4, eotaxin, CCR3, CCR5 and VCAM-1 were all expressed in biopsies of AIN. Using in situ hybridization and immunohistochemistry, EBV was not detected in any of the AIN sections analysed. CONCLUSION: This study has assessed the nature of the interstitial infiltrate in AIN. EBV was not detected in the renal biopsies, suggesting that EBV is not a pathogenetic factor in AIN.

Anti-plasminogen antibodies compromise fibrinolysis and associate with renal histology in ANCA-associated vasculitis.

Antibodies recognizing plasminogen, a key component of the fibrinolytic system, associate with venous thrombotic events in PR3-ANCA vasculitis. Here, we investigated the prevalence and function of anti-plasminogen antibodies in independent UK and Dutch cohorts of patients with ANCA-associated vasculitis (AAV). We screened Ig isolated from patients (AAV-IgG) and healthy controls by ELISA. Eighteen of 74 (24%) UK and 10/38 (26%) Dutch patients with AAV had anti-plasminogen antibodies compared with 0/50 and 1/61 (2%) of controls. We detected anti-plasminogen antibodies in both PR3-ANCA- and MPO-ANCA-positive patients. In addition, we identified anti-tissue plasminogen activator (tPA) antibodies in 13/74 (18%) patients, and these antibodies were more common among patients with anti-plasminogen antibodies (P = 0.011). Eighteen of 74 AAV-IgG (but no control IgG) retarded fibrinolysis in vitro, and this associated with anti-plasminogen and/or anti-tPA antibody positivity. Only 4/18 AAV-IgG retarded fibrinolysis without harboring these antibodies; dual-positive samples retarded fibrinolysis to the greatest extent. Patients with anti-plasminogen antibodies had significantly higher percentages of glomeruli with fibrinoid necrosis (P < 0.05) and cellular crescents (P < 0.001) and had more severely reduced renal function than patients without these antibodies. In conclusion, anti-plasminogen and anti-tPA antibodies occur in AAV and associate with functional inhibition of fibrinolysis in vitro. Seropositivity for anti-plasminogen antibodies correlates with hallmark renal histologic lesions and reduced renal function. Conceivably, therapies that enhance fibrinolysis might benefit a subset of AAV patients.

Depletion of LAG-3+ T Cells Translated to Pharmacology and Improvement in Psoriasis Disease Activity: A Phase I Randomized Study of mAb GSK2831781.

Activated T cells drive a range of immune-mediated inflammatory diseases. LAG-3 is transiently expressed on recently activated CD4+ and CD8+ T cells. We describe the engineering and first-in-human clinical study (NCT02195349) of GSK2831781 (an afucosylated humanized IgG1 monoclonal antibody enhanced with high affinity for Fc receptors and LAG-3 and antibody-dependent cellular cytotoxicity capabilities), which depletes LAG-3 expressing cells. GSK2831781 was tested in a phase I/Ib, double-blind, placebo-controlled clinical study, which randomized 40 healthy participants (part A) and 27 patients with psoriasis (part B) to single doses of GSK2831781 (up to 0.15 and 5 mg/kg, respectively) or placebo. Adverse events were generally balanced across groups, with no safety or tolerability concern identified. LAG-3+ cell depletion in peripheral blood was observed at doses ≥ 0.15 mg/kg and was dose-dependent. In biopsies of psoriasis plaques, a reduction in mean group LAG-3+ and CD3+ T-cell counts was observed following treatment. Downregulation of proinflammatory genes (IL-17A, IL-17F, IFNγ, and S100A12) and upregulation of the epithelial barrier integrity gene, CDHR1, was observed with the 5 mg/kg dose of GSK2831781. Psoriasis disease activity improved up to day 43 at all GSK2831781 doses (0.5, 1.5, and 5 mg/kg) compared with placebo. Depletion of LAG-3-expressing activated T cells is a novel approach, and this first clinical study shows that GSK2831781 is pharmacologically active and provides encouraging early evidence of clinical effects in psoriasis, which warrants further investigation in T-cell-mediated inflammatory diseases.

Patients with Wegener's granulomatosis demonstrate a relative deficiency and functional impairment of T-regulatory cells.

An increased proportion of CD4(+) CD25(+) T cells has been reported in Wegener's granulomatosis (WG) and may represent an accumulation of regulatory T cells (Treg). CD25 is also expressed on recently activated effector T cells. We have determined the relative proportion of these subsets in a large patient cohort. The fraction of Treg in peripheral blood mononuclear cells from patients and healthy controls was determined by assessment of Foxp3 expression on CD4(+) CD25(+) T cells. The functional activity of Treg was determined by their ability to suppress proliferation and cytokine production in response to proteinase-3. Although WG patients demonstrated an increased fraction of CD4(+) CD25(+) T cells, the percentage of Foxp3-positive cells was decreased. In addition, the percentage of Treg was inversely related to the rate of disease relapse. CD4(+) CD25(hi) T cells were able to suppress T-cell proliferation to proteinase-3 in healthy controls and anti-neutrophil cytoplasm antibody (ANCA)- negative patients (at time of sampling) but not in ANCA-positive patients. In patients with active disease, an increased proportion of CD4(+) Foxp3(+) cells was associated with a more rapid disease remission. Patients with WG demonstrate abnormalities in the number and function of Treg and this is most pronounced in those with most active disease. This information is of value in understanding the pathogenesis and potential treatment of this disease.

Pulse versus daily oral cyclophosphamide for induction of remission in antineutrophil cytoplasmic antibody-associated vasculitis: a randomized trial.

BACKGROUND: Current therapies for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis are limited by toxicity. OBJECTIVE: To compare pulse cyclophosphamide with daily oral cyclophosphamide for induction of remission. DESIGN: Randomized, controlled trial. Random assignments were computer-generated; allocation was concealed by faxing centralized treatment assignment to providers at the time of enrollment. Patients, investigators, and assessors of outcomes were not blinded to assignment. SETTING: 42 centers in 12 European countries. PATIENTS: 149 patients who had newly diagnosed generalized ANCA-associated vasculitis with renal involvement but not immediately life-threatening disease. INTERVENTION: Pulse cyclophosphamide, 15 mg/kg every 2 to 3 weeks (76 patients), or daily oral cyclophosphamide, 2 mg/kg per day (73 patients), plus prednisolone. MEASUREMENT: Time to remission (primary outcome); change in renal function, adverse events, and cumulative dose of cyclophosphamide (secondary outcomes). RESULTS: Groups did not differ in time to remission (hazard ratio, 1.098 [95% CI, 0.78 to 1.55]; P = 0.59) or proportion of patients who achieved remission at 9 months (88.1% vs. 87.7%). Thirteen patients in the pulse group and 6 in the daily oral group achieved remission by 9 months and subsequently had relapse. Absolute cumulative cyclophosphamide dose in the daily oral group was greater than that in the pulse group (15.9 g [interquartile range, 11 to 22.5 g] vs. 8.2 g [interquartile range, 5.95 to 10.55 g]; P < 0.001). The pulse group had a lower rate of leukopenia (hazard ratio, 0.41 [CI, 0.23 to 0.71]). LIMITATIONS: The study was not powered to detect a difference in relapse rates between the 2 groups. Duration of follow-up was limited. CONCLUSION: The pulse cyclophosphamide regimen induced remission of ANCA-associated vasculitis as well as the daily oral regimen at a reduced cumulative cyclophosphamide dose and caused fewer cases of leukopenia. PRIMARY FUNDING SOURCE: The European Union.

ANCA-associated vasculitis.

The anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) are a group of disorders involving severe, systemic, small-vessel vasculitis and are characterized by the development of autoantibodies to the neutrophil proteins leukocyte proteinase 3 (PR3-ANCA) or myeloperoxidase (MPO-ANCA). The three AAV subgroups, namely granulomatosis with polyangiitis (GPA), microscopic polyangiitis and eosinophilic GPA (EGPA), are defined according to clinical features. However, genetic and other clinical findings suggest that these clinical syndromes may be better classified as PR3-positive AAV (PR3-AAV), MPO-positive AAV (MPO-AAV) and, for EGPA, by the presence or absence of ANCA (ANCA+ or ANCA-, respectively). Although any tissue can be involved in AAV, the upper and lower respiratory tract and kidneys are most commonly and severely affected. AAVs have a complex and unique pathogenesis, with evidence for a loss of tolerance to neutrophil proteins, which leads to ANCA-mediated neutrophil activation, recruitment and injury, with effector T cells also involved. Without therapy, prognosis is poor but treatments, typically immunosuppressants, have improved survival, albeit with considerable morbidity from glucocorticoids and other immunosuppressive medications. Current challenges include improving the measures of disease activity and risk of relapse, uncertainty about optimal therapy duration and a need for targeted therapies with fewer adverse effects. Meeting these challenges requires a more detailed knowledge of the fundamental biology of AAV as well as cooperative international research and clinical trials with meaningful input from patients.

Chimeric IgG4 PR3-ANCA induces selective inflammatory responses from neutrophils through engagement of Fcgamma receptors.

Anti-proteinase 3 antibodies are implicated in the pathogenesis of small vessel vasculitis. These are primarily immunoglobulin G (IgG), with different subclasses predominating at different stages of disease. However, little is known of their respective roles in pathogenesis. We have previously shown that patient IgG4 was able to induce superoxide release from human neutrophils. To circumvent difficulties in separating the subclasses and additional differences in polyclonal patient antibodies we have generated monoclonal mouse/human IgG1 and IgG4 anti-proteinase 3 antibodies. Using these antibodies we have compared effects of IgG1 and IgG4 on human neutrophils in terms of superoxide release, cytokine production, degranulation and adhesion. Additionally we have investigated the interaction of the subclasses with Fc receptors expressed by the neutrophil. Chimeric antibodies were generated using human constant regions of each subclass and a variable region taken from a monoclonal antibody directed against proteinase 3. Superoxide release from neutrophils was measured by the reduction of ferricytochrome C, degranulation by the conversion of a synthetic colour substrate, cytokine release by interleukin-8 enzyme-linked immunosorbent assay, and adhesion by a flow-based adhesion assay. Fc receptor binding was assessed using blocking antibodies. The IgG4 anti-proteinase 3 was able to induce a dose-dependent release of superoxide, degranulation and adhesion. The antibody was not able to stimulate the secretion of interleukin-8. Fc receptors were essential for neutrophil stimulation and the constitutive Fc receptors were necessary for different stimulatory pathways. The IgG4 anti-proteinase 3 antibodies are able to stimulate neutrophils to undergo a pro-inflammatory response and may play a role in the pathogenesis of small vessel vasculitis.

CT60 and +49 polymorphisms of CTLA 4 are associated with ANCA-positive small vessel vasculitis.

OBJECTIVE: To investigate whether single nucleotide polymorphisms (SNPs) within cytotoxic T-lymphocyte antigen-4 (CTLA-4) are associated with ANCA-associated small vessel vasculitis (SVV). METHODS: The CTLA-4 CT60 (exon 4), +49 (exon 1) and -318 (promoter region) genotypes were determined by PCR and restriction fragment length polymorphism (RFLP) in 222 white Caucasians of UK origin with SVV and 670 ethnically matched controls. RESULTS: The CTLA-4 exon 1 (+49) and 4 (CT60) polymorphisms are associated with SVV (+49: chi(2) = 10.965, P = 0.004; CT60: chi(2) = 12.017, P = 0.002). Both disease-susceptible and disease-protective haplotypes have been identified in this cohort, and their frequencies are similar in the subtypes of WG and microscopic polyangiitis. CONCLUSION: This study provides further evidence that CTLA-4, a susceptibility locus for a number of common autoimmune diseases, may also be involved in the development of ANCA-associated SVV.

Review article: Leukocyte-endothelial dysregulation in systemic small vessel vasculitis.

Aberrant interaction between the leukocyte and the endothelial cell (EC) results in the uncontrolled inflammation seen in systemic small vessel vasculitis. This review discusses our current understanding of this process and includes consideration of the role of adhesion molecules, proteases and the neutrophil respiratory burst. The effects of anti-neutrophil cytoplasm antibodies and anti-endothelial cell antibodies and their pathogenic roles are examined, and we look at experimental disease models. Specificity of disease-targetted endothelial beds and the role of circulating EC are discussed.

Mechanisms of ANCA-mediated leukocyte-endothelial cell interactions in vivo.

Anti-myeloperoxidase (anti-MPO) antibodies have been implicated in the pathogenesis of small-vessel vasculitis, but the molecular mechanisms by which these antibodies contribute to disease are unknown. For determination of how anti-MPO antibodies affect inflammatory cell recruitment in small-vessel vasculitis, intravital microscopy was used to monitor leukocyte behavior in the accessible cremasteric microvessels under various experimental conditions. After local pretreatment of the cremaster muscle with cytokines (TNF-alpha, IL-1beta, or keratinocyte-derived chemokine), administration of anti-MPO IgG to wild-type mice reduced leukocyte rolling in favor of augmented adhesion to and transmigration across the endothelium. This led to a decrease in the number of systemic circulating leukocytes and, similar to the early events in the development of vasculitic lesions, an increase in leukocyte recruitment to renal and pulmonary tissue. TNF-alpha led to the greatest recruitment of inflammatory cells, and IL-1beta led to the least. When anti-CD18 was co-administered, anti-MPO IgG did not affect leukocyte rolling, adhesion, or transmigration; similarly, anti-MPO IgG did not produce these effects in Fc receptor gamma chain-/- mice. This study provides direct in vivo evidence of enhanced leukocyte-endothelial cell interactions in the presence of anti-MPO IgG and highlights the critical roles of Fcgamma receptors and beta2 integrins in mediating these interactions. In addition, it suggests that neutrophils primed by cytokines in the presence of anti-MPO IgG can have systemic effects and target specific vascular beds.

Phenotypic Analysis of Human Lymph Nodes in Subjects With New-Onset Type 1 Diabetes and Healthy Individuals by Flow Cytometry.

Background: Ultrasound guided sampling of human lymph node (LN) combined with advanced flow cytometry allows phenotypic analysis of multiple immune cell subsets. These may provide insights into immune processes and responses to immunotherapies not apparent from analysis of the blood. Methods: Ultrasound guided inguinal LN samples were obtained by both fine needle aspiration (FNA) and core needle biopsy in 10 adults within 8 weeks of diagnosis of type 1 diabetes (T1D) and 12 age-matched healthy controls at two study centers. Peripheral blood mononuclear cells (PBMC) were obtained on the same occasion. Samples were transported same day to the central laboratory and analyzed by multicolour flow cytometry. Results: LN sampling was well-tolerated and yielded sufficient cells for analysis in 95% of cases. We confirmed the segregation of CD69+ cells into LN and the predominance of CD8+ Temra cells in blood previously reported. In addition, we demonstrated clear enrichment of CD8+ naïve, FOXP3+ Treg, class-switched B cells, CD56bright NK cells and plasmacytoid dendritic cells (DC) in LNs as well as CD4+ T cells of the Th2 phenotype and those expressing Helios and Ki67. Conventional NK cells were virtually absent from LNs as were Th22 and Th1Th17 cells. Paired correlation analysis of blood and LN in the same individuals indicated that for many cell subsets, especially those associated with activation: such as CD25+ and proliferating (Ki67+) T cells, activated follicular helper T cells and class-switched B cells, levels in the LN compartment could not be predicted by analysis of blood. We also observed an increase in Th1-like Treg and less proliferating (Ki67+) CD4+ T cells in LN from T1D compared to control LNs, changes which were not reflected in the blood. Conclusions: LN sampling in humans is well-tolerated. We provide the first detailed "roadmap" comparing immune subsets in LN vs. blood emphasizing a role for differentiated effector T cells in the blood and T cell regulation, B cell activation and memory in the LN. For many subsets, frequencies in blood, did not correlate with LN, suggesting that LN sampling would be valuable for monitoring immuno-therapies where these subsets may be impacted.