A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress

Recent observations using multiphoton intravital microscopy (MP-IVM) have uncovered an unexpectedly high lymphocyte motility within peripheral lymph nodes (PLNs). Lymphocyte-expressed intracellular signaling molecules governing interstitial movement remain largely unknown. Here, we used MP-IVM of murine PLNs to examine interstitial motility of lymphocytes lacking the Rac guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3K)gamma, signaling molecules that act downstream of G protein-coupled receptors, including chemokine receptors (CKRs). T and B cells lacking DOCK2 alone or DOCK2 and PI3Kgamma displayed markedly reduced motility inside T cell area and B cell follicle, respectively. Lack of PI3Kgamma alone had no effect on migration velocity but resulted in increased turning angles of T cells. As lymphocyte egress from PLNs requires the sphingosine-1-phosphate (S1P) receptor 1, a G(alphai) protein-coupled receptor similar to CKR, we further analyzed whether DOCK2 and PI3Kgamma contributed to S1P-triggered signaling events. S1P-induced cell migration was significantly reduced in T and B cells lacking DOCK2, whereas T cell-expressed PI3Kgamma contributed to F-actin polymerization and protein kinase B phosphorylation but not migration. These findings correlated with delayed lymphocyte egress from PLNs in the absence of DOCK2 but not PI3Kgamma, and a markedly reduced cell motility of DOCK2-deficient T cells in close proximity to efferent lymphatic vessels. In summary, our data support a central role for DOCK2, and to a lesser extent T cell-expressed PI3Kgamma, for signal transduction during interstitial lymphocyte migration and S1P-mediated egress.

Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis

ABSTRACT: INTRODUCTION: In transgenic animal models of sepsis, members of the Bcl-2-family of proteins regulate lymphocyte apoptosis and survival of sepsis. This study investigates the gene regulation of pro- and anti-apoptotic members of the Bcl-2-family of proteins in patients with early stage severe sepsis. METHODS: In this prospective case-control study patients were recruited from three intensive care units in a university hospital. Sixteen patients were enrolled as soon as they fulfilled the criteria of severe sepsis. Ten critically ill but non-septic patients and eleven healthy volunteers served as controls. Blood samples were immediately obtained at inclusion. To confirm the presence of accelerated apoptosis in the patient groups, caspase-3 activation and phosphatidylserine (PS) externalization in CD4+, CD8+ and CD19+ lymphocyte subsets were assessed by flow cytometry. Specific mRNA's of Bcl-2 family members were quantified from whole blood by real-time polymerase chain reaction. To test for statistical significance, Kruskal-Wallis testing with Dunn's multiple comparison test for post hoc testing was performed. RESULTS: In all lymphocyte populations caspase-3 (p<0.05) was activated, which was reflected in an increased PS externalization (p<0.05). Accordingly, lymphocyte counts were decreased in early severe sepsis. In CD4+ T-cells (p<005) and in B-cells (p<0.001) the Bcl-2 protein was decreased in severe sepsis. Gene expression of the BH3-only Bim was massively upregulated as compared to critically ill patients (p<0.001) and 51.6 fold as compared to healthy controls (p<0.05). Bid was increased 12.9 fold compared to critically ill (p<0.001). In the group of the mitochondrial apoptosis-inducers, Bak was upregulated 5.6 fold, while the expression of Bax showed no significant variations. By contrast, the pro-survival members Bcl-2 and Bcl-xl were both downregulated in severe sepsis (p<0.001, p<0.05). CONCLUSIONS: In early severe sepsis a gene expression pattern with induction of the pro-apoptotic Bcl-2 family members Bim, Bid and Bak and a downregulation of the anti-apoptotic Bcl-2 and Bcl-xl was observed in peripheral blood. This constellation may affect cellular susceptibility to apoptosis and complex immune dysfunction in sepsis.

"Exterritorial" lymphocytic hypophysitis within an ovarian teratoma during pregnancy: a possible sentinel lesion?

Pituitary tissue is rarely to be found among the constituents of ovarian teratomas (dermoid cysts). In some exceptional cases, however, such ectopic pituitary anlagen may even give rise to secondary organ-specific pathologies. Akin to those of the pituitary in its natural location, these tend to be adenomas. We describe a unique example of lymphocytic hypophysitis incidentally encountered in a mature left ovarian teratoma from a 30-year-old woman in the 19th week of pregnancy. Amidst various fully differentiated derivatives of all three embryonic layers, the cyst wall also included a miniature replica of the anterior pituitary lobe 0.5 cm in diameter. While a full set of adenohypophyseal hormone-producing cell types could be identified, there was characteristic pregnancy-related hyperplasia of lactotrophs. This was further overlaid by prominent mononuclear inflammation, including infiltration by T lymphocytes, follicular aggregates of B cells, and attendant destruction of parenchyma. There was no significant inflammatory reaction elsewhere. Discounting the non-standard location, the ensemble of the clinical setting and histology were felt to be indistinguishable from the classical paradigm of lymphocytic hypophysitis complicating pregnancy. To date, lymphocytic thyroiditis is the sole form of organ-specific inflammatory process within an ovarian teratoma on record. By analogy, we hypothesize that this ectopic manifestation of immune-mediated inflammation of pituitary parenchyma may possibly be read as a preclinical sentinel lesion of lymphocytic hypophysitis.

Reactivation of rheumatoid arthritis after pregnancy: increased phagocyte and recurring lymphocyte gene activity

OBJECTIVE: Pregnancy is associated with reduced disease activity in rheumatoid arthritis (RA) and frequently with disease exacerbation after delivery. This study was undertaken to generate a systematic overview of the molecular mechanisms related to disease remission and postpartum reactivation. METHODS: Transcriptomes of peripheral blood mononuclear cells (PBMCs) were generated from RA patients and healthy women by transcription profiling during the third trimester and 24 weeks after delivery. For functional interpretation, signatures of highly purified immune cells as well as Kyoto Encyclopedia of Genes and Genomes pathway annotations were used as a reference. RESULTS: Only minor differences in gene expression in PBMCs during pregnancy were found between RA patients and controls. In contrast, RA postpartum profiles presented the most dominant changes. Systematic comparison with expression signatures of monocytes, T cells, and B cells in healthy donors revealed reduced lymphocyte and elevated monocyte gene activity during pregnancy in patients with RA and in controls. Monocyte activity decreased after delivery in controls but persisted in RA patients. Furthermore, analysis of 32 immunologically relevant cellular pathways demonstrated a significant additional activation of genes related to adhesion, migration, defense of pathogens, and cell activation, including Notch, phosphatidylinositol, mTOR, Wnt, and MAPK signaling, in RA patients postpartum. CONCLUSION: Our findings indicate that innate immune functions play an important role in postpartum reactivation of arthritis. However, this may depend not only on the monocyte itself, but also on the recurrence of lymphocyte functions postpartum and thus on a critical interaction between both arms of the immune system.

Anti-CD20 (rituximab) treatment improves atopic eczema

BACKGROUND: Atopic eczema (AE) is a chronic inflammatory skin disorder characterized by eczematous skin lesions, pruritus, and typical histopathologic features. OBJECTIVE: We asked whether depletion of B cells by monoclonal anti-CD20 antibody therapy (rituximab) would improve severe AE. METHODS: Six patients (4 women and 2 men) with severe AE received 2 intravenous applications of rituximab, each 1000 mg, 2 weeks apart. To evaluate the efficacy of rituximab, we monitored clinical parameters (eczema area and severity index, pruritus), total and allergen-specific IgE levels, skin histology, and inflammatory cells and cytokine expression in the skin and peripheral blood before and after therapy. RESULTS: All patients showed an improvement of their skin symptoms within 4 to 8 weeks. The eczema area and severity index significantly decreased (before therapy, 29.4 +/- 4.3; week 8, 8.4 +/- 3.6; P < .001). Histologic alterations such as spongiosis, acanthosis, and dermal infiltrate, including T and B cell numbers, also dramatically improved. However, whereas blood B cells were below detectable levels as a consequence of rituximab administration, skin B cells were reduced by approximately 50% only. Expression of IL-5 and IL-13 was reduced after therapy. Moreover, whereas allergen-specific IgE levels were not altered, we observed a slight reduction in total IgE concentrations in blood. CONCLUSIONS: B cells play a major role in AE pathogenesis. Treatment with an anti-CD20 antibody leads to an impressive improvement of AE in patients with severe disease.

Toll-like receptor 2 is highly expressed in lesions of acne inversa and colocalizes with C-type lectin receptor

BACKGROUND: Acne inversa (hidradenitis suppurativa) is a chronic inflammatory and cicatricial disorder that affects skin areas rich in apocrine glands and terminal hairs, such as perineum and axillae. The exact pathogenesis of the disease is not well understood and the mechanisms by which bacterial superinfection contributes to the disease progression are not clear. Toll-like receptors (TLRs) expressed by inflammatory cells play a crucial role in the innate immune response to bacteria. OBJECTIVES: We sought to investigate the role of TLR2 in the pathogenesis of acne inversa. METHODS: We investigated the expression of TLR2 using real-time polymerase chain reaction analysis and immunohistochemical stainings of tissue samples from patients with acne inversa. Furthermore, we phenotypically characterized the infiltrating cells and their expression of TLR2. RESULTS: Compared with normal skin, a highly increased in situ expression of TLR2 in acne inversa skin lesions was found at both the mRNA and the protein level. The most abundant cells in the dermal infiltrate of acne inversa were CD68+ macrophages, CD209+ dendritic cells (DCs) and CD3+ T cells. CD19+ B cells and CD56+ natural killer cells were found only in small numbers. Double staining with fluorescence-labelled antibodies showed that TLR2 was expressed by infiltrating macrophages (CD68+) and DCs (CD209+). Flow cytometric analysis of isolated infiltrating cells further confirmed surface expression of TLR2 by macrophages and DCs. CONCLUSIONS: These data indicate that the enhanced expression of TLR2 by infiltrating macrophages and DCs may contribute to the pathogenesis of inflammatory lesions of acne inversa.

The immune geography of IgA induction and function

The production of immunoglobulin A (IgA) in mammals exceeds all other isotypes, and it is mostly exported across mucous membranes. The discovery of IgA and the realization that it dominates humoral mucosal immunity, in contrast to the IgG dominance of the systemic immune system, was early evidence for the distinct nature of mucosal immunology. It is now clear that IgA can function in high-affinity modes for neutralization of toxins and pathogenic microbes, and as a low-affinity system to contain the dense commensal microbiota within the intestinal lumen. The basic map of induction of IgA B cells in the Peyer's patches, which then circulate through the lymph and bloodstream to seed the mucosa with precursors of plasma cells that produce dimeric IgA for export through the intestinal epithelium, has been known for more than 30 years. In this review, we discuss the mechanisms underlying selective IgA induction of mucosal B cells for IgA production and the immune geography of their homing characteristics. We also review the functionality of secretory IgA directed against both commensal organisms and pathogens.

6th International Immunoglobulin Symposium: poster presentations

The posters presented at the 6th International Immunoglobulin Symposium covered a wide range of fields and included both basic science and clinical research. From the abstracts accepted for poster presentation, 12 abstracts were selected for oral presentations in three parallel sessions on immunodeficiencies, autoimmunity and basic research. The immunodeficiency presentations dealt with novel, rare class-switch recombination (CSR) deficiencies, attenuation of adverse events following IVIg treatment, association of immunoglobulin (Ig)G trough levels and protection against acute infection in patients with X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVID), and the reduction of class-switched memory B cells in patients with specific antibody deficiency (SAD). The impact of intravenous immunoglobulin on fetal alloimmune thrombocytopenia, pregnancy and postpartum-related relapses in multiple sclerosis and refractory myositis, as well as experiences with subcutaneous immunoglobulin in patients with multi-focal motor neuropathy, were the topics presented in the autoimmunity session. The interaction of dendritic cell (DC)-SIGN and alpha2,6-sialylated IgG Fc and its impact on human DCs, the enrichment of sialylated IgG in plasma-derived IgG, as wells as prion surveillance and monitoring of anti-measles titres in immunoglobulin products, were covered in the basic science session. In summary, the presentations illustrated the breadth of immunoglobulin therapy usage and highlighted the progress that is being made in diverse areas of basic and clinical research, extending our understanding of the mechanisms of immunoglobulin action and contributing to improved patient care.

CC chemokines and the receptors CCR3 and CCR5 are differentially expressed in the nonneoplastic leukocytic infiltrates of Hodgkin disease

Lymph nodes with Hodgkin disease (HD) harbor few neoplastic cells in a marked leukocytic infiltrate. Since chemokines are likely to be involved in the recruitment of these leukocytes, the expression of potentially relevant chemokines and chemokine receptors were studied in lymph nodes from 24 patients with HD and in 5 control lymph nodes. The expression of regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta was analyzed by in situ hybridization and that of CCR3 and CCR5 by immunohistochemistry and flow cytometry. It was found that, overall, the expression of all 4 chemokines was markedly enhanced, but the cellular source was different. RANTES was expressed almost exclusively by T cells whereas the expression of MCP-1, MIP-1alpha, and MIP-1beta was confined largely to macrophages. In control lymph nodes, chemokine expression was low, with the exception of MIP-1alpha in macrophages. CCR3 and CCR5 were highly expressed in T cells of HD involved but not of control lymph nodes. CCR3 was equally distributed in CD4+ and CD8+ cells, but CCR5 was associated largely with CD4+ cells. In HD lymph nodes, CCR3 and CCR5 were also expressed in B cells, which normally do not express these receptors. All these chemokines and receptors studied, by contrast, were absent in the neoplastic cells. It was concluded that chemokines are involved in the formation of the HD nonneoplastic leukocytic infiltrate. Expression of CCR3 and CCR5 appears to be characteristic of HD, but the roles of these receptors' up-regulation for the disease process remain unclear.

Maternal transfer of IgE and subsequent development of IgE responses in the horse (Equus callabus)

Immunoglobulin E (IgE) mediates the immune response to parasites, but can also cause allergies. In humans maternal IgE is not transferred to cord blood and high levels of cord blood IgE are associated with subsequent allergy. In horses, both maternal IgG and IgE are transferred via colostrum; the IgE levels in the mare's serum, the colostrum and the foal's serum are correlated but the consequences of IgE transfer to foals are not known. By about 6 weeks of age the levels of IgE in foal serum have dropped to a nadir, at 6 months of age the level of IgE has risen only very slightly and is no longer correlated with the levels seen at birth, IgE(+) B-cells could be detected in lymphoid follicles of some foals at this age. Surprisingly, the levels of total IgE detected in a foals serum at 6 months of age are significantly correlated with the level in its serum at 1, 2 and even 3 years of age suggesting that by 6 months of age the foals are synthesizing IgE and that a pattern of relatively higher or lower total serum IgE has been established. The neonatal intestinal mucosa contained connective tissue mast cells which stained for bound IgE in foals up to 9 weeks of age but not mucosal mast cells, thereafter, the intestinal mast cells were IgE negative until 6 months of age. IgE antibodies to Culicoides nubeculosus salivary antigens were detected in Swiss born foals from imported Icelandic mares allergic to Culicoides spp. yet the foals showed no signs of skin sensitization and such second generation foals are known not to have an increased risk of developing allergy to Culicoides. Overall this evidence suggests there is a minimal effector role of maternal IgE also that maternal IgE has waned prior to the onset of IgE synthesis in foals and does not support maternal priming of IgE responses in foals. Furthermore the total levels of IgE in any given foal are seen to be relatively high or low from soon after the onset of IgE synthesis, and most likely they are determined by genetic factors.

Clinical and pathological analysis of epidural inflammation in intervertebral disk extrusion in dogs

BACKGROUND Little is known about the pathologic changes in the epidural space after intervertebral disk (IVD) extrusion in the dog. OBJECTIVES To analyze the pathology of the epidural inflammatory response, and to search for correlations between this process and clinical findings. METHODS Clinical data from 105 chondrodystrophic (CD) and nonchondrodystrophic (NCD) dogs with IVD extrusion were recorded. Epidural material from these dogs was examined histopathologically and immunohistochemically. Using statistical analysis, we searched for correlations between severity of epidural inflammation and various clinical and pathologic variables. RESULTS Most dogs exhibited an epidural inflammatory response, ranging from acute invasion of neutrophils to formation of chronic granulation tissue. The mononuclear inflammatory infiltrates consisted mostly of monocytes and macrophages and only few T and B cells. Surprisingly, chronic inflammatory patterns also were found in animals with an acute clinical history. Severity of the epidural inflammation correlated with degree of the epidural hemorrhage and nucleus pulposus calcification (P = .003 and .040), but not with age, chondrodystrophic phenotype, neurologic grade, back pain, pretreatment, or duration. The degree of inflammation was statistically (P = .021) inversely correlated with the ability to regain ambulation. CONCLUSION AND CLINICAL IMPORTANCE Epidural inflammation occurs in the majority of dogs with IVD extrusion and may develop long before the onset of clinical signs. Presence of calcified IVD material and hemorrhage in the epidural space may be the triggers of this lesion rather than an adaptive immune response to the nucleus pulposus as suggested in previous studies. Because epidural inflammation may affect outcome, further research is warranted.

Evaluation of histological, serological and clinical changes in response to abatacept treatment of primary Sjögren's syndrome: A pilot study

Objectives To prospectively evaluate histopathologic, blood cellular, serological and clinical changes in response to abatacept treatment in patients with primary Sjögren's syndrome (pSS). Methods Blood, saliva and minor salivary gland biopsies were obtained before and after the last of 8 doses of abatacept in 11 pSS patients. The histologic data evaluated the number of lymphocytic foci and of B- and T-cell subtypes (CD20(+) , CD3(+) , CD4(+) , CD8(+) ). The numbers of FoxP3(+) regulatory T-cells were measured and the FoxP3 /CD 3 ratio was calculated. Histologic data were compared with results from peripheral blood and with changes in saliva secretion. Results The numbers of lymphocytic foci decreased significantly (p=0.041). Numbers of local FoxP3(+) T-cells decreased significantly in percentage of total lymphocytic infiltrates (p=0.037). In the peripheral blood B-cells increased (p=0.038). This was due to an expansion of the naïve B cell pool (p=0.034). When adjusting for disease duration, an increase was also noted for total lymphocytes (p=0.044) and for CD 4 cells (p=0.009). Gamma globulins decreased significantly (p=0.005), but IgG reduction did not reach significance. Adjusted for disease duration, saliva production increased significantly (p=0.029). Conclusions CTLA4-Ig treatment significantly reduces glandular inflammation in pSS, induces several celluar changes and increases saliva production. Remarkably, this increase in saliva production is significantly influenced by disease duration.

Transport and uptake of immunogenic lipids

An increasing number of lipid mediators have been identified as key modulators of immunity. Among these is a family of glycolipids capable of cellular uptake, loading onto the MHC-like molecule CD1d and stimulation of NKT cells. NKT cells are particularly interesting because they bridge innate and adaptive immunity by coordinating the early events of dendritic cell maturation, recruitment of NK cells, CD4 and CD8 T cells, and B cells at the site of microbial injury. As such, their therapeutic manipulation could be of the greatest interest in vaccine design or active immunotherapy. However, the use of NKT cells as cellular adjuvant of immunity in the clinic will require a better knowledge of the pharmacology of lipid agonists in order to optimize their action and avoid potential unseen off-target effects. We have been studying extracellular transport and cellular uptake of NKT agonists for the past few years. This field is confronted to a very limited prior knowledge and a small set of usable tools. New technology must be put in place and adapted to answering basic immunology questions related to NKT cells. The intimate link between the pharmacology of glycolipids and lipid metabolism makes us believe that great variations of bioactivity could be seen in the general population when NKT agonists are used therapeutically.

Pathophysiology of Hidradenitis suppurativa

Only limited data are available about the precise mechanism leading to tissue inflammation and damage in patients with hidradenits suppurativa (HS). The central pathogenetic event in HS is the occlusion of the upper parts of the hair follicle leading to a perifollicular lympho-histiocytic inflammation. In early lesions, neutrophilic abscess formation and influx of mainly macrophages, monocytes and dendritic cells predominate. In chronic disease, the infiltrate expand with increased frequencies of B cells and plasma cells. In the inflammatory infiltrates toll like receptor 2 (TLR2) was highly expressed by infiltrating macrophages and dendritic cells indicating that stimulation of inflammatory cells by TLR2 activating microbial products may be important trigger factors in the chronic inflammatory process. Furthermore, the pro inflammatory cytokines IL-12 and IL-23 are abundantly expressed by macrophages infiltrating papillary and reticular dermis of HS skin. Both of these cytokines are believed to be important mediators in autoimmune tissue destruction and its blocking by biologics has been shown to be effective in the treatment of psoriasis. Especially IL-23 has been shown to be involved in the induction of a T helper cell subset producing IL-17, therefore, named Th17, which is distinct from the classical Th1/Th2 subsets. In chronic HS lesions IL-17-producing T helper cells were found to infiltrate the dermis. An overexpression of various other cytokines like IL-1beta, CYCL9 (MIG), IL-10 , IL-11 and BLC has been described in HS lesion whereas IL-20 and IL-22 have been shown to be down regulated. Similar to psoriasis also in HS the antimicrobial peptides beta defensin 2 and psoriasin are highly upregulated. This may at least in part explain the clinical finding that HS patients suffer only rarely from skin infections. Taken together the inflammatory reaction leading to HS are only poorly understood, but they show many similarity with other inflammatory reactions as e.g. in psoriasis.

Intestinal Microbial Diversity during Early-Life Colonization Shapes Long-Term IgE Levels

Microbial exposure following birth profoundly impacts mammalian immune system development. Microbiota alterations are associated with increased incidence of allergic and autoimmune disorders with elevated serum IgE as a hallmark. The previously reported abnormally high serum IgE levels in germ-free mice suggests that immunoregulatory signals from microbiota are required to control basal IgE levels. We report that germ-free mice and those with low-diversity microbiota develop elevated serum IgE levels in early life. B cells in neonatal germ-free mice undergo isotype switching to IgE at mucosal sites in a CD4 T-cell- and IL-4-dependent manner. A critical level of microbial diversity following birth is required in order to inhibit IgE induction. Elevated IgE levels in germ-free mice lead to increased mast-cell-surface-bound IgE and exaggerated oral-induced systemic anaphylaxis. Thus, appropriate intestinal microbial stimuli during early life are critical for inducing an immunoregulatory network that protects from induction of IgE at mucosal sites.