Search

The expression of Bruton tyrosine kinase (BTK) in B cells and innate immune cells provides essential downstream signaling for BCR, Fc receptors, and other innate immune cell pathways. The topical covalent BTK inhibitor PRN473 has shown durable, reversible BTK occupancy with rapid on-rate and slow off-rate binding kinetics and long residence time, resulting in prolonged, localized efficacy with low systemic exposure in vivo. Mechanisms of PRN473 include inhibition of IgE (FcεR)–mediated activation of mast cells and basophils, IgG (FcγR)–mediated activation of monocytes, and neutrophil migration. In vivo, oral PRN473 was efficacious and well tolerated in the treatment of canine pemphigus foliaceus. In this study, we evaluated in vitro selectivity and functionality, in vivo skin Ab inflammatory responses, and systemic pharmacology with topically administered PRN473. Significant dose-dependent inhibition of IgG-mediated passive Arthus reaction in rats was observed with topical PRN473 and was maintained when given 16 h prior to challenge, reinforcing extended activity with once-daily administration. Similarly, topical PRN473 resulted in significant dose-dependent inhibition of the mouse passive cutaneous anaphylaxis IgE-mediated reaction. Multiday treatment with topical PRN473 in rodents resulted in low-to-no systemic accumulation, suggesting that efficacy was mainly due to localized exposure. Reduced skin Ab inflammatory activity was also confirmed with oral PRN473. These preclinical studies provide a strong biologic basis for targeting innate immune cell responses locally in the skin, with rapid onset of action following once-daily topical PRN473 administration and minimal systemic exposure. Dose-dependent inhibition in these preclinical models of immune-mediated skin diseases support future clinical studies.

Zizyphus mauritiana Lam. seeds (ZMS) have been used medicinally as sedative or hypnotic drugs in most of Asian countries. ZMS has significant benefits to the human health. Therefore, we have evaluated immunomodulatory effect of lectin extracted from these ZMSL in both in vitro and in vivo study. Anaphylaxis is a severe life-threatening allergic reaction and Arthus reaction is deposition of immune complex and complement system activation, so we hypothesized that if ZMSL can protect these severe allergic diseases. We have studied the effect of ZMSL on macrophages and Wistar albino rats and confirmed its protective effect against anaphylaxis and Arthus reaction. Results of this study suggest ZMSL have immunostimulatory and antiallergic activity.

Although neutrophils play important roles in immunity and inflammation, their analysis is strongly hindered by their short-lived and terminally differentiated nature. Prior studies reported conditional immortalization of myeloid progenitors using retroviral expression of an estrogen-dependent fusion protein of the HoxB8 transcription factor. This approach allowed the long-term culture of mouse myeloid progenitors (HoxB8 progenitors) in estrogen-containing media, followed by differentiation toward neutrophils upon estrogen withdrawal. Although several reports confirmed the in vitro functional responsiveness of the resulting differentiated cells (HoxB8 neutrophils), little is known about their capacity to perform in vivo neutrophil functions. We have addressed this issue by an in vivo transplantation approach. In vitro–generated HoxB8 neutrophils showed a neutrophil-like phenotype and were able to perform conventional neutrophil functions, like respiratory burst, chemotaxis, and phagocytosis. The i.v. injection of HoxB8 progenitors into lethally irradiated recipients resulted in the appearance of circulating donor-derived HoxB8 neutrophils. In vivo–differentiated HoxB8 neutrophils were able to migrate to the inflamed peritoneum and to phagocytose heat-killed Candida particles. The reverse passive Arthus reaction could be induced in HoxB8 chimeras but not in irradiated, nontransplanted control animals. Repeated injection of HoxB8 progenitors also allowed us to maintain stable circulating HoxB8 neutrophil counts for several days. Injection of arthritogenic K/B×N serum triggered robust arthritis in HoxB8 chimeras, but not in irradiated, nontransplanted control mice. Taken together, our results indicate that HoxB8 progenitor–derived neutrophils are capable of performing various in vivo neutrophil functions, providing a framework for using the HoxB8 system for the in vivo analysis of neutrophil function.

Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune thrombocytopenia. Additionally, rilzabrutinib inhibited IgE-mediated, FcεR-dependent immune mechanisms in human basophils and mast cell–dependent mouse models. In canines with naturally occurring pemphigus, rilzabrutinib treatment resulted in rapid clinical improvement demonstrated by anti-inflammatory effects visible within 2 wk and all animals proceeding to complete or substantial disease control. Rilzabrutinib is characterized by reversible covalent BTK binding, long BTK residence time with low systemic exposure, and multiple mechanistic and biological effects on immune cells. Rilzabrutinib’s unique characteristics and promising efficacy and safety profile support clinical development of rilzabrutinib for a broad array of immune-mediated diseases.

Store-operated calcium entry (SOCE) is the most common mode of calcium influx in non-excitable cells, including immune cells. The two STIM isoforms mediate SOCE as well as Fc receptor (FcR)-downstream activation of macrophages and mast cells-which appears to be relevant in vivo, in models of antibody-dependent tissue injury and allergy. Hence, the pathway of SOCE may be a therapeutic target for treatment of immune complex (IC)-mediated autoimmunity and allergic asthma. The pyrazole derivative, BTP2 is an efficient inhibitor of SOCE, which has already been shown to attenuate allergic inflammation. However, its effect on Fc gamma receptor (FcγR) signaling and IC-induced tissue injury had not yet been studied. Here, we show that BTP2 is a potent inhibitor of SOCE in primary macrophages, blocking FcγR-mediated responses. To investigate the effect of inhibition of SOCE in IC-mediated tissue injury, we induced reverse passive Arthus reaction to IgG immune complexes in the skin and lungs of BTP2- or control-treated mice. Treatment with BTP2 resulted in markedly attenuated inflammation in both the skin and the lungs. Our findings indicate the involvement of SOCE in FcγR-mediated responses in vitro and in vivo and suggest that BTP2-mediated inhibition of SOCE may have a therapeutic potential on IC-mediated autoimmunity.

The present study reports the evaluation of immunomodulatory and therapeutic potential of a purified Aspergillus panamensis lectin. The immunomodulatory potential of the purified lectin was determined in swiss albino mice by studying its effect on anaphylaxis reaction, arthus reaction, respiratory burst activity, nitric oxide production and quantification of cytokine levels. The therapeutic potential of the lectin was evaluated in male wistar rat models by studying its curative effect on ulcerative colitis. The purified lectin inhibited systemic anaphylaxis and arthus reaction. It enhanced the functional ability of macrophages which was evident from increase in reduction of nitroblue tetrazolium dye and nitric oxide production. It also stimulated the production of Th-1 cytokine IFN-γ and Th-2 cytokine IL-6. Maximum immunomodulatory effect was seen at lectin concentration of 1.5 mg/kg body weight. The lectin also showed curative effect against trinitrobenzene sulphonic acid induced ulcerative colitis. The results of this study adequately reflect the role of purified A. panamensis lectin in improving the immune status of mice models. They also show the effect of lectin in reducing the severity of incidence and decrease in clinical symptoms of ulcerative colitis.

Background:IgG4-related disease (IgG4-RD) is an immune-mediated disorder causing fibro-inflammatory lesions. Although the cause remains unknown, it may be driven by interactions between B lymphocytes and CD4+ cytotoxic and regulatory T cells and is characterized by an increase in short-lived plasmablasts, circulating antibodies, and macrophages. Standard therapy mainly includes glucocorticoids (GC), limited by toxicity with long-term use (> 6 mo), and to a lesser extent, immunosuppressives (eg, rituximab). Bruton tyrosine kinase (BTK) plays an important role in the activation of multiple immune effector cells such as B cells, mast cells, eosinophils, basophils, monocytes/macrophages, and neutrophils. Dysregulation of the activation of these immune cells results in autoimmune inflammation, tissue damage, and development of fibrosis. Rilzabrutinib is a highly selective oral BTK inhibitor that targets multiple pathways of innate and adaptive immunity (with direct effects on B-cell and FcR pathways) and has the potential to inhibit antigen presentation to autoreactive T cells.Objectives:To provide the biological rationale for rilzabrutinib in IgG4-RD.Methods:Rilzabrutinib has been evaluated in biochemical, in vitro studies, and in vivo models of inflammatory diseases. Additional support is provided by the phase 2 trial for oral rilzabrutinib in patients with pemphigus vulgaris and the phase 2 trial for oral rilzabrutinib in patients with immune thrombocytopenia (ITP).Results:Rilzabrutinib inhibited the activity of BTK and B-cell receptor in B cells (IC50 5-123 nM) and Fc gamma receptor in IgG/Fc gamma receptor-stimulated monocytes (IC50 56 nM) and blocked IgG- and IgM-mediated antibody production in enriched B cells when stimulated in T-cell dependent (anti-CD40+IL-21) and T-cell independent (TLR-9/CpG and TNP-LPS) pathways. The impact of rilzabrutinib on innate cell pathways was further confirmed by significant dose-dependent inhibition of macrophage and neutrophil-driven passive rat Arthus reaction (P < 0.01 vs vehicle) and antibody-induced murine ITP (P < 0.05 vs vehicle). In a 12-week phase 2 pemphigus vulgaris trial, 54% of patients achieved the primary endpoint, control of disease activity (CDA) on low-dose corticosteroids by week 4, and 73% achieved it by week 12. In the phase 2 trial of ITP patients (median 6 prior therapies), rilzabrutinib 400 mg bid showed rapid and sustained improvement in platelet counts and only grade 1/2-related adverse events1. In responders, platelet counts increased as early as day 8, potentially due to innate immune mechanisms. Collectively, results in both B and innate immune cells provide an initial basis for evaluating rilzabrutinib in IgG4-RD. The ongoing phase 2a study (NCT04520451) is investigating rilzabrutinib 400 mg bid (+tapered GC) vs GC control (3:1) for 12 weeks in IgG4-RD patients refractory to rituximab. The primary objective is to evaluate the safety and ability of rilzabrutinib to induce GC-free remission at week 12. Coupled with known preclinical/clinical findings, mechanistic analyses in this ongoing IgG4-RD study will profile B and other immune cell effects pre-/post-rilzabrutinib dosing to enhance the clinical understanding of rilzabrutinib in IgG4-RD.Conclusion:Studies of rilzabrutinib that show beneficial effects on both B-cell and innate cell pathways provide support for its therapeutic role in immune-mediated diseases and for targeting the underlying pathophysiological effects of IgG4-RD. Effective and safe therapies that rapidly induce and maintain clinical responses, while minimizing the need for continuous GC treatment, remain an unmet need for patients with IgG4-RD.References:[1]Kuter et al. Res Pract Thromb Haemost. 2020;4(suppl 1): PB1318.Disclosure of Interests:Li Long Employee of: Principia Biopharma, a Sanofi Company, Matthew Baker: None declared, Mollie Carruthers: None declared, Alireza Meysami: None declared, Robert Spiera Consultant of: research funding and personal fees for consulting from Chemocentryx, Formation Biologics, Roche-Genentech, and Sanofi, Grant/research support from: research funding fees from BMS, Boehringer Ingelheim, Corbus, GSK, and Inflarx; personal fees from AbbVie, CSL Behring, GSK, and Janssen, Mamatha Reddy Employee of: Principia Biopharma, a Sanofi Company, Marianne Kavanagh Employee of: Principia Biopharma, a Sanofi Company, Michelle Francesco Employee of: Principia Biopharma, a Sanofi Company, Claire Langrish Employee of: Principia Biopharma, a Sanofi Company, Ann Neale Employee of: Principia Biopharma, a Sanofi Company, Puneet Arora Employee of: Principia Biopharma, a Sanofi Company, John H. Stone Consultant of: research funding and personal fees for consulting from Principia and Sanofi

Background: Beta-sitosterol (BS) is a compound discovered to be present in numerous plants. A number of interesting biomedical properties have been attributed to BS, including immuno-modulating and anti-inflammatory activities. Therefore, the aim of this report was to evaluate its anti-inflammatory capacity by applying various rodent experimental tests.Methods. To carry out the objective of the study we applied the methods indicated here. Two of the adopted methods were based on the passive reverse Arthus reaction: the rat paw edema test and the rat pleurisy assay. We also applied two methods related with the non-specific acute inflammation: the mouse ear edema test, and the mouse mieloperoxidase activity assay.Results. The results obtained in all tests established a significant anti-inflammatory potential of BS. In the rat paw edema test we found an inhibitory effect which goes from 50-70%; in the rat pleurisy assay our findings with respect to the volume of pleural exuded showed a reduction of 46%, as well as a 20% low amount of neutrophils in comparison with the level of the control group. In the mouse ear edema test we found a mean inflammatory inhibition of 75%, and with respect to mieloproxidase activity the results showed a significant inhibition induced by the three doses of BS.Conclusions. In the present study we determined a potent anti-inflammatory capacity of BS in specific and nonspecific types of acute inflammation in rodents.Keywords: Beta-sitosterol, anti-inflammatory assays, mouse, rat

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.

Background Local type III hypersensitivity reactions are acute inflammatory events induced by immune complex (IC) deposition. CD22 and CD72 are B cell-specific cell surface molecules that negatively regulate B cell function. Objective To elucidate the roles of CD22 and CD72 in the development of IgG-mediated type III hypersensitivity reactions. Method The reverse Arthus reaction model in the skin was induced in mice lacking CD22 (CD22 -/- ), CD72 (CD72 -/- ), and both of them (CD22 -/- /CD72 -/- ). Edema at 4 hours and hemorrhage at 8 hours after IC challenge were evaluated. Inflammatory cell infiltration and cytokine and chemokine expression were also examined. Results Edema and hemorrhage were significantly reduced in CD22 -/- /CD72 -/- mice compared with wild-type mice. The loss of both membrane proteins resulted in a greater decrease in edema at 4 hours, but not hemorrhage at 8 hours, than the loss of each protein alone. Infiltration of neutrophils, macrophages, and T cells, and the expression of TNF-α, IL-6, MIP-1α, and CCR5 mRNA were also diminished in the knockout mice compared to wild-type mice, and most significantly reduced in CD22 -/- /CD72 -/- mice. Regulatory T (Treg) cells in the spleen were significantly increased in all knockout mice at 4 hours. Significant differences in the severity of edema and hemorrhage between wild-type and knockout mice were lost when Treg cells were depleted in the knockout mice. Conclusion These results demonstrate that CD22 and CD72 expression contribute to the development of the reverse Arthus reaction model and CD22 and CD72 might be therapeutic targets for human IC-mediated diseases.

Aryl hydrocarbon receptor (AhR), which was discovered as a receptor for environmental concomitants, plays an important role widely in the immune system. In this study, we assessed AhR involvement in immune-complex-mediated vascular injury by examining the reverse-passive Arthus reaction in AhR heterozygous knockout (AhR+/−) mice. In the cutaneous Arthus reaction, dermal edema was severer in AhR+/− mice than in wild-type mice. The number of infiltrating neutrophils and mRNA expression levels of CXC chemokine ligand 1 and IL-6 were also increased in AhR+/− mice. Similarly, in the peritoneal Arthus reaction, infiltration of neutrophils was increased in AhR+/− mice. Peritoneal macrophages from AhR+/− mice expressed higher levels of Fcγ receptor III and produced higher levels of CXC chemokine ligand 1 and IL-6 after immune complex treatment. In addition, AhR occupied the promoter regions of Fcγ receptor III gene in peritoneal macrophages in a ligand-dependent manner. Depletion of macrophages reduced the cutaneous Arthus reaction in AhR+/− mice, and adoptive transfer of AhR+/− mice macrophages into wild-type mice exacerbated the peritoneal Arthus reaction. Furthermore, AhR expression was decreased and Fcγ receptor III expression was increased in CD14+ monocytes in peripheral blood from patients with immune-complex-mediated vasculitis compared with those from healthy controls. These results suggest that downregulation of AhR is associated with the exacerbation of immune-complex-mediated vascular injury.

Immune complex (IC) deposition causes significant tissue injury associated with various autoimmune diseases such as vasculitis. In the cascade of inflammation, cell-to-cell and cell-to-matrix adhesion via adhesion molecules are essential. To assess the role of αE and β7 integrin in IC-mediated tissue injury, peritoneal and cutaneous reverse-passive Arthus reaction was examined in mice lacking αE integrin (αE−/−) or β7 integrin (β7−/−). Both αE−/− and β7−/− mice exhibited significantly attenuated neutrophil infiltration in the peritoneal and cutaneous Arthus reaction. β7 integrin deficiency, not αE integrin deficiency, significantly reduced the number of mast cells in the peritoneal cavity, which was consistent with the result that mast cells expressed only α4β7 integrin, not αEβ7 integrin. αE−/− mice instead revealed the reduction of CD8+ T cells in the peritoneal cavity, and nearly half of them in wild-type mice expressed αE integrin. These αE+CD8+ T cells produced more proinflammatory cytokines than αE−CD8+ T cells, and adoptive transfer of αE+CD8+ T cell into αE−/− recipients restored cutaneous and peritoneal Arthus reaction. These results suggest that in the peritoneal and cutaneous reverse-passive Arthus reaction, α4β7 integrin is involved in the migration of mast cells for initial IC recognition. αEβ7 integrin, in contrast, contributes by recruiting αE+CD8+ T cells, which produce more proinflammatory cytokines than αE−CD8+ T cells and amplify IC-mediated inflammation.

We aimed to study the inhibitory effects of topical cyclosporine A (CsA) 0.05 % on immune-mediated corneal neovascularization, and to compare its efficacy with those of dexamethasone 0.1 % and bevacizumab 0.5 %. Immune-mediated corneal neovascularization was created in 36 right eyes of 36 rabbits. The rabbits were then randomized into four groups. Group I received CsA 0.05 %, Group II received dexamethasone 0.1 %, Group III received bevacizumab 0.5 %, and Group IV received isotonic saline twice a day for 14 days. The corneal surface covered with neovascular vessels was measured on the photographs. The rabbits were then sacrificed and the corneas excised. Paraffin-embedded sections were stained with hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The means of percent area of corneal neovascularization in Group I, II, III, and IV were 24.4 %, 5.9 %, 37.1 %, and 44.1 %, respectively. The inhibitory effect of CsA 0.05 % was found to be better than the effect found in the bevacizumab 0.5 % and control groups (p = 0.03 and p = 0.02, respectively). CsA 0.05 % was found to have significantly lesser inhibitory effects on corneal neovascularization than dexamethasone 0.1 % (p

H2S donor attenuates inflammatory responses through the L-selectin involved pathway, in the cutaneous reverse passive Arthus reaction. H2S has been highlighted recently as an endogenous, gaseous signaling molecule, especially in inflammations. The deposition of IC induces an acute inflammatory response with tissue injury. To assess the roles of H2S in the IC-induced diseases, the cutaneous, reverse passive Arthus reaction was conducted using NaHS as a H2S donor. Furthermore, we conducted similar experiments using selectin−/− mice to determine the involvement of selectin molecules in the H2S-mediated pathway. Exogenous application of NaHS dramatically attenuated inflammatory reactions in WT mice associated with Arthus reaction. Namely, mRNA expressions of TNF-α, IFN-γ, and neutrophil numbers were reduced significantly in the lesional skins of NaHS-treated WT mice relative to untreated ones. NaHS treatment significantly reduced these three parameters in the lesional skins of E- and P-selectin−/− mice but not in those of L-selectin−/− mice. Quite similar results were obtained in the blocking study using WT mice injected with mAb to E-, P-, and L-selectin. Our results indicated that the exogenous application of NaHS attenuates inflammatory responses in reverse passive Arthus reaction through a L-selectin-involved pathway but not through E- or P-selectin pathways.

Lectins are non-immune proteins or glycoproteins with the ability to induce cell agglutination and mediate cellular and molecular recognition processes in a variety of biological interactions. Recently, several mushroom lectins have been reported to show immunomodulatory activities. The immunomodulatory potential of purified A. gorakhpurensis lectin was determined by evaluating its effect on systemic anaphylaxis reaction, arthus reaction, respiratory burst activity, nitric oxide production and levels of cytokines IFN-γ and IL-6. Administration of different doses of purified lectin was found to stimulate immunological activity. Lectin prevented BSA induced Arthus reaction and systemic anaphylaxis. The enhanced functional ability of macrophages was evident from respiratory burst activity and nitric oxide production in splenocyte cultures. Interferon-γ and interleukin-6 levels were significantly up-regulated in lectin treated groups. Maximum modulatory effect was observed at the lectin dose of 3.0 mg/kg body weight. The results of the experiments conducted adequately reflect the immunostimulatory effect of purified A. gorakhpurensis lectin.

IgG antibodies are potent inducers of proinflammatory responses by cross-linking Fc receptors on innate immune effector cells resulting in tissue injury. The recently discovered enzymes endoglycosidase S (EndoS) and IgG-degrading enzyme (IdeS) of Streptococcus pyogenes are able to modulate the interaction between IgG antibodies and the Fc receptors, by hydrolysis of the glycan associated with the heavy chain of the IgG molecule (EndoS), or cleavage in the hinge region of the heavy IgG chain (IdeS). In this work, we investigated their ability to inhibit damage mediated by skin-bound antibodies in vivo in two different experimental models, the Arthus reaction, and epidermolysis bullosa acquisita, an autoimmune blistering skin disease associated with autoantibodies against type VII collagen. We demonstrate that both enzymes efficiently interfere with IgG-mediated proinflammatory processes, offering a great asset to specifically target pathological IgG antibodies in the skin and holding great promise for future applications in human therapy.

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.

Objective: Lectins are complex and heterogenous group of carbohydrate binding proteins, commonly found in all types of organisms with striking biological activities. Many anti-inflammatory synthetic medicines are available, but they show many side effects. In this context, anti-inflammatory compounds free from any adverse effects are the requirement of time. In the present study, the most active lectin isolated from seeds of Zizyphus oenoplia is tested for its anti-allergic and anti-inflammatory activities. Methods: The most active lectin was isolated from seeds of Zizyphus oenoplia. The procedure of isolation involved extraction of lectins from dried seeds in sodium phosphate buffered saline (0.02 M, pH 7) followed by precipitation with ammonium sulphate (20%-60%) and dialysis. Later, affinity chromatography and ion-exchange chromatography were performed for further purification. The purified fraction was estimated for homogeneity and molecular weight determination on 10% SDS-PAGE. The isolated most active lectin was named as ZOSL (Zizyphus oenoplia seed lectin) and was tested for its anti-allergic and anti-inflammatory activity in vivo through Arthus reaction and anaphylactic shock on Wistar albino rats. Results: ZOSL was found to be monomeric with molecular weight 25 kD. It was seen that regular oral administration of ZOSL to Wistar rats can prevent anaphylactic shock as well as Arthus reaction. Conclusion: A novel monomeric lectin (ZOSL), with anti-allergic and anti-inflammatory activity, was isolated from the seeds of Zizyphus oenoplia.

Background The pathogenic role of IgE has been implicated in a variety of allergic and inflammatory diseases. We have previously established an IgE-mediated cutaneous reverse passive Arthus model in which eosinophil infiltration is a prominent feature. This uniquely provides a model of type III hypersensitivity in which Fc classes of Ig that forms immune complex differentially determine the disease manifestation. Objective To investigate the mechanisms of how mast cells and basophils regulate this IgE-mediated Arthus reaction. Methods IgE-mediated cutaneous reverse passive Arthus reaction was induced in wild-type C57BL/6 or WBB6F1-+/+ mice and mast-cell-deficient WBB6F1-W/W v mice by intradermal injection of IgE anti-trinitrophenyl antibodies followed immediately by intravenous administration of trinitrophenyl bovine serum albumin. Basophils were depleted in vivo using anti-CD200R3 monoclonal antibody prior to the IC challenge. Results Hemorrhage and infiltration of eosinophils, neutrophils, and basophils were significantly reduced but were not completely abrogated in WBB6F1-W/W v mice compared with those in wild-type WBB6F1-+/+ mice. Wild-type C57BL/6 mice treated by basophil-depleting mAb also showed significantly decreased hemorrhage and inflammatory cell infiltration, especially that of eosinophils, compared with control mice. Furthermore, basophil depletion in WBB6F1-W/W v mice led to nearly complete inhibition of eosinophil recruitment. By contrast, basophil depletion did not further decrease neutrophil infiltration in WBB6F1-W/W v mice. Conclusion While mast cells play a central role, basophils also have an important function, especially for eosinophil recruitment, in IgE-mediated cutaneous reverse passive Arthus reaction.

Therapeutic use and function of recombinant molecules can be studied by the expression of foreign genes in mice. In this study, we have expressed human Fcγ receptor-Ig fusion molecules (FcγR-Igs) in mice by administering FcγR-Ig plasmid DNAs hydrodynamically and compared their effectiveness with purified molecules in blocking immune-complex (IC)-mediated inflammation in mice. The concentration of hydrodynamically expressed FcγR-Igs (CD16A(F)-Ig, CD32A(R)-Ig and CD32A(H)-Ig) reached a maximum of 130 μg ml(-1) of blood within 24 h after plasmid DNA administration. The in vivo half-life of FcγR-Igs was found to be 9-16 days and western blot analysis showed that the FcγR-Igs were expressed as a homodimer. The hydrodynamically expressed FcγR-Igs blocked 50-80% of IC-mediated inflammation up to 3 days in a reverse passive Arthus reaction model. Comparative analysis with purified molecules showed that hydrodynamically expressed FcγR-Igs are more efficient than purified molecules in blocking IC-mediated inflammation and had a higher half-life. In summary, these results suggest that the administration of a plasmid vector with the FcγR-Ig gene can be used to study the consequences of blocking IC binding to FcγRs during the development of inflammatory diseases. This approach may have potential therapeutic value in treating IC-mediated inflammatory autoimmune diseases such as lupus, arthritis and autoimmune vasculitis.

The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.

Lectins bind to surface receptors on target cells, and activate a cascade of events, eventually leading to altered immune status of host. The immunomodulatory potential of purified lectin from Aspergillus nidulans was evaluated in Swiss albino mice treated intraperitoneally with seven different doses of purified lectin. Lectin prevented BSA-induced Arthus reaction and systemic anaphylaxis. The enhanced functional ability of macrophages was evident from respiratory burst activity and nitric oxide production in splenocyte cultures. Interferon-gamma and interleukin-6 levels were significantly up-regulated in treated groups. Maximum stimulatory effect was observed at the dose of 1.5 mg/kg body weight. Therapeutic potential of A. nidulans lectin was assessed against trinitrobenzene sulfonic acid-induced ulcerative colitis in male Wistar rats. Rats pre-treated with 80 mg/kg body weight of purified lectin intraperitoneally prior to colitis induction showed lesser disease severity and recovery within 7 days, while rats post-treated with the same dose showed recovery in 11 days. The results demonstrate immunomodulatory effects of A. nidulans lectin in Swiss albino mice, resulting in improved immune status of the animals and unfold its curative effect against ulcerative colitis in rat model. This is the first report on immunomodulatory and therapeutic potential of a lectin from microfungi.

Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice, the development of granulocytes is favored at the expense of monocytes. However, Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras, we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils, Btk plays a role in GM-CSF– and Toll-like receptor–induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα, C/EBPβ, and PU.1, depends on Btk. In addition, expression of several granule proteins, including myeloperoxidase, neutrophilic granule protein, gelatinase and neutrophil elastase, is Btk-dependent. In the Arthus reaction, an acute inflammatory response, neutrophil migration into tissues, edema formation, and hemorrhage are significantly reduced in Btk-deficient animals. Together, our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo.

FcγRIV is a relatively new IgG Fc receptor (FcγR) that is reported to contribute to the pathogenesis of autoimmune diseases, although its specific role in relation to FcγRIII, complement and IgG2 subclasses remains uncertain. Here we define FcγRIV on macrophages as a receptor for soluble IgG2a/b complexes but not for cellular bound IgG2a and show that simultaneous activation of FcγRIV and FcγRIII is critical to mediate certain type II/III autoimmune responses. FcγRIII-deficient mice display compensatory enhanced FcγRIV expression, are protected from lung inflammation after deposition of IgG complexes, and show reduced sensitivity to IgG2a/b-mediated hemolytic anemia, indicating that increased FcγRIV alone is not sufficient to trigger these diseases in the absence of FcγRIII. Importantly, however, blockade of FcγRIV is also effective in inhibiting phagocytosis and cytokine production in IgG2b-induced anemia and acute lung injury, processes that display a further dependence on C5a anaphylatoxin receptor. Using gene deletion and functional inhibition studies, we found that FcγRIII and FcγRIV are each essential to trigger an FcRγ-linker for activation of T-cell-dependent signal that drives C5a production in the Arthus reaction. Together, the results demonstrate a combined requirement for FcγRIII and FcγRIV in autoimmune injury, and identify the linker for activation of T cells adaptor as an integral component of linked FcγR and C5a anaphylatoxin receptor activation to generate inflammation.

Antibody-antigen complex mediated inflammation is integral to the pathogenesis of many autoimmune diseases. Mice deficient in the γ-chain of Fc-receptors are protected in IgG-mediated glomerulonephritis and the Arthus reaction and FcR-bearing mast cells and macrophages have been assigned primary roles in these processes. Here we demonstrate that neutrophil selective transgenic expression of the two uniquely human activating FcγRs, FcγRIIA and FcγRIIIB was sufficient to restore susceptibility to progressive anti-glomerular basement membrane (GBM) nephritis and the cutaneous Reverse Passive Arthus (RPA) reaction in γ-chain deficient mice. Both FcγRIIA and FcγRIIIB mediated robust neutrophil accumulation in tissues suggesting direct roles for these human receptors in IC-induced neutrophil recruitment, while FcγRIIA alone mediated organ injury. In an acute model of anti-GBM nephritis, both FcγRIIIB and FcγRIIA promoted initial neutrophil recruitment to glomerular immune-complexes (ICs) accessible to circulating cells, while FcγRIIA further sustained accumulation. In a model of soluble ICs deposited strictly within the post-capillary venules of the cremaster muscle, FcγRIIIB was solely responsible for converting initial selectin-dependent tethers to slow rolling and adhesion. However, in the cremaster RPA reaction, dependent on vascular and tissue accumulation of soluble ICs, FcγRIIA predominated in neutrophil recruitment that was dependent on G-protein coupled receptor activation. Thus, human FcγRs on neutrophils serve as the primary molecular links between ICs and immunological disease with FcγRIIA promoting tissue injury, and FcγRIIIB and FcγRIIA displaying specialized context-dependent functions in IC-induced neutrophil recruitment.

Antigen-antibody (Ab) interactions that lead to the formation of immune complexes (ICs) are subtle biochemical processes determining health or disease according to the outcome. Good laboratory practice (GLP)-acknowledged IC detection methods reveal that plasma levels of up to 15 microg/mL heat-aggregated immunoglobulin G (IgG) equivalents are normal, indicating the physiological role of ICs. Among the major variables that influence the equilibrium association constant Ka, are specificity and epitope density of the antigen, Ig class/subclass of the Ab, IC complement (C)-activating capacity, Fc receptor (FcR) interaction, and cytokine activation pattern. The Ka of antigen-Ab binding at approximately 20 degrees C ranges from low affinity (105) to high affinity (107-1011). Beneficial ICs serve to remove and/or neutralize infectious or toxic antigens, following an infectious attack in immune and vaccinated hosts. Circulating ICs are more prone for benefit than tissue-bound ICs, which reflect in situ formation and/or undesired deposition in tissues due to overflow from insufficient reticuloendothelial system (RES) removal. The classical textbook topic on ICs still holds true but is under revision because of the improved knowledge of effector systems, such as C, cytokine, and FcR apparatus. Therapeutic options to treat IC-associated diseases include intravenous immunoglobulins (IVIG) at their onset and monoclonal antibodies (mAb) directed at C activation products and/or cytokines.

Objectives: Systemic lupus erythematosus (SLE) is a classical autoimmune disorder characterised by the production of IgG autoantibodies against double-stranded DNA (dsDNA). Activation of FcγR-bearing effector cells by immune complexes (ICs) is a key event in SLE pathogenesis as lupus-prone NZB/NZW F 1 hybrids lacking activating Fcγ receptors (FcγR) are protected against inflammatory kidney damage despite glomerular deposition of ICs. Moreover, soluble FcγRs inhibit IC-caused Arthus reaction in vivo . Therefore, recombinant human soluble FcγRII (CD32) was evaluated as a novel therapeutic strategy in lupus-like disease in NZB/NZW F 1 hybrids. Methods: Binding of husCD32 to murine IgG was studied in vitro by binding to IgG-coated erythrocytes and inhibition of phagocytosis of IgG-opsonised murine erythrocytes. In order to examine therapeutic impact of husCD32 in vivo , female NZB/NZW F 1 mice were treated either from week 16 to 20 (“prophylactic”, 150 μg/week husCD32) or continuously from week 24 (“therapeutic”; 100 μg/week husCD32) by subcutaneous injections. Controls received buffered saline. Results: In vitro investigations of husCD32 revealed binding to murine erythrocytes coated with murine IgG. Moreover, husCD32 substantially diminished phagocytosis of murine IgG-opsonised murine red blood cells by peritoneal macrophages indicating disruption of IgG–FcγR interaction. There was a therapeutic efficacy of husCD32 to attenuate lupus pathology indicated by significantly delayed onset of proteinuria and weight loss, reduced histopathological findings, delayed development of anaemia and improved survival by prophylactical application. Therapeutic treatment did not reverse nephritis but significantly prolonged survival despite apparent kidney damage. B cell count, concentration of IgG anti-dsDNA autoantibodies and deposition of glomerular ICs was not significantly affected by the application of husCD32. Conclusions: The results demonstrate binding properties of husCD32 to ICs in vitro and as a proof-of-principle therapeutic efficacy in inhibiting chronic murine lupus pathology in vivo .

On October 20, 1997, the U.S. Food and Drug Administration (FDA) licensed Purified Chick Embryo Cell (PCEC, RabAvert®) vaccine against rabies in humans following clinical trials demonstrating safety and efficacy. From October 1997 through December 2005, the Vaccine Adverse Event Reporting System (VAERS) received 336 reports of adverse events (AEs) following vaccination with PCEC vaccine in the U.S.; there were no death reports. Serious events, including 20 hospitalizations and 13 neurological events, were described in 24 (7%) reports. There was no pattern among the 13 neurological AEs suggesting a plausible relationship to vaccination. A total of 20 AEs, 3 serious, were classified as possible anaphylaxis. There were 312 non-serious AEs (93%). Nineteen reports (6%) described that the vaccination series was discontinued because of non-serious AEs. Most reported AEs are non-serious and consistent with pre-licensure safety data. The rabies risk must be carefully considered before vaccine discontinuation.

金沢大学大学院医学系研究科血管分子科学, Immune complex (IC)-induced inflammation is mediated by inflammatory cell infiltration, a process that is highly regulated by expression of multiple adhesion molecules. The roles and interactions of ICAM-1 and VCAM-1, the major regulators of leukocyte firm adhesion, were examined in the cutaneous reverse-passive Arthus reaction using ICAM-1-deficient (ICAM-1-/-) mice and blocking mAb against VCAM-1. Within 8 h, IC challenge of wild-type mice induced edema, hemorrhage, interstitial accumulation of neutrophils and mast cells, as well as production of TNF-α and IL-6. All of these inflammatory parameters were reduced significantly in ICAM-1-/- mice. The blockade of VCAM-1 in wild-type mice did not affect any inflammatory parameters. In contrast, ICAM-1-/- mice treated with anti-VCAM-1 mAb had significantly reduced edema, hemorrhage, and neutrophil infiltration. Furthermore, VCAM-1 blockade in ICAM-1-/- mice suppressed cutaneous TNF-α and IL-6 production. Thus, VCAM-1 plays a complementary role to ICAM-1 in the cutaneous Arthus reaction by regulating leukocyte accumulation and proinflammatory cytokine production. © Society for Leukocyte Biology.

BACKGROUND Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory cytokine, which is closely associated with the pathogenesis of various types of cutaneous vasculitis (CV). AIM To investigate the therapeutic effects of an anti-TWEAK monoclonal antibody (mAb) in a mouse model of cutaneous reverse passive Arthus (RPA) reaction. METHODS Cutaneous RPA reaction was induced in BALB/c mice by intradermal injection of anti-ovalbumin IgG into the left ear followed immediately by intravenous injection of chicken ovalbumin. After treatment, haemorrhagic lesions in the mouse skin were scored semiquantitatively. The amount of extravasated fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) in the ears was detected spectrophotometrically. Expression of myeloperoxidase (MPO) was detected by immunohistochemical staining, while mRNA expression of TNF-α and interleukin (IL)-6 in lesional skin was detected by real-time quantitative (q)PCR. RESULTS Our results indicated that anti-TWEAK mAb significantly attenuated the clinical and histopathological changes in immune complex (IC)-induced mice, and also reduced the semiquantitative haemorrhage score, FITC-labelled BSA extravasation and MPO activity. Real-time qPCR showed that anti-TWEAK mAb significantly inhibited mRNA expression of TNF-α and IL-6 in lesional skin from IC-induced mice. CONCLUSION These data suggest that anti-TWEAK mAb can block vascular damage and leucocyte infiltration in IC-induced mice. TWEAK might be a candidate immunotherapeutic medicine for suppression of IC-induced CV.

The complement system is important for the host defence against infection as well as for the development of inflammatory diseases. Here we show that C1q/TNF-related protein 6 (CTRP6; gene symbol C1qtnf6) expression is elevated in mouse rheumatoid arthritis (RA) models. C1qtnf6 -/- mice are highly susceptible to induced arthritis due to enhanced complement activation, whereas C1qtnf6-transgenic mice are refractory. The Arthus reaction and the development of experimental autoimmune encephalomyelitis are also enhanced in C1qtnf6 -/- mice and C1qtnf6 -/- embryos are semi-lethal. We find that CTRP6 specifically suppresses the alternative pathway of the complement system by competing with factor B for C3(H 2 O) binding. Furthermore, treatment of arthritis-induced mice with intra-articular injection of recombinant human CTRP6 cures the arthritis. CTRP6 is expressed in human synoviocytes, and CTRP6 levels are increased in RA patients. These results indicate that CTRP6 is an endogenous complement regulator and could be used for the treatment of complement-mediated diseases., Nature Communications, 6, 8483; 2015

Leukocyte adhesion via β2 integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe–induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk–/– neutrophils from wild-type mice reconstituted with Syk–/– bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk–/– bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk–/– neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for β2 integrin–mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.

Staphylococcal protein A (SpA) is representative of a new class of antigens, the B-cell superantigens (SAgs). These antigens bind to the Fab regions of immunoglobulin molecules outside their complementarity-determining regions. SpA, the best-studied B-cell SAg, reacts with the Fabs of most VH3+immunoglobulins, which are expressed on 30 to 60% of human peripheral B cells. Therefore, B-cell SAgs like SpA have great potential to elicit inflammatory responses in vivo. We previously reported that the interaction of SpA with VH3+immunoglobulin molecules leads to activation of the complement cascade and produces a histologic pattern of inflammation in the skin of a rabbit indicative of immune complex injury. To elucidate the cellular and molecular events contributing to this type of unconventional immune complex-mediated inflammation, we established a mouse peritoneal Arthus reaction model. Mice treated intravenously with human polyclonal immunoglobulin G (IgG), followed by intraperitoneal injection of SpA, showed neutrophil influx into the peritoneal cavity with peak numbers appearing at 8 h. This inflammatory reaction was dependent on the interaction of SpA with VH3+IgG. Mast cells, FcγRIII, complement components, and tumor necrosis factor alpha play obligatory roles, and the reaction is associated with the local release of the CXC chemokines macrophage inflammatory protein 2 and KC. The data provide further compelling evidence for the induction of immune complex-mediated injury by a B-cell SAg and highlight important factors contributing to the pathogenesis of this novel type of inflammatory reaction.

Complement and FcgammaR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcgammaRIIB, increased levels of activating FcgammaRIII, and highly induced FcgammaR-mediated TNF-alpha and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of G(i)-type G protein signaling in C5aR-mediated control of the regulatory FcgammaR system in vitro, and analysis of the various C5aR-, FcgammaR-, and G(i)-deficient mice verifies the importance of Galpha(i2)-associated C5aR and the FcgammaRIII-FcgammaRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcgammaRIII-positive cells into C5aR- and FcgammaRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcgammaRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcgammaRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Galpha(i2)-dependent signal for modulating the two opposing FcgammaR, FcgammaRIIB and FcgammaRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.

Replacement of cellular with acellular pertussis (aP) vaccines has considerably reduced the systemic reactions observed with diphtheria-tetanus toxoids-pertussis vaccine but has not eliminated the extensive swelling (sometimes involving an entire limb) observed after the fifth injection of diphtheria-tetanus toxoids-aP (DTaP) vaccine. This local reaction, which is likely an Arthus hypersensitivity reaction caused by high levels of antibodies reacting with DTaP vaccine, could discourage its use in adults, who serve as the major reservoir of pertussis for infants. That a critical level of antibodies to pertussis toxin is both essential and sufficient to prevent infection with Bordetella pertussis is derived from data from animal and clinical studies, including data showing the similarities between the immunity induced by diphtheria and pertussis toxoids. The genetically inactivated diphtheria and pertussis mutant toxins are more immunogenic and, therefore, induce comparable levels of antitoxin at lower protein levels than do the formalin-treated native toxins. Replacement of the diphtheria and aP components with these improved antigens will reduce the amount of protein in DTaP vaccine and, most likely, the incidence and severity of local reactions in teenagers and adults.

The melanocortin-1 receptor (MC1) – being most abundantly expressed in the skin by melanocytes – has a physiological role for melanin pigmentation in many vertebrate species. MC1 has also been implicated in regulation of skin inflammation as this receptor is detectable in the majority of non-melanocytic cell types and its ligand α-melanocyte-stimulating hormone (α-MSH) exerts immunoregulatory and anti-inflammatory effects. However, in vivo studies on mice with targeted disruption of MC1 have been missing in the context of skin inflammation until recently. Wolnicka-Glubisz et al. now reported that the course of ultraviolet (UV)-induced inflammation, contact hypersensitivity, neonatal immune tolerance and UV-induced immunosuppression is similar in MC1 signal-deficient (C57BL/6-Mc1re/e) and wild-type mice. These unexpected findings are supported by own observations in experimentally induced immune-complex-mediated vasculitis: Mc1re/e mice exhibited a similar extent of the reverse passive cutaneous Arthus reaction compared with wild-type animals. Future studies are thus needed to clarify whether these findings are due to limitations in the chosen mouse model and/or point to additional MC subtypes that may regulate inflammatory and immune responses in the skin.

To elucidate the anti-inflammatory action of nicotine-induced corticosterone elevation on the passive skin Arthus reaction (PSAR), we investigated the inflammatory process in the PSAR. The polymorphonuclear leukocyte (PMNs) infiltration was observed just before as well as after elicitation by measuring extractable myeloperoxidase. The plasma exudation was significantly inhibited by anti-rat tumor necrosis factor (TNF)-α antibody (5 μg/site, i.d.) at the time of sensitization or by superoxide dismutase (52500 units/kg, i.p.) 1 h before elicitation or NG-nitro-L-arginine-methyl ester (100 mg/kg, i.v.) just at elicitation. Pretreatment with a single injection of nicotine (0.8 mg/kg, i.p.) 30 min before elicitation suppressed the plasma exudation but not the PMNs infiltration. This nicotine-induced decreasing effect was abolished in animals supplemented with L-arginine (300 mg/kg, i.v.) just at elicitation. The production of nitric oxide (NO) in peritoneal PMNs derived from an animal injected peritoneally with oyster glycogen was significantly suppressed by pretreatment with nicotine (0.8 mg/kg, i.v.) 30 min prior to harvesting. This inhibitory action of nicotine was abolished in animals pretreated with mifepristone (30 mg/kg, s.c.), a glucocorticoid receptor antagonist. These findings indicate that a single systematic administration of nicotine may attenuate the plasma exudation in the PSAR by suppressing the production of NO in the PMNs primed with TNF-α via nicotine-induced endogenous glucocorticoid. Keywords:: passive skin Arthus reaction, nicotine, tumor necrosis factor-α, superoxide, nitric oxide

Many adhesion molecule pathways have been invoked as mediating leukocyte recruitment during immune complex-induced inflammation. However the individual roles of these molecules have not been identified via direct visualization of an affected microvasculature. Therefore, to identify the specific adhesion molecules responsible for leukocyte rolling and adhesion in immune complex-dependent inflammation we used intravital microscopy to examine postcapillary venules in the mouse cremaster muscle. Wild-type mice underwent an intrascrotal reverse-passive Arthus model of immune complex-dependent inflammation and subsequently, leukocyte-endothelial cell interactions and P- and E-selectin expression were assessed in cremasteric postcapillary venules. At 4 hours, the reverse-passive Arthus response induced a significant reduction in leukocyte rolling velocity and significant increases in adhesion and emigration. P-selectin expression was increased above constitutive levels whereas E-selectin showed a transient induction of expression peaking between 2.5 to 4 hours and declining thereafter. While E-selectin was expressed, rolling could only be eliminated by combined blockade of P- and E-selectin. However, by 8 hours, all rolling was P-selectin-dependent. In contrast, inhibition of vascular cell adhesion molecule-1 had a minimal effect on leukocyte rolling, but significantly reduced both adhesion and emigration. These observations demonstrate that immune complex-mediated leukocyte recruitment in the cremaster muscle involves overlapping roles for the endothelial selectins and vascular cell adhesion molecule-1.

Human IgG receptors (FcgammaR) display considerable heterogeneity, and are crucial immune response modulating molecules. FcgammaRIIA, FcgammaRIIIA, and FcgammaRIIIB display functional biallelic polymorphisms. FcgammaR polymorphisms have been found associated with susceptibility to infectious and autoimmune diseases. Linked transmission of FcgammaR alleles was studied by determining the distribution of FcgammaRIIA-FcgammaRIIIA-FcgammaRIIIB genotype combinations in 514 Dutch Caucasian, and 149 Japanese blood donors. The structure of the FcgammaR locus was studied by radiation hybrid mapping of FcgammaRIA, FcgammaRIIA, FcgammaRIIB, FcgammaRIIIA, FcgammaRIIIB, and adjacent genes from the pentraxin family. In addition, crossing-over frequencies within the FcgammaR locus were determined in 63 Dutch Caucasian families, encompassing 183 individuals. FcgammaRII and FcgammaRIII subclasses were mapped in close proximity (0.47-3.14 cR). Accordingly, crossing-over frequencies within the FcgammaRII-III locus in Dutch families were low. Analysis of combined FcgammaR genotypes strongly suggested non-random distribution of FcgammaRIIA-FcgammaRIIIA-, and FcgammaRIIIA-FcgammaRIIIB genotypes in Dutch donors (P0.001 and P0.00001, respectively), and of FcgammaRIIA-FcgammaRIIIb genotypes in Japanese blood donors (P0.02). Frequencies of FcgammaRII-FcgammaRIII haplotypes differed significantly between Dutch and Japanese (P0.00001). This study provides important information for the interpretation of studies reporting associations of FcgammaR alleles with disease, and underscores the apparent differences in FcgammaR heterogeneity between ethnic groups.

Immune complex-induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. To assess the relative contribution of adhesion molecules, including selectins and ICAM-1, in this pathogenetic process, the cutaneous passive Arthus reaction was examined in mice lacking E-selectin, P-selectin, or both L-selectin and ICAM-1 with anti-P- or E-selectin mAbs. Edema and hemorrhage were significantly reduced in P-selectin(-/-) mice compared with wild-type mice while they were not inhibited in E-selectin(-/-) mice. Combined E- and P-selectin blockade resulted in more significant reduction relative to L-selectin/ICAM-1(-/-) as well as P-selectin(-/-) mice. Remarkably, both E- and P-selectin blockade in L-selectin/ICAM-1(-/-) mice completely abrogated edema and hemorrhage. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells that expressed significant levels of P-selectin glycoprotein ligand-1. Similarly reduced infiltration of neutrophils and mast cells was observed in the peritoneal Arthus reaction and was associated partly with the decreased production of tumor necrosis factor-alpha and interleukin-6. The results of this study indicate that both endothelial selectins contribute predominantly to the Arthus reaction by regulating mast cell and neutrophil infiltration and that the full development of the Arthus reaction is mediated cooperatively by all selectins and ICAM-1.

We examined the capacity of mouse glomerular mesangial cells (MC) to express and function through two different low affinity FcgammaRs, the activating FcgammaRIII and the inhibitory FcgammaRII. Immunohistochemistry identified FcgammaRII as the prominent FcgammaR in the kidney, and low levels of FcgammaRIIb2-specific mRNA were also detected in primary cultures of growth-arrested MC. Activation by tumor necrosis factor-alpha/interleukin-1beta induced substantial FcgammaRII expression in proliferating MC. Importantly, however, stimulation with interferon-gamma (IFN-gamma)/lipopolysaccharide or IFN-gamma alone resulted in a complete down-regulation of FcgammaRII, which was accompanied by a strong increase in FcRgamma chain mRNA and a surface appearance of FcgammaRIII. Activating FcgammaRIII triggered mRNA synthesis for monocyte chemoattractant protein-1 (MCP-1), MCP-5, cytokine-induced neutrophil chemoattractant, and RANTES, whereas FcgammaRIII-deficient MC failed to respond to immune complex (IC) activation as shown by impaired production of MCP-1 mRNA/protein. In a passive model of acute anti-glomerular basement membrane (GBM) nephritis, induction of FcgammaRIII and suppression of FcgammaRII occurred in kidney tissues. Blockade of FcgammaRII, when induced selectively in the kidney, resulted in enhanced inflammation. Taken together, our results define a novel regulatory pathway with opposite regulation of FcgammaRII (suppressed) and FcgammaRIII (induced) by IFN-gamma on MCs in vitro and anti-GBM IgG in vivo. Herein is provided the first evidence that glomerular FcgammaRII plays an important immunoregulatory role in the initiation of IC glomerulonephritis.

To investigate the pathomechanisms of leukocytoclastic vasculitis (LcV) we compared mouse models of LcV with non-vasculitic irritant contact dermatitis (ICD). Criteria for LcV as met by the immune complex-mediated Arthus reaction (Art-r) were also fulfilled by the localized Shwartzman reaction (Shw-r) and by cutaneous Loxoscelism (Lox) (injection of venom from Loxosceles reclusa containing sphingomyelinase D). After depletion of PMN (by gamma-irradiation) vessel damage could not be elicited in these models, distinguishing them from models of direct endothelial insult (necrotizing ICD). Depletion of complement could only delay, but not inhibit the Art-r, and did not change ICD, Lox or the Shw-r. The Shw-r exclusively revealed a sustained local expression of vascular adhesion molecules for 24 h in the preparatory phase (LPS s.c.), not observed in the Art-r, in Lox or ICD. Subsequent challenge with LPS i.p. was associated with upregulation of Mac-1 and ICAM-1 on PMN, but not of VLA-4 or LFA-1 (FACS analysis). Cytokines which were able to replace LPS in priming for LcV in the Shw-r (TNF-alpha and IL-1beta) also induced sustained expression of adhesion molecules, whereas IL-12 and IFN-gamma did neither. Neutralizing IL-12 or IFN-gamma also inhibited neither LcV nor sustained expression of adhesion molecules, whereas anti-TNF-alpha inhibited both. Anti-TNF-alpha had no marked inhibitory effects in the Art-r, in Lox or ICD. Combined (but not separate) neutralization of both E-selectin and VCAM-1 by antibodies suppressed LcV independent from reducing influx of PMN, proving that their sustained expression is decisive for the Shw-r and interferes with normal diapedesis. Since Loxosceles venom is known to dysregulate diapedesis and degranulation of PMN in vitro, since adherent immune complexes activate PMN at the vessel wall, and since adhesion molecules are dysregulated in the Shw-r, we suggest that LcV develops when activation of PMN coincides with vascular alterations which interfere with normal diapedesis.

In the present study we investigated in the reverse passive Arthus reaction elicited in the rat skin the anti-inflammatory effect of double-stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-kappaB (NF-kappaB) sequence as transcription factor decoys (TFD) to inhibit NF-kappaB binding to native DNA sites. Local administration of wild-type-, but not mutant-decoy ODN, dose-dependently reduced both plasma leakage and neutrophil infiltration in rat skin. Molecular analysis performed on soft tissue obtained from rat skin demonstrated: (1) an inhibition of NF-kappaB/DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-kappaB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-kappaB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-kappaB complexes, as well as ammonium pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB activation, exhibited a similar profile of activity of decoy ODN. Our results indicate that decoy ODN, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate immune reactions.

Neutrophil accumulation is a hallmark of immune complex-mediated inflammatory disorders. Current models of neutrophil recruitment envision the capture of circulating neutrophils by activated endothelial cells. We now demonstrate that immobilized immune complexes alone support the rapid attachment of neutrophils, under physiologic flow conditions. Initial cell tethering requires the low-affinity Fc gamma receptor IIIB (Fc gamma RIIIB), and the beta(2) integrins are additionally required for the subsequent shear-resistant adhesion. The attachment function of Fc gamma RIIIB may be facilitated by its observed presentation on neutrophil microvilli. In vivo, in a model of acute antiglomerular basement membrane nephritis in which immune complexes are accessible to circulating neutrophils, Fc gamma RIII-deficient mice had a significant reduction in neutrophil recruitment. Thus, the interaction of immune complexes with Fc gamma RIII may mediate early neutrophil recruitment in immune complex-mediated inflammation.

Many substances, both natural and manufactured, cause inflammation when introduced into living tissue. Some compounds cause a local inflammatory reaction in all exposed individuals and often are called toxic or irritant reactions. Skin testing with other compounds causes an inflammatory response in a limited number of subjects. Those reactions that occur on the basis of specific immune responses are termed hypersensitivity reactions, and the responding individuals are said to be hypersensitive to that antigen. Intradermal skin testing may induce four or more types of cutaneous hypersensitivity reactions in humans 35 : (1) an immediate wheal and flare reaction related to the presence of antigen-specific immunoglobulin E (IgE) in the skin; (2) an IgE-mediated late-phase reaction that follows the wheal and flare, begins several hours after injection, and peaks at 12 to 24 hours; (3) a local vasculitic reaction peaking at 12 to 24 hours that results from the interaction of complement-fixing antibody and antigen at the injection site (Arthus reaction); and (4) a lymphocyte and macrophage-dependent delayed hypersensitivity reaction (DHR). Erythema and induration at the injection site peaks at 48 hours after the injection. The latter reaction is the subject of this article.

Acute ovalbumin-induced arthritis (OIA), which is mediated by Arthus reaction to ovalbumin (OVA) in the joint space, can be induced by immunization of rats with OVA followed by the intraarticular injection of OVA. Because oral administration of antigen induces immunological unresponsiveness, we studied for the first time the effects of oral administration of OVA on acute OIA. The oral administration of OVA before immunization significantly suppressed the development of acute OIA, in accordance with decreases in both the anti-OVA IgG antibody production and in vitro lymphocyte proliferative responses to OVA. However, the oral administration of OVA after immunization did not show any decrease in antibody production or in vitro lymphocyte proliferation to OVA, or in the severity of acute OIA. These results indicate that the induction of oral tolerance to OVA in OIA is possible by oral administration of OVA before, but not after, immunization with the antigen. This supports the concept of using antigen feeding as a treatment for certain humoral immune-mediated diseases.

The Src family kinase Lyn initiates intracellular signal transduction by associating with a variety of immune receptors such as antigen receptor on B cells and high-affinity Fc receptor (FcR) for immunoglobulin Ig(E) (FcepsilonRI) on mast cells. Involvement of Lyn in the IgE-mediated immediate-type hypersensitivity is well documented, but the physiological significance of Lyn in IgG-dependent, type III low-affinity FcR for IgG (FcgammaRIII)-mediated responses is largely unknown. In this study, we generated a double-mutant mouse strain deficient in both type II FcR for IgG (FcgammaRIIB) and Lyn to exclude any involvement of inhibitory signaling by FcgammaRIIB, which otherwise downregulates FcgammaRIII-mediated cellular responses. FcgammaRIIB-deficient but Lyn-sufficient mice served as controls. The Lyn deficiency attenuated IgG-mediated systemic anaphylaxis in vivo, and significantly reduced calcium mobilization and degranulation responses of bone marrow-derived mast cells (BMMCs) in vitro. However, we found that either interleukin 4 or tumor necrosis factor alpha release by BMMCs was comparable to that from Lyn-deficient and control mice, and the reverse-passive Arthus reaction was equally induced in both mutant mice, indicating that Lyn is not involved in the onset of the IgG-mediated, FcgammaRIII-dependent late phase responses of mast cells. These findings provide us with insight into distinct signaling mechanisms in mast cells underlying the development of diverse pathologies as well as a therapeutic potential for selective treatment of allergic disorders.

Background Recombinant soluble forms of complement regulatory molecules, including the human complement regulatory protein CD46 (rsCD46), have been shown to inhibit hyperacute transplant rejection (HAR) and protect against complement-mediated inflammatory tissue damage. Similarly, recombinant soluble forms of the immunoglobulin receptor FcgammaRII (rsFcgammaRII) can attenuate antibody-mediated inflammatory responses. We have produced and tested the function of novel recombinant chimeric proteins that incorporate the functional domains of both CD46 (membrane cofactor protein, MCP) and the low affinity human IgG receptor FcgammaRII (CD32). Methods Two recombinant soluble chimeric proteins (CD46:FcR and FcR:CD46) were designed and produced using a human cell expression system. Their ability to protect cells against complement-mediated lysis (through the CD46 domain) and bind human IgG (through the Fc receptor domain) was assessed in vitro. They were also tested in vivo in the rat reverse passive Arthus reaction and a murine model of hyperacute cardiac transplant rejection. Results In vitro, the functional domains of the chimeric proteins each retained their activity. In vivo, the serum half-life of the recombinant chimeric proteins in mice was more than either rsCD46 or rsFcgammaRII. In the rat reverse passive Arthus reaction, intradermal injection of each recombinant protein substantially reduced inflammatory skin edema (>50%) and polymorphonuclear neutrophil infiltration (>90%). In the hyperacute rejection model, i.v. treatment with FcR:CD46 prevented complement-mediated rejection, macroscopic bruising, edema, and thrombosis more effectively than rsCD46. Conclusions CD46/FcgammaRII bifunctional proteins have an improved ability to control complement-mediated hyperacute graft rejection and have therapeutic potential in other conditions involving antibody-mediated inflammation.

Murine low-affinity receptors for IgG, FcgammaRII and FcgammaRIII, differ by their distinct capacities in mediating down-regulation or activation of cellular effector functions, respectively. In this study, antibodies detecting the mouse Ly-17.1 / 2 alloantigen system are demonstrated to be specific for FcgammaRII with no cross-reactivities to other FcgammaR, including FcgammaRIII. Using these FcgammaRII-specific monoclonal antibodies (mAb), the significance of FcgammaRII inhibition of FcgammaRIII was examined in two models of autoantibody [autoimmune hemolytic anemia (AIHA)]- and IgG immune complex-induced (Arthus reaction) inflammation in C57BL / 6 mice in comparison with FcgammaRII(- / -) and FcgammaRIII(- / -) mice. Our results demonstrate that both FcgammaRIII and FcgammaRII contributed to the binding of erythrocytes opsonized with the pathogenic IgG1 autoreactive anti-murine red blood cell antibody 105-2H. However, the functional blocking with anti-FcgammaRII mAb in C57BL / 6 mice and the lack of FcgammaRII expression in FcgammaRII(- / -) mice, which both lowered the threshold level of FcgammaRIII-triggered phagocytosis in vitro, did not results in enhanced disease development of 105-2H mAb-induced AIHA in vivo. This was in sharp contrast to cutaneous Arthus reaction, where FcgammaRIII-mediated activation was inhibited by FcgammaRII. Together these results show that murine AIHA is markedly different from other FcgammaR-dependent inflammatory diseases where FcgammaRIII is normally counterregulated by FcgammaRII.