Cutaneous Reverse Passive Arthus Reaction Research Articles

Cooperation Between Platelet β1 and β3 Integrins in the Arrest of Bleeding Under Inflammatory Conditions in Mice-Brief Report.

Platelets prevent bleeding in a variety of inflammatory settings, the adhesion receptors and activation pathways involved being highly context-dependent and functionally redundant. In some situations, platelets recruited to inflammatory sites act independently of aggregation. The mechanisms underlying stable platelet adhesion in inflamed microvessels remain incompletely understood, in particular, whether and if so, how β1 and β3 integrins are involved. The impact of isolated or combined platelet deficiency in β1 and β3 integrins on inflammation-associated hemostasis was investigated in 3 models of acute inflammation: immune complex-based cutaneous reverse passive Arthus reaction, intranasal lipopolysaccharide-induced lung inflammation, and cerebral ischemia-reperfusion following transient (2-hour) occlusion of the middle cerebral artery. Mice with platelet-directed inactivation of Itgb1 (PF4Cre-β1-/-) displayed no bleeding in any of the inflammation models, while mice defective in platelet Itgb3 (PF4Cre-β3-/-) exhibited bleeding in all 3 models. Remarkably, the bleeding phenotype of PF4Cre-β3-/- mice was exacerbated in the reverse passive Arthus model by the concomitant deletion of β1 integrins, PF4Cre-β1-/-/β3-/- animals presenting increased bleeding. Intravital microscopy in reverse passive Arthus experiments highlighted a major defect in the adhesion of PF4Cre-β1-/-/β3-/- platelets to inflamed microvessels. Unlike PF4Cre-β1-/- and PF4Cre-β3-/- mice, PF4Cre-β1-/-/β3-/- animals developed early hemorrhagic transformation 6 hours after transient middle cerebral artery occlusion. PF4Cre-β1-/-/β3-/- mice displayed no more bleeding in lipopolysaccharide-induced lung inflammation than PF4Cre-β3-/- animals. Altogether, these results show that the requirement for and degree of functional redundancy between platelet β1 and β3 integrins in inflammation-associated hemostasis vary with the inflammatory situation.

Inhibitory role of interleukin-10 in the cutaneous reverse Arthus reaction.

The formation and deposition of immune complexes (IC) containing immunoglobulin (Ig)G antibodies induces an acute inflammatory response with tissue injury. One of the experimental models of IC-related vasculitis is the cutaneous reverse passive Arthus reaction, in which IgG antibodies are injected i.d., followed immediately by the i.v. application of the corresponding antigen. This reaction is characterized by edema, hemorrhage and neutrophil infiltration. To assess the role of the anti-inflammatory cytokine interleukin (IL)-10 in IC-related vasculitis, we investigated the cutaneous Arthus reaction using IL-10 knockout (IL-10KO) mice. Edema, which was quantified macroscopically by measuring the vascular leakage of Evans blue dye at 4h after IC challenge, was significantly increased in IL-10KO mice compared with wild-type mice. In addition, hemorrhage, which was assessed by the average diameter of purpuric spots at 8h after IC challenge, was enhanced significantly in IL-10KO mice compared with wild-type mice. Histological examination showed that the number of extravascular neutrophils was significantly increased in IL-10KO mice compared with wild-type mice at 4 and 8h after IC challenge. Analysis of pro-inflammatory cytokine mRNA expression showed that IL-6 mRNA levels were significantly increased in IL-10KO mice compared with wild-type mice at 4 and 8h after IC challenge. These results showed that IC-induced inflammation and vascular damage were significantly enhanced in the absence of IL-10. Thus, IL-10 may limit tissue disruption by suppressing the excessive infiltration of neutrophils and cytokine expression in a mouse model of type III vasculitis.

Anti-TWEAK monoclonal antibodies reduce vascular damage and leucocyte infiltration in a mouse model of cutaneous reverse passive Arthus reaction.

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). To investigate the therapeutic effects of an anti-TWEAK monoclonal antibody (mAb) in a mouse model of cutaneous reverse passive Arthus (RPA) reaction. 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. 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. 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.

Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2

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.

β7 Integrin Controls Mast Cell Recruitment, whereas αE Integrin Modulates the Number and Function of CD8+ T Cells in Immune Complex–Mediated Tissue 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.

Exogenous application of hydrogen sulfide donor attenuates inflammatory reactions 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.

Basophils and mast cells play critical roles for leukocyte recruitment in IgE-mediated cutaneous reverse passive Arthus reaction

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. To investigate the mechanisms of how mast cells and basophils regulate this IgE-mediated Arthus reaction. 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. 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. 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.

Establishment of Experimental Eosinophilic Vasculitis by IgE-Mediated Cutaneous Reverse Passive Arthus Reaction

Prominent eosinophil infiltration is a characteristic of some forms of vasculitis, such as Churg-Strauss syndrome, also known as allergic granulomatous vasculitis. In the current study, we established a mouse model of cutaneous eosinophilic vasculitis by the cutaneous reverse passive Arthus reaction using IgE injection instead of IgG. Wild-type C57BL/6 mice were injected with IgE anti-trinitrophenyl antibodies, followed immediately by intravenous administration of trinitrophenyl bovine serum albumin. IgE-mediated immune complex challenge induced substantial hemorrhage with marked infiltration of eosinophils in which neutrophils, mast cells, and macrophages were also mixed. This finding contrasted remarkably with the neutrophil-dominant infiltration pattern in IgG-mediated immune complex challenge. In the lesion, the expression level of monocyte chemotactic protein-3 was increased, and anti-monocyte chemotactic protein-3 treatment resulted in a significant but incomplete blockade of eosinophil recruitment. Furthermore, mice lacking E-selectin, P-selectin, L-selectin, or intercellular adhesion molecule-1, as well as wild-type mice that received anti-vascular cell adhesion molecule-1-blocking antibodies were assessed for the IgE-mediated Arthus reaction. After 24 hours, the loss of P-selectin resulted in a significant reduction in eosinophil accumulation compared with both wild-type mice and other mouse mutants. Collectively, the Fc class of immunoglobulins, which forms these immune complexes, critically determines the disease manifestation of vasculitis. The IgE-mediated cutaneous reverse passive Arthus reaction may serve as an experimental model for cutaneous eosinophilic infiltration in vasculitis as well as in other diseases.

Human Neutrophil Fcγ Receptors Initiate and Play Specialized Nonredundant Roles in Antibody-Mediated Inflammatory Diseases

Inflammation mediated by antibody-antigen complexes contributes to autoimmune diseases. Mice deficient in the common Fcgamma-chain are protected from IgG-mediated glomerulonephritis and the reverse passive Arthus (RPA) reaction and FcR-bearing macrophages, and mast cells have been assigned primary roles in these processes. Here we demonstrate that neutrophil-selective transgenic expression of the two uniquely human neutrophil Fc gamma receptors (FcgammaRs), FcgammaRIIA and FcgammaRIIIB, in Fcgamma-chain-deficient mice restored susceptibility to progressive glomerulonephritis and the cutaneous RPA reaction. FcgammaRIIIB and FcgammaRIIA mediated neutrophil accumulation, whereas FcgammaRIIA alone promoted organ injury. In a model of soluble immune complexes deposited within the vasculature, FcgammaRIIIB was responsible for neutrophil slow rolling and adhesion whereas in the cremaster RPA, induced by both vascular and tissue soluble immune complexes, FcgammaRIIA predominated. Thus, human FcgammaRs on neutrophils serve as molecular links between antibody and immunological disease, with FcgammaRIIA promoting tissue injury and FcgammaRIIIB and FcgammaRIIA displaying specialized context-dependent functions in neutrophil recruitment.

Involvement of gaseous low molecular monoxides in the cutaneous reverse passive Arthus reaction: cytoprotective action of carbon monoxide

The deposition of immune complexes (IC) induces an acute inflammatory response with tissue injury, for which the involvement of nitric oxide (NO) and carbon monoxide (CO) has been suggested. NO is induced by NO synthase (NOS) and CO is generated by haeme oxygenase (HO). Among HO isoenzymes, HO-1 is an induced type. To assess the role of NO and CO in the pathogenic process, the cutaneous reverse passive Arthus reaction was examined using NOS inhibitor, HO-1 stimulator and HO-1 inhibitor. To evaluate the reaction we considered oedema, tumour necrosis factor-alpha, interleukin-6, and neutrophil number. The values of these four parameters were significantly reduced in mice treated with HO-1 stimulator as compared with the positive control mice. Quite the reverse was observed in mice treated with HO-1 inhibitor. These results suggest that the HO-1/CO signalling pathway is a therapeutic target for human IC-mediated disease.

Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 cooperatively contribute to the cutaneous Arthus reaction

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-alpha 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-alpha 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.

Establishment of an animal model for allergic granulomatous vasculitis by IgE-mediated cutaneous reverse passive arthus reaction: contribution of selectins and ICAM-1

Establishment of an animal model for allergic granulomatous vasculitis by IgE-mediated cutaneous reverse passive arthus reaction: contribution of selectins and ICAM-1

P-selectin glycoprotein ligand-1 is required for the development of cutaneous vasculitis induced by immune complex deposition.

Immune complex (IC)-induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. To assess the contribution of P-selectin glycoprotein ligand-1 (PSGL-1) and selectins in the pathogenetic process, the cutaneous reverse-passive Arthus reaction was examined in mice treated with monoclonal antibodies (mAb) to PSGL-1 or P- and/or E-selectin. Edema and hemorrhage were significantly reduced in mice treated with anti-P-selectin mAb compared with control mice while they were not inhibited in mice treated with anti-E-selectin mAb. It is remarkable that blocking PSGL-1 by mAb resulted in significant, further reduction in edema and hemorrhage compared with blocking anti-P- or anti-E-selectin. However, blockade of E- and P-selectins exhibited more significant reduction relative to PSGL-1 blockade. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells. Reduced infiltration of neutrophils and mast cells was observed in the peritoneal Arthus reaction and was associated with the decreased production of tumor necrosis factor alpha and interleukin-6. The results of this study indicate that PSGL-1 contributes to the Arthus reaction mainly as a ligand of P-selectin and partly as a ligand of E- and/or L-selectin by regulating neutrophil and mast-cell recruitment and that PSGL-1 would be a therapeutic target for human IC-mediated diseases.

The cutaneous reverse Arthus reaction requires intercellular adhesion molecule 1 and L-selectin expression.

The deposition of immune complexes (IC) induces an acute inflammatory response with tissue injury. IC-induced inflammation is mediated by inflammatory cell infiltration, a process highly regulated by expression of multiple adhesion molecules. To assess the role of L-selectin and ICAM-1 in this pathogenetic process, the cutaneous reverse passive Arthus reaction was examined in mice lacking L-selectin (L-selectin(-/-)), ICAM-1 (ICAM-1(-/-)), or both (L-selectin/ICAM-1(-/-)). Edema and hemorrhage, which peaked 4 and 8 h after IC challenge, respectively, were significantly reduced in L-selectin(-/-), ICAM-1(-/-), and L-selectin/ICAM-1(-/-) mice compared with wild-type littermates. In general, edema and hemorrhage were more significantly inhibited in ICAM-1(-/-) mice than in L-selectin(-/-) mice, but were most significantly reduced in L-selectin/ICAM-1(-/-) mice compared with ICAM-1(-/-) or L-selectin(-/-) mice. Decreased edema and hemorrhage correlated with reduced neutrophil and mast cell infiltration in all adhesion molecule-deficient mice, but leukocyte infiltration was most affected in L-selectin/ICAM-1(-/-) mice. Reduced neutrophil and mast cell infiltration was also observed for all mutant mice in the peritoneal Arthus reaction. Furthermore, cutaneous TNF-alpha production was inhibited in each deficient mouse, which paralleled the reductions in cutaneous inflammation. These results indicate that ICAM-1 and L-selectin cooperatively contribute to the cutaneous Arthus reaction by regulating neutrophil and mast cell recruitment and suggest that ICAM-1 and L-selectin are therapeutic targets for human IC-mediated disease.