A comparative study of the expression of cytotoxic proteins in allergic contact dermatitis and psoriasis: spongiotic skin lesions in allergic contact dermatitis are highly infiltrated by T cells expressing perforin and granzyme B

Recent reports indicate that cytotoxic T cells are critically involved in contact hypersensitivity reactions in animals. In this study we sought to investigate the in vivo expression of cytotoxic granule proteins in the elicitation phase of allergic contact dermatitis in humans. Skin biopsy specimens were obtained from patients with allergic contact dermatitis (n = 8) and psoriasis (n = 6) and from controls with normal skin (n = 6). Expression of perforin and granzyme B was investigated by in situ hybridization and immunohistochemistry. In contrast to normal skin and psoriasis, a significant enhancement of perforin and granzyme B gene expression and immunoreactivity was observed in the mononuclear cell infiltrate of allergic contact dermatitis. Immunoreactivity for perforin and granzyme B was mainly found in the cytoplasm of lymphocytic cells, which were located in the dense perivascular infiltrate as well as at sites of marked spongiosis in the epidermis. Double immunostaining revealed that both CD4+ and CD8+ T cells are capable of expressing perforin and granzyme B. In conclusion, our data suggest that T-cell-mediated mechanisms involving cytotoxic granule proteins may elicit epidermal cell injury in vivo and thereby strongly contribute to the development of allergic contact dermatitis in humans.

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

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

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

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

Living and dying for inflammation: neutrophils, eosinophils, basophils

Neutrophils, eosinophils, and basophils play essential roles during microbe-induced and sterile inflammation. The severity of such inflammatory processes is controlled, at least in part, by factors that regulate cell death and survival of granulocytes. In recent years, major progress has been made in understanding the molecular mechanisms of granulocyte cell death and in identifying novel damage- and pathogen-associated molecular patterns as well as regulatory cytokines impacting granulocyte viability. Furthermore, an increased interest in innate immunity has boosted our overall understanding of granulocyte biology. In this review, we describe and compare factors and mechanisms regulating neutrophil, eosinophil, and basophil lifespan. Because dysregulation of death pathways in granulocytes can contribute to inflammation-associated immunopathology, targeting granulocyte lifespan could be therapeutically promising.

Extracellular DNA traps in allergic, infectzious, and autoimmune diseases

Extracellular DNA traps are part of the innate immune response and are seen with many infectious, allergic, and autoimmune diseases. They can be generated by several different leukocytes, including neutrophils, eosinophils, and monocytes, as well as mast cells. Here, we review the composition of these extracellular DNA-containing structures as well as potential mechanisms for their production and function. In general, extracellular DNA traps have been described as binding to and killing pathogens, particularly bacteria, fungi, but also parasites. On the other hand, it is possible that DNA traps contribute to immunopathology in chronic inflammatory diseases, such as bronchial asthma. In addition, it has been demonstrated that they can initiate and/or potentiate autoimmune diseases. Extracellular DNA traps represent a frequently observed phenomenon in inflammatory diseases, and they appear to participate in the cross-talk between different immune cells. These new insights into the pathogenesis of inflammatory diseases may open new avenues for targeted therapies.

IL-33 is a mediator rather than a trigger of the acute allergic response in humans

BACKGROUND IL-33 enhances FcεRI-induced mediator release in human basophils without inducing degranulation itself. In contrast, studies in mice suggested that in the presence of high IgE levels, IL-33 triggers degranulation and anaphylaxis of similar severity as specific allergen. Consistent with this view, sera of atopic patients contain elevated levels of IL-33 after anaphylaxis. In this study, we determined whether IL-33 is potentially anaphylactogenic in humans with high IgE levels by regulating exocytosis independent of FcεRI cross-linking. Furthermore, we investigated whether IL-33 is released upon allergen provocation in vivo. METHODS In subjects with high serum IgE levels, we measured IL-33-induced histamine/LTC4 in vitro, CD63 translocation ex vivo, and responsiveness of mast cells in vivo by skin prick test (SPT). In asthma patients, release of IL-33 and its correlation with early (tryptase)- and late-phase markers (IL-13 levels, eosinophil numbers) of the allergic response were assessed in bronchoalveolar lavage fluids (BALFs) after allergen challenge. RESULTS IL-33 itself does not trigger basophil degranulation in vitro and ex vivo, even in subjects with high serum IgE levels, and negative SPTs demonstrate that skin mast cells do not degranulate in response to IL-33. However, in response to allergen challenge, IL-33 is rapidly released into BALFs at levels that do not correlate with other immediate- and late-phase parameters. CONCLUSION IL-33 is unlikely an independent trigger of anaphylaxis even in subjects with high IgE levels. However, the rapid release of IL-33 upon allergen provocation in vivo supports its role as a mediator of immediate allergic responses.

Deciphering microRNA code in pain and inflammation: lessons from bladder pain syndrome

MicroRNAs (miRNAs), a novel class of molecules regulating gene expression, have been hailed as modulators of many biological processes and disease states. Recent studies demonstrated an important role of miRNAs in the processes of inflammation and cancer, however, there are little data implicating miRNAs in peripheral pain. Bladder pain syndrome/interstitial cystitis (BPS/IC) is a clinical syndrome of pelvic pain and urinary urgency/frequency in the absence of a specific cause. BPS is a chronic inflammatory condition that might share some of the pathogenetic mechanisms with its common co-morbidities inflammatory bowel disease (IBD), asthma and autoimmune diseases. Using miRNA profiling in BPS and the information about validated miRNA targets, we delineated the signaling pathways activated in this and other inflammatory pain disorders. This review projects the miRNA profiling and functional data originating from the research in bladder cancer and immune-mediated diseases on the BPS-specific miRNAs with the aim to gain new insight into the pathogenesis of this enigmatic disorder, and highlighting the common regulatory mechanisms of pain and inflammation.

Cytokines and chemokines in irritant contact dermatitis

Irritant contact dermatitis is a result of activated innate immune response to various external stimuli and consists of complex interplay which involves skin barrier disruption, cellular changes, and release of proinflammatory mediators. In this review, we will focus on key cytokines and chemokines involved in the pathogenesis of irritant contact dermatitis and also contrast the differences between allergic contact dermatitis and irritant contact dermatitis.

Interferon-α antagonizes IL-3-mediated immunoregulatory functions of human basophils

Human basophils are major inflammatory cells in maintaining chronic allergic asthma. It has been published that interferon-α (IFN-α) improves clinical symptoms of asthma patients. In contrast, IL-3 exacerbates airway inflammation by inducing IL-4, IL-8 and IL-13 secretion from human basophils thus regulating their immunoregulatory functions. Furthermore, IL-3 exceptionally promotes survival of basophils. Here, we assessed cellular response of human basophils treated with IFN-α alone or in combination with IL-3. Our data show that IFN-α enhances apoptosis in purified human blood basophils compared to spontaneous apoptosis of controls or IFN-γ treated cells. Furthermore, we demonstrate that both IFN-α and FasL enhance apoptosis in human basophils with similar efficiency in a rather additive than synergistic way. IFN-α inhibits IL-3-induced survival to a minor degree. Particularly however, it suppresses IL-3-induced de-novo production of IL-8 and IL-13 up to 80%. In contrast, the production of IL-4 is not affected. Analyses of signaling pathways reveal that IFN-α promotes prolonged phosphorylation of STAT1/STAT2. By using a pan-JAK inhibitor the phosphorylation of STAT1/STAT2 is inhibited and most importantly the pro-apoptotic effect of IFN-α is abolished. Although the phosphorylation of p38-MAPK in IFN-α-treated cells is comparable to non-treated cells, inhibition of p-p38 activity abrogates IFN-α-enhanced apoptosis as well. We conclude that IFN-α-enhanced apoptosis is tightly regulated by the cooperation of JAK/STAT and p38-MAPK pathways. Our study identifies IFN-α as a novel inhibitor of IL-3-induced IL-8 and IL-13 production of human basophils. Taken together our study may explain the improved clinical symptoms of asthma patients treated with IFN-α.

Fatty acid-induced mitochondrial uncoupling elicits inflammasome-independent IL-1α and sterile vascular inflammation in atherosclerosis

Chronic inflammation is a fundamental aspect of metabolic disorders such as obesity, diabetes and cardiovascular disease. Cholesterol crystals are metabolic signals that trigger sterile inflammation in atherosclerosis, presumably by activating inflammasomes for IL-1β production. We found here that atherogenesis was mediated by IL-1α and we identified fatty acids as potent inducers of IL-1α-driven vascular inflammation. Fatty acids selectively stimulated the release of IL-1α but not of IL-1β by uncoupling mitochondrial respiration. Fatty acid-induced mitochondrial uncoupling abrogated IL-1β secretion, which deviated the cholesterol crystal-elicited response toward selective production of IL-1α. Our findings delineate a previously unknown pathway for vascular immunopathology that links the cellular response to metabolic stress with innate inflammation, and suggest that IL-1α, not IL-1β, should be targeted in patients with cardiovascular disease.

Combined inhibition of complement C5 and CD14 markedly attenuates inflammation, thrombogenicity, and hemodynamic changes in porcine sepsis

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli-induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin-antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology

Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (<3% and <1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs.

Inflammation and symptoms of depression and anxiety in patients with acute coronary heart disease

Depression following an acute coronary syndrome (ACS, including myocardial infarction or unstable angina) is associated with recurrent cardiovascular events, but the depressive symptoms that are cardiotoxic appear to have particular characteristics: they are 'incident' rather than being a continuation of prior depression, and they are somatic rather than cognitive in nature. We tested the hypothesis that the magnitude of inflammatory responses during the ACS would predict somatic symptoms of depression 3 weeks and 6 months later, specifically in patients without a history of depressive illness. White cell count and C-reactive protein were measured on the day after admission in 216 ACS patients. ACS was associated with very high levels of inflammation, averaging 13.23×10(9)/l and 17.06 mg/l for white cell count and C-reactive protein respectively. White cell count during ACS predicted somatic symptom intensity on the Beck Depression Inventory 3 weeks later (β=0.122, 95% C.I. 0.015-0.230, p=0.025) independently of age, sex, ethnicity, socioeconomic status, marital status, smoking, cardiac arrest during admission and clinical cardiac risk, but only in patients without a history of depression. At 6 months, white cell count during ACS was associated with elevated anxiety on the Hospital Anxiety and Depression Scale independently of covariates including anxiety measured at 3 weeks (adjusted odds ratio 1.08, 95% C.I. 1.01-1.15, p=0.022). An unpredicted relationship between white cell count during ACS and cognitive symptoms of depression at 6 months was also observed. The study provides some support for the hypothesis that the marked inflammation during ACS contributes to later depression in a subset of patients, but the evidence is not conclusive.

Immunopathogenesis of eosinophilic esophagitis

Background: Eosinophilic esophagitis (EoE) is a chronic inflammatory disease of the esophagus associated with dysphagia in adults and refractory reflux syndromes in children. Methods: Immunological and genetic approaches have been used to better understand the pathophysiology of the underlying inflammation. Results and Conclusions: Evidence has accumulated that EoE represents a T-helper (Th) 2-type inflammatory disease, in which allergens play a role in triggering the disease. The majority of the patients suffer from concurrent allergic rhinitis, asthma, and eczema, and have a history of atopy. The chronic inflammatory response in EoE is associated with tissue damage and remodeling, both of which lead to esophageal dysfunction and bolus impaction. The new insights into the pathophysiology have resulted in the development of the first pharmacological therapies of EoE.