GPR91 deficiency exacerbates allergic contact dermatitis while reducing arthritic disease in mice.

BACKGROUND: Succinate, in addition to its role as an intermediary of the citric acid cycle, acts as an alarmin, initiating and propagating danger signals resulting from tissue injury or inflammatory stimuli. The contribution of this immune sensing pathway to the development of allergic and inflammatory responses is unknown. METHODS: Ear thickness of wild-type (wt) and Sucnr1-deficient (Sucnr1-/- ) mice, sensitized and challenged with oxazolone, was used as a criterion to assess the relevance of SUCNR1/GPR91 expression mediating allergic contact dermatitis (ACD). Results obtained in this system were contrasted with data generated using passive cutaneous anaphylaxis, ovalbumin-induced asthma and arthritis models. RESULTS: We found augmented ACD reactions in Sucnr1-/- mice. This observation correlated with increased mast cell activation in vitro and in vivo. However, exacerbated mast cell activation in Sucnr1-/- mice did not contribute to the enhancement of asthma or arthritis and seemed to be due to alterations during mast cell development as augmented mast cell responses could be recapitulated in wt mast cells differentiated in the absence of succinate. CONCLUSIONS: A deficiency in succinate sensing during mast cell development confers these cells with a hyperactive phenotype. Such a phenomenon does not translate into exacerbation of asthma or mast cell-dependent arthritis. On the contrary, the fact that Sucnr1-/- mice developed reduced arthritic disease, using two different in vivo models, indicates that GPR91 antagonists may have therapeutic potential for the treatment of allergic and autoimmune diseases.

CCL18 is expressed in patients with bullous pemphigoid and parallels disease course.

BACKGROUND: The autoimmune skin disease bullous pemphigoid (BP) is characterized by subepidermal blister formation and a strong dermal infiltrate of mononuclear cells and eosinophils as well as a T-helper (Th) 2-dominated cytokine milieu. CCL18 is a chemokine, with unknown receptor counterpart, frequently associated with inflammatory Th2-type responses. OBJECTIVES: The study was performed to investigate an association of CCL18 with BP. METHODS: CCL18 was determined by enzyme-linked immunosorbent assay in serum and blister fluid of patients with BP, pemphigus vulgaris and healthy individuals. In vitro chemotaxis assays were performed to demonstrate migration of peripheral blood mononuclear cells to BP blister fluid. Immunohistology and immunofluorescence staining were used to evaluate CCL18 expression in skin. RESULTS: We have found that the levels of CCL18 in sera from patients with BP are 84% higher than those normally observed in healthy individuals. In addition, blister fluid of patients with BP is extremely rich in CCL18, reaching concentrations which are fivefold and sevenfold higher than those found in the sera of patients with BP and healthy individuals, respectively. Using immunofluorescence techniques we identified Langerhans cells, antigen-presenting cells of the dermis and eosinophils as producers of CCL18 in BP skin. We studied the possibility of using CCL18 expression as a biomarker linked to BP by monitoring the serum levels of CCL18 and the disease course of nine patients with BP over a maximum period of 54 months. In this study, CCL18 levels correlated with the disease course in most of the patients. CONCLUSIONS: Our data implicate CCL18 as a functionally relevant chemokine in BP, mediating recruitment of blood mononuclear cells into the hallmark infiltrated skin lesion. The high correlation of CCL18 expression and BP disease suggests that blood levels of this chemokine can be used as an easy method to monitor disease progression and/or efficacy of therapeutic interventions.

The intensity of T cell receptor engagement determines the cytokine pattern of human allergen-specific T helper cells.

Enhanced production of T helper (Th)2 cytokines by allergen-specific Th cells plays a major role in the induction and maintenance of IgE-mediated allergic disorders. The mechanism that triggers this type of response in atopic individuals is not fully understood. Allergen-specific human Th cell clones produce interleukin (IL)-4 and low or undetectable levels of interferon (IFN)-gamma after stimulation with low concentrations of antigen. However, these Th cell clones are capable of generating significant amounts of IFN-gamma after optimal activation through their T cell receptor (TcR). Allergen-specific Th cell clones isolated from allergic individuals required higher doses of antigen to reach the plateau of proliferation and to generate Th0 cytokine responses than their counterparts isolated from nonallergic subjects. On the other hand, if allergen was replaced by anti-CD3 monoclonal antibody (mAb), both allergic and nonallergic Th cell clones attained the highest level of proliferation and significant IFN-gamma production in response to equivalent concentrations of anti-CD3 mAb. These results indicate that the strength of T cell ligation, which can be modulated by the availability of the TcR ligand, controls the balance of Thl/Th2 cytokines produced by memory Th cells in vitro. In the particular case of bee venom phospholipase A2, it is shown that the expression of allergen-specific surface Ig on antigen-presenting B cells has little influence on antigen uptake and therefore in determining the levels of T cell activation and cytokine production. Alternatively, the affinity of particular major histocompatibility complex class II molecules on antigen-presenting cells for allergen-derived peptides might determine the amount of specific ligand presented to the Th cells and play a decisive role skewing the Th cell cytokine production towards Th1 or Th2 phenotypes. These findings, which are consistent with the changes in cytokine patterns observed following clinical hyposensitization, suggest that polarized human Th2 cell subsets still retain the capacity to modulate their cytokine pattern.

Regulation of IgE and IgG4 responses by allergen specific T-cell clones to bee venom phospholipase A2 in vitro.

An in vitro antibody response to bee venom phospholipase A2 (PLA) from peripheral blood mononuclear cells of bee sting-sensitized individuals was achieved after stimulation with PLA and pokeweed mitogen. This stimulation resulted in a secretion of TH1-associated cytokines and induced PLA-specific and nonspecific IgG4 antibody production but not IgE production. The addition of interleukin-4 (IL-4) to this system decreased the secretion of IgG antibodies, whereas secretion of polyspecific IgE was induced. The mitogen was not required if peripheral blood mononuclear cells were enriched with autologous, PLA-specific, resting T-cell clones in the presence of the antigen. In these experiments the cytokine profile of the particular clone determined the antibody class generated. Low ratios of IL-4 to interferon-gamma, induced by the antigen alone or obtained by neutralizing anti-IL-4 antibodies, enhanced IgG4 antibody formation, whereas IgE levels increased at high ratios of IL-4 to interferon-gamma. These results suggest a complementary regulation of the main isotypes, IgE and IgG4, implicated in allergic and protective hyperimmune responses.

Antigen-specific cytotoxic T cells mediate human fetal pancreas allograft rejection in SCID-hu mice.

Human allograft rejection was studied in SCID mice transplanted with human fetal liver and thymus tissue (SCID-hu mice). These SCID-hu mice have functional, mature T cells with a polyclonal TCR repertoire. Within 12 to 36 wk after construction, SCID-hu mice were transplanted with an HLA-mismatched human fetal pancreas. In contrast to control SCID mice transplanted with pancreas alone, cellular infiltration, induction of HLA-DR on pancreatic epithelial cells, and tissue destruction of the allogenic pancreata were observed in SCID-hu mice. In addition, human insulin was not detected in the serum of SCID-hu mice in which pancreas rejection occurred. The infiltrating cells were mainly human CD3+ T lymphocytes of thymic origin, expressing the CD45RO isoform. T cell lines and CD4+ T cell clones obtained from the rejected tissues proliferated vigorously when stimulated with EBV-transformed B cell lines of pancreas donor origin. Furthermore, the majority of these CD4+ T cell clones displayed strong allospecific cytotoxicity. In addition, CD8+ T cell clones cytotoxic for EBV-transformed B cell lines of pancreas donors were isolated. Blocking experiments with anti-HLA mAbs and panel studies with HLA-matched cell lines showed that these CD4+ and CD8+ T cell clones were specific for the HLA class II and class I molecules, respectively, expressed by the pancreas donor. These data indicate that human T lymphocytes developing in SCID-hu mice are able to mount in vivo responses against allogenic organs, resulting in tissue infiltration and rejection. In addition, they show that both CD4(+)- and CD8(+)-allospecific CTL can be isolated from rejected allogenic pancreata.

Differential induction of T cell cytokine mRNA in Epstein-Barr virus-transformed B cell clones: constitutive and inducible expression of interleukin-4 mRNA.

Clones of human B lymphocytes, obtained after immortalization with Epstein-Barr virus (EBV) of single CD19+ B cells and expansion in the absence of human T lymphocytes, produced mRNA for the T cell cytokines interleukin(IL)-2, IL-4, and interferon (IFN)-gamma. As detected by reverse transcriptase-polymerase chain reaction, IL-2 mRNA was expressed only after stimulation with the combination of phorbol 12-myristate 13-acetate (PMA) plus ionomycin. IL-4 mRNA was constitutively detectable in all (10/10) EBV-transformed B cell clones, and the mRNA for IFN-gamma was constitutively present in half of the clones. In contrast to IL-2 mRNA, the expression of IL-4 and IFN-gamma mRNA could be increased by PMA alone. Most of the clones produced IL-2 bioactivity and immunoreactive protein, but neither IL-4 nor IFN-gamma protein secretion was detected. The intriguing question raised by these results is whether IL-2 secretion could contribute to the immune control of EBV-infected B lymphocytes by cytolytic T cells, and whether normal B lymphocytes can potentially be induced to express certain cytokines including IL-4 in response to the appropriate activation signals.

Bee venom phospholipase A2-specific T cell clones from human allergic and non-allergic individuals: cytokine patterns change in response to the antigen concentration.

Protein antigens with both allergenic and immunoprotective properties represent appropriate molecules to study IgE and IgG regulation. We have established a panel of T cell clones specific to bee venom phospholipase A2 (PLA) from human individuals allergic, hyposensitized or immune (protected) to bee sting. All clones obtained were CD3+, CD4+ and expressed alpha, beta T cell receptor. Depending on the T cell clone, maximal stimulation required 1 to 100 micrograms/ml of PLA, and the addition of interleukin (IL)-2 and/or IL-4 increased their antigen-dependent proliferation. Following antigen stimulation, the clones produced IL-4, interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor. Most clones also produced tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor beta (TNF-beta), and some produced IL-5 and/or IL-2. Both absolute and relative amounts of secreted cytokines depended on the antigen concentration. At low antigen doses, IL-4 was produced but little or not IFN-gamma, whereas at higher PLA concentrations significant amounts of both IL-4 and IFN-gamma were obtained. Thus, these PLA-specific T cell clones could be classified according to the changes in the ratio of IL-4/IFN-gamma production in response to increasing antigen concentrations. Clones derived from allergic and hyposensitized individuals required higher critical amounts of antigen for IFN-gamma induction, and expressed increasing IL-4/IFN-gamma ratios with increasing concentrations of PLA. Modulation of cytokine patterns by the dose of the antigen may be a driving force for IgE or IgG formation resulting in allergy or immunoprotection.

Induction and function of eosinophil intercellular adhesion molecule-1 and HLA-DR.

We have previously established that eosinophils studied ex vivo from the sputum of asthmatics express intercellular adhesion molecule-1 (ICAM-1) and HLA-DR, whereas peripheral blood eosinophils do not express these surface proteins. On incubation of highly purified (greater than 99.5% pure) blood eosinophils from normal subjects with T cell supernatants, eosinophil ICAM-1 was induced in 24 h, whereas HLA-DR was maximally induced within 48 h. Recombinant cytokines that enable eosinophil survival (IL-5, IL-3, and granulocyte macrophage-CSF) were found to be unable to induce ICAM-1 or HLA-DR, even when pooled at concentrations individually required for eosinophil survival. However, synergy between these eosinophil survival factors and TNF (-alpha and -beta) was found mainly responsible for ICAM-1 induction, whereas synergy between IL-3 and IFN-gamma occurred for HLA-DR induction. Culture of eosinophils in the presence of cytokines and cycloheximide prevented expression of ICAM-1 and HLA-DR, showing that de novo eosinophil protein synthesis is occurring. At a functional level we demonstrate that ICAM-1-bearing eosinophils have increased adhesion capacity for autologous T cells. In contrast, HLA-DR-expressing eosinophils mediated Ag-specific proliferation of an autologous HLA-DR-restricted T cell clone that was inhibitable by anti-HLA-DR and anti-ICAM-1 mAb. Since eosinophil-mediated Ag presentation was inhibitable by treatment of eosinophils with glutaraldehyde or chloroquine, this suggests that eosinophils participate in Ag uptake, processing, and presentation and have accessory functions. Thus, through the induction of ICAM-1 and HLA-DR on tissue eosinophils, eosinophils have the capacity to interact with leukocytes and present Ag to T cells.

T cell epitope specificity in human allergic and nonallergic subjects to bee venom phospholipase A2.

Phospholipase A2 (PLA) is a biochemically fully defined glycoprotein, representing the main allergen of bee venom. We have established CD4+ T cell clones specific to PLA from subjects allergic and nonallergic to bee sting. By screening the epitope specificity of these clones with 62 synthetic overlapping dodecapeptides representing the PLA molecule, two immunogenic epitopes, PLA81-92 and PLA113-124, were identified. Additional screening, using longer peptides of up to 18 residues, revealed a third epitope at position PLA 45-62. These epitopes are recognized by specific T cell clones in association with HLA-DP and -DQ molecules, although HLA-DR-associated responses to PLA exist. Primary in vitro proliferation of unfractionated PBMC from bee sting allergic, hyposensitized, or hyperimmune subjects to PLA-derived peptides revealed the same immunogenic sites as found in the T cell clones. Among the different groups of individuals, proliferative responses to the PLA molecule and fragments thereof were generally higher in allergic patients than in nonallergic subjects. Thus, at least three linear epitopes are involved in T cell recognition of PLA, independently whether or not subjects are allergic.