Marco Londei

Prevention of Arthritis by Interleukin 10–producing B Cells

In this study we have shown that activation of arthritogenic splenocytes with antigen and agonistic anti-CD40 gives raise to a B cell population that produce high levels of interleukin (IL)-10 and low levels of interferon (IFN)-γ. Transfer of these B cells into DBA/1-TcR-β-Tg mice, immunized with bovine collagen (CII) emulsified in complete Freunds adjuvant inhibited T helper type 1 differentiation, prevented arthritis development, and was also effective in ameliorating established disease. IL-10 is essential for the regulatory function of this subset of B cells, as the B cells population isolated from IL-10 knockout mice failed to mediate this protective function. Furthermore, B cells isolated from arthritogenic splenocytes treated in vitro with anti–IL-10/anti–IL-10R were unable to protect recipient mice from developing arthritis. Our results suggest a new role of a subset of B cells in controlling T cell differentiation and autoimmune disorders.

Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease

BACKGROUND The adaptive immune system is central to the development of coeliac disease. Adaptive immune responses are, however, controlled by a preceding activation of the innate immune system. We investigated whether gliadin, a protein present in wheat flour, could activate an innate as well as an adaptive immune response in patients with coeliac disease. METHODS Duodenal biopsy samples from 42 patients with untreated coeliac disease, 37 treated patients, and 18 controls, were cultured in vitro for 3 h or 24 h, in the presence of either immunodominant gliadin epitopes (p(alpha)-2 and p(alpha)-9) or a non-immunodominant peptide (p31-43) known to induce small intestine damage in coeliac disease. We also incubated biopsy samples from nine untreated and six treated patients with a non-immunodominant peptide for 3 h, before incubation with immunodominant gliadin epitopes. Different combinations of interleukin-15 or signal transduction inhibitors were added to selected incubations. FINDINGS Only the non-immunodominant peptide induced rapid expression of interleukin-15, CD83, cyclo-oxygenase (COX)-2, and CD25 by CD3- cells (p=0.005 vs medium alone) and enterocyte apoptosis (p<0.0001). Only the non-immunodominant peptide induced p38 MAP kinase activation in CD3- cells. Pre-incubation with the non-immunodominant peptide enabled immunodominant epitopes to induce T-cell activation (p=0.001) and enterocyte apoptosis. Inhibition of interleukin-15 or of p38 MAP kinase controlled such activity. INTERPRETATION A gliadin fragment can activate the innate immune system, affecting the in situ T-cell recognition of dominant gliadin epitopes. Although our findings emphasise the key role of gliadin-specific T cells, they suggest a complex pathogenic situation, and show that inhibition of interleukin-15 or p38 MAP kinase might have the potential to control coeliac disease.

Chronic exposure to tumor necrosis factor (TNF) in vitro impairs the activation of T cells through the T cell receptor/CD3 complex; reversal in vivo by anti-TNF antibodies in patients with rheumatoid arthritis.

Experiments were designed to test the hypothesis that chronic exposure to tumor necrosis factor alpha (TNF) alters the function of activated T lymphocytes. Pretreatment of tetanus toxoid-specific T cell clones with TNF for up to 16 d impaired rechallenge proliferative responses to antigen in a dose- and time-dependent fashion. IL-2 and PHA responses were preserved. Prolonged treatment with TNF impaired production of IL-2, IL-10, IFN gamma, TNF, and lymphotoxin (LT) following stimulation with immobilized OKT3, and resulted in suboptimal expression of the IL-2R alpha chain (Tac) but not CD3, CD4, or HLA-DR antigens, when compared to untreated control cells. By contrast, pretreatment of T cells for prolonged periods in vitro with neutralizing anti-TNF monoclonal antibodies (mAb) enhanced proliferative responses, increased lymphokine production, and upregulated Tac expression following stimulation with OKT3. To determine whether TNF exerts immunosuppressive effects on T cells in vivo, we studied cell-mediated immunity in patients with active rheumatoid arthritis (RA), before and after treatment with a chimeric anti-TNF mAb. Treatment with anti-TNF restored the diminished proliferative responses of PBMC to mitogens and recall antigens towards normal in all patients tested. These data demonstrate that persistent expression of TNF in vitro and in vivo impairs cell-mediated immune responses.

Production of antiendomysial antibodies after in-vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease

BACKGROUND Diagnosis of many immune-mediated diseases is helped by detection of antibodies directed against the pathogenetic (self or foreign) antigen. In coeliac disease (CD), we have a situation in which antiendomysial antibodies (EMA), which are not specific for the pathogenetic antigen, reach a specificity and sensitivity of detection of CD approaching 100%, whereas detection of antibodies against gliadin (AGA), the pathogenetic antigen, is far less specific and sensitive in diagnosis. No direct evidence of a relation between gluten/gliadin challenge and EMA production exists. We tried to establish whether the small intestine of CD patients is the site of EMA production and whether gliadin challenge could induce their release. METHODS Small intestine biopsy samples from treated (23) and untreated (16) CD patients and controls (18) were cultured in vitro for 24-48 h in the presence of gliadin, another alimentary antigen, or medium. EMA and AGA were detected in the supernatants of these organ culture biopsy samples by ELISA and immunofluorescence technique, respectively. FINDINGS No EMA were found in the culture supernatants of biopsy samples of 18 controls, whereas they were detected in the culture supernatants of all 16 untreated CD patients irrespective of gliadin challenge. Conversely, EMA were not detected in supernatants of biopsy samples cultured in medium only from 23 treated CD patients, but were detected in 17 of the 23 biopsy samples challenged with gliadin. INTERPRETATION Our results suggest that a more complex pathogenetic mechanism than normally accepted is involved in CD. Furthermore, our findings raise the possibility that EMA, or the antigen recognised by them, are involved directly in the pathogenesis of CD.

Therapeutic activity of agonistic monoclonal antibodies against CD40 in a chronic autoimmune inflammatory process.

The use of agonistic monoclonal antibody against CD40 has emerged as one the most effective ways to boost immune responses against infectious agents or to fight cancer. Here, we report that the same monoclonal antibodies against CD40 (FGK45 and 3/23) previously used to elicit protective immune responses treated the autoimmune inflammatory process of chronic collagen-induced arthritis in DBA/1–TCR-β transgenic mice, as well as collagen-induced arthritis in DBA/1 mice, both animal models of rheumatoid arthritis. This study indicates that agonistic monoclonal antibody against CD40 can potentially be used to treat chronic autoimmune inflammatory processes.

Immunodominant epitopes of glutamic acid decarboxylase 65 and 67 in insulin-dependent diabetes mellitus

To find the dominant epitopes of a major autoantigen in insulin-dependent diabetes mellitus (IDDM), glutamic acid decarboxylase (GAD), we studied the reactivity of peripheral blood T lymphocytes with peptides covering both major isoforms. A significant response to GAD 65 or GAD 67 peptides was detected in 13 of 15 IDDM patients and in 9 of 10 normal controls. Controls most frequently recognised the central region of GAD 65 (residues 161-243). IDDM patients preferentially recognised residues 473-555 (p < 0.03). T-cell responses to GAD 67 peptides were similar in IDDM patients and controls. T lymphocytes from IDDM patients recognise a distinct dominant epitope of GAD, which may be an important target for the disease process.

CAMPYLOBACTER PYLORI IN ABATTOIR WORKERS: IS IT A ZOONOSIS?

Sera from 98 abattoir workers were tested for IgG to Campylobacter pylori, C jejuni, and klebsiella. Clerical workers had significantly lower C pylori and C jejuni IgG titres than any of the groups in direct contact with freshly cut animal parts. No difference was found for antibodies to klebsiella. 28 non-clerical workers with high-titre C pylori IgG consented to upper gastrointestinal endoscopy. C pylori associated gastritis was found in all 28, and four weeks of colloidal bismuth subcitrate (240 mg twice daily) was prescribed. On repeat testing at three months all showed a decrease in IgG titres to C pylori but not to C jejuni, whereas 18 untreated non-endoscoped workers showed no change. These findings raise the possibility that C pylori infection is a zoonosis.

Evidence of chronic inflammation in morphologically normal small intestine of cystic fibrosis patients.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator gene and characteristically leads to prominent lung and pancreatic malfunctions. Although an inflammatory reaction is normally observed in the CF airways, no studies have been performed to establish whether a chronic inflammatory response is also present in the CF intestine. We have investigated whether immunologic alterations and signs of inflammation are observed in CF small intestine. Fourteen CF, 20 negative, and four disease controls underwent duodenal endoscopy for diagnostic purposes. Two CF patients were rebiopsied, one after 3 mo of an elemental diet and the other after 2 wk of pancreatic enzyme withdrawal. In three CF and 10 controls, in vitro small intestine organ cultures were also performed. Expression of ICAM-1, IL-2 receptor, IL-2, IFN-γ, CD80, and transferrin receptor was studied by immunohistochemistry before and after in vitro organ culture. In CF small intestine, an increased number of lamina propria mononuclear cells express ICAM-1 [mean 114 (SD 82.8), p < 0.001 versus controls], CD25 [20.2 (18.7), p < 0.01], IL-2 [23.6 (13.7), p < 0.05], and IFN-γ [19 (15.9), p < 0.05], whereas villus enterocytes highly express transferrin receptor. Reduced expression of immunologic markers was observed after 24 h of in vitro culture in all three CF patients as well as in the patient kept on elemental diet for 3 mo. These results indicate that chronic inflammation is observed in CF duodenum and suggest that the perturbation of local mucosal immune response may contribute to the overall clinical picture in CF patients.

Immune reactivity to glutamic acid decarboxylase 65 in stiff-man syndrome and type 1 diabetes mellitus

BACKGROUND The immune response to an isoform of glutamic acid decarboxylase (GAD), GAD65, is associated with two clinically distinct diseases, stiff-man syndrome (SMS) and type 1 (insulin-dependent) diabetes mellitus. We sought to identify differences in the cellular and humoral immune responses to GAD in these two diseases. METHODS We compared T-cell responses in 14 SMS patients with axial disease and 17 patients with type 1 diabetes. FINDINGS Peripheral blood T cells of eight SMS patients recognised different immunodominant epitopes of GAD65 compared with T cells from 17 patients with type 1 diabetes. GAD regions 81-171 and 313-403 induced a dominant T-cell response in six of eight patients with SMS but in only one of 17 patients with type 1 diabetes (p=0.001). No SMS patients responded dominantly to GAD fragments 161-243 and 473-555 compared with ten patients with type 1 diabetes (p=0.008). GAD antibodies were detected in 11 of 14 SMS patients (seven with diabetes) and 11 of 17 patients with type 1 diabetes; IgG1 was dominant in both groups. SMS patients, however, were more likely than patients with diabetes to have isotypes other than IgG1 (p=0.03), in particular, IgG4 or IgE isotypes, which were not detected in patients with type 1 diabetes (p=0.012). INTERPRETATION Our findings indicate differences between patients with SMS and type 1 diabetes in cellular (epitope recognition) and humoral (isotype pattern) responses to GAD65. Thus the same autoantigen can elicit distinct immune responses in patients with SMS, even when associated with diabetes, compared with patients with type 1 diabetes.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases define the migratory characteristics of human monocyte-derived dendritic cells.

Dendritic cells (DCs) have an essential role in the initiation of immune responses as they deliver antigen/epitope and the appropriate signals to activate naïve T cells and thus start an immune response. In order to fulfil their function, DCs have to patrol different part of the body, thus migrating through the extracellular matrix to sample the local ‘antigenic’ environment. In the present study, we have investigated which enzymes might be involved in this process using the Matrigel trans‐well migration assay, an in vitro model of extracellular matrix migration. In this assay we analysed the migratory ability of interleukin‐4 (IL‐4)/granulocyte macrophage–colony‐stimulating factor (GM‐CSF)‐derived immature DCs as well as mature DCs, induced by tumour necrosis factor‐α (TNF‐α) and modified vaccinia virus Ankara (MVA). The ‘mature’ DCs showed an increased migration through Matrigel, which was significantly inhibited by inhibitors of matrix metalloproteinases (MMP). We also observed that the dominant MMP involved in this process was MMP‐9, and a concomitant decrease of the endogenous tissue inhibitors of metalloproteinases (TIMP)‐1 and TIMP‐2 was also observed. Collectively these data suggest that the balance between MMP/TIMP determines the net migratory capacity of human DCs. Surprisingly, TIMP‐3 was significantly increased in mature DC. Our data thus indicate that MMP and TIMP play a role in the migratory ability of human DCs. Our results also suggest that TIMP‐3 expression might represent a new marker of maturation of human DCs.