Background: Concentrations of proinflammatory cytokines are increased in the intestinal mucosa of patients with active Crohn's disease (CD). In a prospective study we investigated whether cytokines ...can predict long‐term remission (>6 months) in patients with steroid‐refractory CD receiving treatment with infliximab or cyclophosphamide, followed by azathioprine or methotrexate.
Methods: Cytokine transcripts were quantified using real‐time polymerase chain reaction (PCR) in mucosal biopsies from 19 patients with active, steroid‐refractory CD before and 8 weeks after initiation of therapy. Patients were treated with cyclophosphamide (monthly treatment of 750 mg cyclophosphamide intravenously) or infliximab (5 mg/kg body weight) and were followed until relapse of the disease. Statistical analysis was performed to identify predictive factors to discriminate between patients with or without long‐term remission.
Results: Seventeen out of 19 patients achieved remission of the disease, two patients were nonresponders, while six out of 17 patients exhibited an early recurrence. Pretreatment TNF‐α, IL‐18, MRP‐14, and IL‐8 transcripts were significantly correlated with long‐term remission. While several cytokines, most importantly MMP‐1, determined after 8 weeks were able to predict patients achieving long‐term remission, only a decrease of TNF‐α levels after 8 weeks was predictive. Overall, statistical analysis identified lower pretreatment TNF‐α levels as the strongest predictor of long‐term remission among baseline variables.
Conclusions: Quantification of mucosal TNF‐α transcripts prior to therapy allows identification of patients achieving long‐term remission upon immunosuppression with infliximab or cyclophosphamide. Real‐time PCR might have considerable potential in the analysis of disease activity and subsequent clinical management of patients with immunosuppressive therapies.
(Inflamm Bowel Dis 2007;13:65–70)
Summary
Recently, we established a pharmacodynamic assay to monitor immunosuppressive effectiveness of cyclosporine A (CsA) in patients on standard CsA regimen. The aim of the present study was to ...extend this correlation to reduced CsA regimen and to compare pharmacodynamic and kinetic parameters to allow prediction of rejections and infections. In 53 heart allograft recipients, nuclear factor of activated T cells (NFAT)‐regulated gene expression was quantified at trough (C0) and 2‐h post‐CsA dose (C2). Gene expression at C2 was calculated relative to C0 (residual gene expression, RGE) or relative to a healthy reference group (absolute gene expression, AGE). RGE correlated with CsA C2‐levels in bimodal fashion: above 575 ng/ml correlation was seen with flat regression gradient. Below 575 ng/ml, correlation was excellent with markedly steeper gradient. At C0 in the low‐C2 group (<575 ng/ml), AGE remained unchanged, whereas in the high‐C2 group (>575 ng/ml) AGE was markedly reduced. In both groups, AGE at C2 was strongly inhibited. In patients contracting infection during follow‐up, RGE was lower than in those without infections independent of CsA levels. CsA‐monitoring by quantitation of NFAT‐regulated gene expression is feasible with standard and reduced CsA regimens. It correlates better with the incidence of infections than measurement of CsA concentrations and might help in avoiding over‐immunosuppression.
Treatment with the anti-tumour necrosis factor alpha chimeric monoclonal antibody infliximab is highly effective in the treatment of refractory and fistulising Crohn's disease. Infliximab has been ...tolerated well, with minimal and short-lived adverse effects. The likelihood of severe reactions to infliximab, such as acute and delayed hypersensitivity infusion reactions, is small; nevertheless, if they do occur, they are life-threatening. We report a case of an anaphylaxis-like reaction in a 22-year-old female with long-standing Crohn's disease. The patient was treated successfully with adalimumab, a fully human anti-tumour necrosis factor alpha monoclonal antibody. Follow-up demonstrated mucosal healing and normalisation of elevated pro-inflammatory cytokine transcripts.
Background & Aims: T-cell receptor reactivity of intestinal lamina propria T cells (LP-T) critically depends on the capacity of local accessory cells to secrete cysteine. For T cells, cysteine is the ...limiting precursor for glutathione synthesis, a prerequisite for antigen-dependent proliferation. We aimed to determine the role of the redoxactive microenvironment for hyporeactivity of LP-T in normal human gut vs hyperreactivity of LP-T in inflammatory bowel disease. Methods: Parameters relevant to cysteine production, determined as acid-soluble thiol, by intestinal lamina propria macrophages (LP-MO) vs peripheral blood monocytes were investigated (L-35 Scystine uptake via system xc− , messenger RNA, and protein expression of the cystine transporter subunit xCT). Glutathione levels in LP-T and peripheral blood T cells were analyzed both spectrophotometrically and by immunofluorescent staining in situ and in vitro. Results: LP-MO from normal gut, unlike peripheral blood monocytes, are unable to take up cystine, which is due to a deficient expression of the transporter xCT in situ and in vitro. As a consequence, LP-MO do not secrete cysteine. The glutathione content in LP-T from normal gut is <50% of that in autologous peripheral blood T cells. In contrast, in inflammatory bowel disease, CD14+ CD68+ LP-MO express xCT and secrete substantial amounts of cysteine upon stimulation, which results in high glutathione levels and full T-cell receptor reactivity in LP-T. Conclusions: The antioxidative microenvironment of LP-T in inflammatory bowel disease and the prooxidative microenvironment in normal gut explain the differential T-cell receptor reactivities.
Thioredoxin (TRX) is a ubiquitous oxidoreductase with strong co‐cytokine, chemoattractant and anti‐apoptotic activities. TRX expression was found to be particularly elevated in the intestinal mucosa, ...where its physiologic function is entirely unknown. Here, we demonstrate a high level of TRX expression in lamina propria T cells (LP‐T) as opposed to autologous peripheral blood T lymphocytes (PB‐T). Addition of recombinant human TRX (rhTRX) to PB‐T enhances TRX gene expression. This autoregulation involves the calcineurin signaling pathway, as rhTRX antagonizes the cyclosporine A (CsA)‐ and tacrolimus‐mediated suppression of TRX gene expression. Similarly, rhTRX reverses the suppression of IL‐2 mRNA production by CsA and enhances cytokine production preferentially in prestimulated cells. The differential TRX expression in LP‐T versus PB‐T may thus contribute to the high‐level, CsA‐resistant IL‐2 production characteristic for CD2‐stimulated LP‐T. Inversely, inactivation of TRX in LP‐T through inhibition of TRX reductase abolishes cytokine gene expression. TRX may play a key role in the specialized intestinal microenvironment in amplifying immediate immune responses of LP‐T whenever appropriate costimulation of LP‐T is provided.
Signal transduction processes in T-cells and other cell types alter the phosphorylation state of cofilin, an actin-binding
phosphoprotein. Whether reversible phosphorylation is responsible for the ...regulation of the functional activities of cofilin
is not clear at present. Here we have identified the phosphoacceptor site of cofilin and analyzed the role of cofilin phosphorylation
with respect to its subcellular localization. Site-directed mutagenesis studies show that phosphorylation occurs exclusively
on Ser-3. Expression of non-phosphorylatable mutant cofilin proteins in NIH3T3 cells and determination of their subcellular
localization by confocal laser scanning microscopy reveal that non-phosphorylated cofilin accumulates within nuclei. This
analysis shows that the subcellular localization of cofilin depends on the phosphorylation state of Ser-3.
Oxidative compounds that are physiologically generated in vivo can induce natural defense mechanisms to enhance the elimination of pathogens and to limit inflammatory tissue damage in the course of ...inflammation. Here, we have investigated WF10, a chlorite-based non-toxic compound for its functional activities on human PBMC in vitro. WF10 exerts potent immune-modulatory effects through generating endogenous oxidative compounds such as taurine chloramine. Proliferation and IL-2 production of anti-CD3 stimulated PBMC were inhibited by WF10, as was the nuclear translocation of the transcription factor NFATc. In PBMC and monocytes, however, WF10 induced pro-inflammatory cytokines like IL-1β, IL-8, and TNF-α. In the monocytic cell line THP-1, the activation of the transcription factors AP-1 and NFκB by WF10 was demonstrated. Inhibition of NFAT regulated genes in activated lymphocytes in concert with the induction of several myeloid cell associated pro-inflammatory genes in monocytes represents a novel mechanism of immune modulation.
At least two membrane receptors have been defined through which human T lymphocytes can be induced to proliferate and differentiate, namely the CD3-Ti antigen receptor complex and the CD2 molecule. ...Monoclonal antibodies directed at either CD2 or CD3 induce intracellular second messenger production and subsequent protein phosphorylation. On most human non-B lymphocytes, CD3-Ti and CD2 are coexpressed and seem to be functionally interrelated. But there are minor subpopulations in which these receptor systems can transduce signals despite a mutually exclusive expression, indicating that CD3-Ti and CD2 can act independently of each other. This view is supported by the finding that most monoclonal antibodies directed at the CD45 molecules are strongly co-mitogenic with CD2 but not CD3 monoclonal antibodies. As the intracytoplasmic domains of CD45 have tyrosine phosphatase activity these functional effects could be explained by a physical association between CD2 and CD45. Using chemical crosslinking techniques, we now show that CD45 is linked to CD2 on the surface of human T lymphocytes.
Human CD8+ lymphocyte subpopulations were analyzed for their expression of CD8 alpha and CD8 beta subunits. Investigations with uncloned peripheral blood lymphocytes as well as cloned human natural ...killer and T cell subpopulations demonstrate that CD3- natural killer cells, T cell receptor gamma/delta, and CD4+CD8+ T cell clones express exclusively CD8 alpha gene products. Structural analysis of CD8 molecules demonstrates that CD8 alpha+/beta- T lymphocytes surface express 75-kDa CD8 alpha/alpha homodimers whereas CD8 alpha/beta lymphocytes express concomittantly two CD8 isoforms of different molecular masses (67 kDa and 75 kDa, respectively). Peptide mapping of these latter two isoforms suggests that CD8 is expressed as alpha/alpha homodimers and alpha/beta heterodimers on CD8 alpha/beta+ cells. Importantly, we found that the two CD8 isoforms behave functionally different. Thus, in contrast to CD8 alpha/beta+/CD8 alpha/alpha+ T lymphocytes, cytolytic activity of CD8 alpha/beta-/CD8 alpha/alpha+ T cell clones was not inhibited by anti-CD8 monoclonal antibodies and the latter were not induced to proliferate following CD3/CD8 cross-linking.
A characteristic of lamina propria lymphocytes (LPL) is their low proliferative response to stimuli of the CD3 pathway. β1 integrins were expressed on LPL; however, their functionis unknown. ...Therefore, we determined whether β1 integrins contribute to T cell responses by providing costimulatory signals. Integrins on CD4+ LPL of controls and patients with inflammatory bowel disease were characterized by flow cytometry. Cells were stimulated by anti‐CD3 or anti‐CD2 antibodies either alone or in combination with a stimulatory β1 integrinantibody (12G10). Proliferation and apoptosis were measured by 3Hthymidine pulsing or flow cytometry. Cytokine mRNA and apoptosis‐related transcripts were quantified by reverse transcriptase‐PCR. We demonstrated that β1 integrin costimulation restored CD3‐induced proliferation of CD4+ LPL and reduced activation‐induced apoptosis. Activation of β1 integrins by addition of 12G10 antibody to CD3‐stimulated cells restored their capacity to express proinflammatory cytokine transcripts. Further, expression of the activated form of β1 integrins was significantly elevated on LPL from inflamed mucosa. These studies demonstrate that β1 integrin costimulation modulates the response of LPL after TCR stimulation. An increased expression of activated β1 integrins on LPL in intestinal inflammation may abolish their unresponsiveness to antigens and perpetuate the inflammatory process.
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