Dissecting the contribution of innate and antigen-specific pathways to the breach of self-tolerance observed in a murine model of arthritis.

BACKGROUND: The relative roles of innate immunity and antigen-specific T cells in rheumatoid arthritis remain controversial. Previous studies demonstrated that T-helper type 1 cells of irrelevant antigen specificity (ovalbumin) induced a transient arthritis in BALB/c mice, which recapitulates many of the pre-articular and articular features of human disease and is associated with the emergence of autoreactive T and B-cell responses to joint-specific antigens. However, the mechanisms underlying this phenomenon were unclear. OBJECTIVES: The aim of this study was to dissect the relative contribution of innate and heterologous antigen-specific pathways to the breach of self-tolerance and pathology observed in this model and how this may result from modified T and B-cell interactions. METHODS: To address this issue, experimental arthritis was elicited either by a non-specific inflammatory stimulus alone, by activation of T cells of an irrelevant specificity or a combination of both. RESULTS: The non-specific inflammatory response generated by lipopolysaccharide led to articular inflammation and cartilage erosion, but did not break tolerance to joint-specific antigens. In contrast, local activation of T cells of an irrelevant specificity produced a similar pathological picture but, in addition, induced T-cell responses to unrelated joint-specific antigens with associated activation of autoreactive B cells. These effects could be further potentiated by the addition of lipopolysaccharide. CONCLUSION: These data demonstrate that non-specific inflammation alone is insufficient to breach self-tolerance. In contrast, T cells of an irrelevant specificity, when triggered locally in an antigen-specific manner, can breach self-tolerance leading to arthritis and autoantibody production, which can then be amplified in a non-specific manner.

An investigation of the impact of the location and timing of antigen-specific T cell division on airways inflammation.

It is widely accepted that allergic asthma is orchestrated by T helper type 2 lymphocytes specific for inhaled allergen. However, it remains unclear where and when T cell activation and division occurs after allergen challenge, and whether these factors have a significant impact on airways inflammation. We therefore employed a CD4-T cell receptor transgenic adoptive transfer model in conjunction with laser scanning cytometry to characterize the location and timing of T cell division in asthma in vivo. Thus, for the first time we have directly assessed the division of antigen-specific T cells in situ. We found that accumulation of divided antigen-specific T cells in the lungs appeared to occur in two waves. The first very early wave was apparent before dividing T cells could be detected in the lymph node (LN) and coincided with neutrophil influx. The second wave of divided T cells accumulating in lung followed the appearance of these cells in LN and coincided with peak eosinophilia. Furthermore, accumulation of antigen-specific T cells in the draining LN and lung tissue, together with accompanying pathology, was reduced by intervention with the sphingosine 1-phosphate receptor agonist FTY720 2 days after challenge. These findings provide greater insight into the timing and location of antigen-specific T cell division in airways inflammation, indicate that distinct phases and locations of antigen presentation may be associated with different aspects of pathology and that therapeutics targeted against leukocyte migration may be useful in these conditions.

Tumour necrosis factor-alpha blockade suppresses murine allergic airways inflammation.

Asthma is a heterogeneous disease that has been increasing in incidence throughout western societies and cytokines, including proinflammatory tumour necrosis factor alpha (TNF-alpha), have been implicated in the pathogenesis of asthma. Anti-TNF-alpha therapies have been established successfully in the clinic for diseases such as rheumatoid arthritis and Crohn's disease. TNF-alpha-blocking strategies are now being trialled in asthma; however, their mode of action is poorly understood. Based on the observation that TNF-alpha induces lymph node hypertrophy we have attempted to investigate this as a mechanism of action of TNF-alpha in airway inflammation by employing two models of murine airway inflammation, that we have termed short and long models, representing severe and mild/moderate asthma, respectively. The models differ by their immunization schedules. In the short model, characterized by eosinophilic and neutrophilic airway inflammation the effect of TNF-alpha blockade was a reduction in draining lymph node (DLN) hypertrophy, eosinophilia, interleukin (IL)-5 production and immunoglobulin E (IgE) production. In the long model, characterized by eosinophilic inflammation, TNF-alpha blockade produced a reduction in DLN hypertrophy and IL-5 production but had limited effects on eosinophilia and IgE production. These results indicate that anti-TNF-alpha can suppress DLN hypertrophy and decrease airway inflammation. Further investigations showed that anti-TNF-alpha-induced inhibition of DLN hypertrophy cannot be explained by preventing l-selectin-dependent capture of lymphocytes into the DLN. Given that overall TNF blockade was able to suppress the short model (severe) more effectively than the long model (mild/moderate), the results suggest that TNF-alpha blocking therapies may be more effective in the treatment of severe asthma.

Peripheral blood based T cell-containing and T cell-depleted culture systems for human IgE synthesis: the role of T cells.

BACKGROUND: Comparable T cell-containing and T cell-depleted culture systems for human IgE synthesis are currently not available. OBJECTIVE: This has prompted us to develop peripheral blood mononuclear cell (PBMC) based culture systems for human IgE synthesis in the presence and absence of T cells. METHODS: In this paper we describe simplified conditions for in vitro synthesis of high levels of IgE by human peripheral blood B cells, both in T cell-containing cultures and in anti-CD40 stimulated T cell-depleted cultures. RESULTS: T cell-depleted cultures released approximately 20 times more IgE [range 410-2220 ng/mliter (mean 1270 ng/mliter); based on six experiments using cells from three donors] than did T cell-containing cultures [range 23-105 ng/mliter (mean 58 ng/mliter); based on 15 experiments using cells from three donors]. Reconstitution experiments were performed to investigate the role of T cells on IgE synthesis. Adding T cells back to the anti-CD40 stimulated T cell-depleted cultures resulted in a dose-dependent inhibition of IgE production. In the absence of anti-CD40 low numbers of T cells stimulated, while high numbers suppressed, IgE production: the optimal ratio of T cells to non-T cells for maximal IgE production was found to be 1:1. At this ratio, irradiated (non-replicating) T cells supported a much greater IgE synthesis than did non-irradiated T cells. CONCLUSION: The development of these systems provides directly comparable T cell-containing and T cell-depleted cultures for human IgE synthesis from peripheral blood, allowing further study of the role of T cells in IgE regulation. These systems will also be of use for determining whether potential modulators of IgE synthesis act on the T cells or on other cell types.

Potentiation of in vitro synthesis of human IgE by cyclosporin A (CsA).

In this study, we investigated the modulatory effects of CsA on in vitro synthesis of IgE, IgG1 and IgG4 by human peripheral blood mononuclear cells (PBMC). In contrast to its known immunosuppressive effect, we have demonstrated that a low dose of CsA (10(-7) M, 120 ng/ml) potentiated IgE production by up to 40-fold (i.e. from 33 +/- 4.5 to 1346 +/- 290 ng/ml). This potentiation was specific for IgE since no such effect was demonstrable with IgG1 and IgG4. Potentiation of IgE synthesis by CsA in the PBMC cultures was partly due to CsA acting on T cells, as demonstrated by the addition of CsA-treated T cells to T cell-depleted cultures. However, potentiation was also demonstrable in a T cell-depleted, anti-CD40-stimulated culture (four-fold increase from 400 +/- 48 to 1606 +/- 127 ng/ml). Our data therefore suggest that there are at least two mechanisms for CsA-induced potentiation of IgE synthesis, one T cell-dependent and the other T cell-independent. The clinical implications of these findings are discussed with regard to the use of CsA in the treatment of Th2-mediated diseases.

The effect of cyclosporin A, FK506, and rapamycin on the murine chronic graft-versus-host response--an in vivo model of Th2-like activity.

We have evaluated the effects of three potent immunosuppressive agents: cyclosporin A, FK506, and rapamycin, on a murine chronic graft-versus-host response (chronic GVHR). The chronic GVHR has previously been described to be a Th2-like response, and is characterized by a marked splenomegaly and hyper-IgE production in the early stages of the response. The effects of the immunosuppressive agents on both splenomegaly and hyper-IgE were measured 3 weeks after the induction of the chronic GVHR. Rapamycin was found to inhibit both splenomegaly and the hyper-IgE response in a dose-dependent manner. Unexpectedly cyclosporin A and FK506 were found to potentiate markedly both the splenomegaly and hyper-IgE response at low doses before exhibiting an inhibitory effect at higher doses. We propose the differences of activity seen with rapamycin compared with cyclosporin A and FK506 may be explained by their different mechanisms of action, and also by the selectivity of low dose cyclosporin A and FK506 for Th1-like lymphocytes. The implications of these observations are discussed in relation to the use of these immunosuppressives for the treatment of Th2-like diseases.

The effects of CP 17193, an immunosuppressive pyrazaloquinoline, on the development of spontaneous lupus disease in NZBW F1 hybrid mice.

The effects of the immunosuppressive agent CP 17193 on the development of spontaneous lupus disease in female NZBW F1 hybrid mice were investigated. Long term dosing with CP 17193 markedly delayed the onset of mortality but did not extend the long term survival of the mice. CP 17193 significantly inhibited immune complex deposition in the glomeruli of 30- and 35-week-old mice and also reduced the levels of proteinuria in the 35-week-old mice. There was a slight reduction in the levels of circulating antinuclear antibody to ds DNA in CP 17193-treated mice but this was not statistically significant. Studies on immune cell function of 35-week-old mice dosed with CP 17193 showed significant reduction in the total numbers of spontaneous polyclonal antibody producing cells. Analysis of the results revealed these effects to result from a marked reduction in total spleen cell numbers in CP 17193-treated mice. When results were expressed as activity per cell unit the differences between drug-treated and control mice were small. Spleen cells from mice given a shorter dosing schedule of 7 weeks with CP 17193 showed an augmentation of IL-2 production and responsiveness. These results show CP 17193 having interesting selective immunomodulating activity on the immunopathogenesis of spontaneous murine lupus disease. Furthermore, compounds with this profile of activity may have a potential role in the treatment of some autoimmune diseases.

Comparison of the effects of FK-506, cyclosporin A and rapamycin on IL-2 production.

The immunosuppressive compounds FK-506, cyclosporin A (CsA) and rapamycin inhibit both the human and mouse mixed lymphocyte reactions (MLR) with IC50s of 2-5 x 10(-10) M for FK-506 and rapamycin and 10(-8) M for CsA. FK-506 and CsA were also potent inhibitors of A23187/PMA-stimulated IL-2 production by Jurkat and HuT-78 cells but had no effect on the response of mouse CTLL cells to IL-2. IC50 values for inhibition of IL-2 production closely matched those for inhibition of the MLR and both drugs were active only during the first 4-6 hr following stimulation. In contrast, rapamycin was a poor inhibitor of IL-2 production, although it inhibited cellular responses to IL-2. The IC50 values for these two activities indicated that neither alone accounted for rapamycin inhibition of the MLR. FK-506 and CsA affected IL-2 gene transcription in Jurkat cells by the same mechanism. Both inhibited the appearance of the transcription factor, NFAT, whereas rapamycin did not. The appearance of another transcription factor, NFK beta, was unaffected by all three drugs. The effects of FK-506 and CsA on IL-2 gene expression, therefore, are similar even though the two drugs act through distinct cytosolic receptors.

Cerebrospinal fluid interleukin 1 like activity during chronic relapsing experimental allergic encephalomyelitis.

Cerebrospinal fluid (CSF) and plasma samples were taken from strain 13 guinea pigs in various stages of chronic relapsing experimental allergic encephalomyelitis (CREAE). The samples were assayed for interleukin 1 (IL-1) in the C3H/Hej mouse thymocyte assay. After removal of inhibitors, IL-1 was detectable in low amounts in plasma (5 U/ml) throughout the course of the disease but was raised in the acute phase (12 U/ml). CSF IL-1 was, however, only present in low amounts (6 U/ml) during the acute phase but was elevated (18 U/ml) during the chronic stages of CREAE. During the relapse phase levels of IL-1 correlated with the total leucocyte count in the CSF. On gel filtration of CSF, IL-1 activity eluted at approximately 15 kD and could not be attributed to leakage of plasma IL-1 during CSF puncture or IL-2 activity.

Chronic relapsing experimental allergic encephalomyelitis. Inhibition of lymphocyte mitogenesis by disease-related serum factors.

Peripheral blood mononuclear cells (PBMC) from strain 13 guinea pigs at various stages of chronic relapsing experimental allergic encephalomyelitis (CREAE) showed significantly reduced reactivity to the polyclonal T cell mitogens, phytohaemagglutinin and concanavalin A. Serum taken at the same time revealed an increase in inhibitors of lymphocyte mitogenesis. The factors identified appeared to inhibit the initial stages of lymphocyte proliferation: they were not cytotoxic for lymphocytes nor were they complement-dependent.

The activity of an anti-allergic compound, proxicromil, on models of immunity and inflammation.

A tricyclic chromone, proxicromil (sodium 6,7,8,9-tetrahydro-5-hydroxy-4-oxo-10-propyl-naphtho (2,3-b) pyran-2-carboxylate), has been tested for activity against certain immunological and inflammatory reactions. When given parenterally it suppressed the development of delayed hypersensitivity reactions in sensitized mice and guinea-pigs but did not affect the rejection of skin allografts in mice. The compound had no activity against certain in vitro correlates of delayed hypersensitivity reactions (lymphocyte transformation and lymphokine activity), but did have an inhibitory effect on lymphokine (MIF) productions at 10(-4) M but not at 10(-5) M. Proxicromil was also found to be active in non-immunologically mediated models of inflammation and in models having an immunological component which are known to be sensitive to non-steroidal anti-inflammatory drugs (adjuvant arthritis, reversed passive Arthus reaction). The activity of this compound was enhanced when administered in arachis oil when compared to its activity in saline. Proxicromil has not direct activity on the development of immune responsiveness but appear to suppress the expression of delayed hypersensitivity and immune complex mediated hypersensitivity reactions by virtue and its anti-inflammatory properties. This activity is not associated with inhibition of cyclo-oxygenase.

Synergistic effect of cortisol and prostaglandin E2 on the PHA response. Relation to immunosuppression induced by trauma.

Surgical and thermal trauma in man are followed by depressed immunological responses in vivo and reduced lymphocyte reactivity in vitro. The possibility that these are related to trauma-induced rises in tissue levels of cortisol and prostaglandins was examined by studying the effect of a wide range of concentrations of cortisol and prostaglandin E2 (PGE2), separately and together on the phytohaemagglutinin (PHA) response of human peripheral blood lymphocytes. These effects were plotted on two-dimensional dose:effect graphs; the shapes of the curves connecting combinations of equal effect (isoboles) showed that these agents acted with marked synergy in suppressing the response, provided they were present while the response was taking place. Synergy was also shown by using a simple equation relating the concentrations of the agents producing a given effect when used in combination to the concentrations needed to produce the same effect when used separately. Cortisol at concentrations reached in the peripheral blood after trauma in man (1-4 X 10(-6)M) and PGE2 at concentrations to be expected in traumatized tissues (up to 4 X 10(-7)M) each suppressed the response only slightly. The former reduced the response to 0-7 of controls and the latter 0-5 (means of seven subjects). When both were present together at these concentrations, the response was markedly depressed (mean 0-06, range 0-02--0-13 of controls). However, when lymphocytes were incubated at 37 degrees C with cortisol and PGE2 for 20 hr and then washed before exposure to PHA, the response was not inhibited, even by substantially higher concentrations than the above, and was usually moderately enhanced. Therefore, these in vitro experiments do not explain the depressed PHA response observed in peripheral blood lymphocytes after trauma. It is possible, however, that raised cortisol and prostaglandin levels depress the reactivity of lymphocytes while they remain in the traumatized region and its lymph drainage area.

The growth of human tumours in immunosuppressed mice and their response to chemotherapy.

One hundred and sixteen human tumours were transplanted to thymectomized, irradiated, antilymphocyte serum-treated mice. In 12 cases the recipient mice died rapidly, presumably from infection. With the remaining 104 tumours, three-quarters grew to a varying extent, retaining the characteristic histological features of the primary tumours. Implant nodules varied widely in composition, from solid tumour and stroma to dense fibrous tissue without recognizable tumour cells. There was no relation between degree of malignancy and ability to grow, and also some benign tumours grew.In 44 cases, mice were treated with the drug or drugs most likely to be used in the patients and the effects on the implants were assessed histologically. Two tumours were largely destroyed and one showed marked metaphase arrest. Three other tumours showed lesser changes that were attributable to the drug but were of equivocal significance.There appeared to be differences in drug sensitivity between structurally different clones of the same tumour, and some tumours treated with two alkylating agents were damaged by one and not the other, suggesting that this model may have substantial discriminatory power. Assays such as this should not be used to guide treatment of the patient without prior validation. The practical and ethical difficulties of validation by clinical trial may be insurmountable, and an alternative approach to validation is proposed which does not raise these difficulties.