Ragnar Mattsson

Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species

Reduced capacity to produce ROS increases the severity of T cell-dependent arthritis in both mice and rats with polymorphisms in neutrophil cytosolic factor 1 (Ncf1) (p47phox). Since T cells cannot exert oxidative burst, we hypothesized that T cell responsiveness is downregulated by ROS produced by APCs. Macrophages have the highest burst capacity among APCs, so to study the effect of macrophage ROS on T cell activation, we developed transgenic mice expressing functional Ncf1 restricted to macrophages. Macrophage-restricted expression of functional Ncf1 restored arthritis resistance to the level of that of wild-type mice in a collagen-induced arthritis model but not in a T cell-independent anti-collagen antibody-induced arthritis model. T cell activation was downregulated and skewed toward Th2 in transgenic mice. In vitro, IL-2 production and T cell proliferation were suppressed by macrophage ROS, irrespective of T cell origin. IFN-gamma production, however, was independent of macrophage ROS but dependent on T cell origin. These effects were antigen dependent but not restricted to collagen type II. In conclusion, macrophage-derived ROS play a role in T cell selection, maturation, and differentiation, and also a suppressive role in T cell activation, and thereby mediate protection against autoimmune diseases like arthritis.

IFN-beta Gene Deletion Leads to Augmented and Chronic Demyelinating Experimental Autoimmune Encephalomyelitis.

Since the basic mechanisms behind the beneficial effects of IFN-β in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN-β gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN-β knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells to proliferate or produce IFN-γ in response to recall Ag. Consequently, we addressed the effect of IFN-β on encephalitogenic T cell development and the disease initiation phase by passive transfer of autoreactive T cells from KO or wt littermates to both groups of mice. Interestingly, IFN-β KO mice acquired a higher incidence and augmented EAE regardless of the source of T cells. This shows that the anti-inflammatory effect of endogenous IFN-β is predominantly exerted on the effector phase of the disease. Histopathological investigations of CNS in the effector phase revealed an extensive microglia activation and TNF-α production in IFN-β KO mice; this was virtually absent in wt littermates. This coincided with an increase in effector functions of T cells in IFN-β KO mice, as measured by IFN-γ and IL-4 production. We suggest that lack of endogenous IFN-β in CNS leads to augmented microglia activation, resulting in a sustained inflammation, cytokine production, and tissue damage with consequent chronic neurological deficits.

Expression of a novel member of estrogen response element-binding nuclear receptors is restricted to the early stages of chorion formation during mouse embryogenesis

Members of the nuclear hormone receptor gene family of transcription factors have been shown to be expressed in characteristic patterns during mouse organogenesis and postnatal development. Using an RT-PCR based screening assay, we have identified nuclear receptors expressed in embryonal carcinoma stem cells. One of the cDNAs characterized, mERR-2, was found to be expressed exclusively during a narrow developmental window in trophoblast progenitor cells between days 6.5 and 7.5 post coitum (p.c.). From 8.5 days p.c. and onwards, the mERR-2 gene activity evaded detection as analysed by in situ hybridization. We also show that the mERR-2 gene product and the estrogen receptor share a common target DNA-sequence recognition specificity unique among members of the gene family. Furthermore, efficient homodimerization and DNA-binding of the orphan receptor mERR-2 was found to be dependent on interaction with the heat shock protein 90, a molecular chaperone hitherto recognized to interact only with the steroid hormone receptor subgroup of nuclear receptors. Based on our results we suggest that the mouse orphan receptor mERR-2 has the potential to regulate overlapping gene networks with the estrogen receptor and may participate in signal transduction pathways during a short developmental period coinciding with the formation of the chorion.

The Th2 cytokines IL-4 and IL-10 are not crucial for the completion of allogeneic pregnancy in mice.

The physiological protection from fetal rejection is believed to be dependent on a Th2 type of inflammatory response at the maternal-fetal interface and the cytokines IL-4 and IL-10 have been suggested to play a critical role. We here present data from breeding experiments with IL-10 and IL-4 double-deficient mice indicating that neither maternal nor feto-placental deficiency of these cytokines are crucial for fetal or neonatal survival. The present study does not analyse possible developmental effects of maternal or fetal IL-10 and IL-4 double-deficiency in detail, but shows that an apparently normal breeding can be achieved in different crossings, providing that the mice are kept under very clean conditions.

Maintained pregnancy levels of oestrogen afford complete protection from post-partum exacerbation of collagen-induced arthritis

Pregnancy is known to influence the course of rheumatoid arthritis (RA) in women, as well as type II collagen‐induced arthritis (CIA) in DBA/1 mice. A characteristic feature is the remission during gestation and the exacerbation of the diseases during the post‐partum period. In the case of CIA in DBA/1 mice, two hormonal changes have been assumed to be critical for the induction of the post‐partum flare: (i) the fall in steroid hormone levels from those present during pregnancy; and (ii) surges of prolactin (PRL) release at and after delivery. Our results show that treatment with oestradiol during a short period immediately after parturition protects the mouse from a post‐partum flare of the disease, and that treatment with bromocriptine, a drug known to inhibit the endogenous PRL release, has a significant though less marked effect. Studies of lactating (i.e. animals with physiological stimulation of endogenous PRL release) and non‐lactating arthritic mice revealed no clear‐cut differences, indicating that PRL is of minor importance for the induction of the post‐partum flare. Some steroids other than oestradiol, which may be implicated in the exacerbation of arthritis, namely progesterone and hydrocortisone, had no clinical effect, Analyses of agalactosyl IgG levels in mice with CIA, and anti‐collagen II antibodies in sera collected at the end of the experiments revealed no significant differences between the oestradiol and the control groups. The successful oestradiol treatment of the mice indicates that the drop in endogenous oestradiol levels prior to delivery ends the oestrogen‐mediated protection against arthritis during pregnancy.

The pineal hormone melatonin exaggerates development of collagen-induced arthritis in mice

We have previously shown that constant darkness enhances autoimmunity to type II collagen (CII) and exaggerates development of collagen-induced arthritis (CIA) in DBA/1 mice. This effect was suggested to be mediated via the major hormone of the pineal gland, melatonin, since this hormone (1) is known to be strongly dependent on environmental lighting and (2) has been reported to affect the immune function. The present study was performed in order to clarify if melatonin could account for the previous observation that mice kept in darkness had a more pronounced development of CIA. First, DBA/1 mice kept in constant darkness were analysed for serum melatonin levels. An increase in background levels in comparison to mice kept in a normal dark/light cycle or in constant light was recorded. Then, different groups of mice (kept in constant light in order to minimize endogenous melatonin levels) were immunised with rat CII to induce arthritis and injected with melatonin. Melatonin injections were performed daily (1 mg/kg b.w.) in the afternoons (at 4 p.m.) for 10 days at two different periods: day 1-10 after collagen injection, or at the onset of the disease (day 30-39). Mice injected with melatonin at day 1-10 developed a more severe arthritis while those injected at onset did not differ significantly from the corresponding controls. Our results support the hypothesis that the pineal gland can exaggerate the development of CIA via a high release of melatonin, probably via enhancement of T-cell priming.

Estrogen induced suppression of collagen arthritis V: Physiological level of estrogen in DBA1 mice is therapeutic on established arthritis, suppresses anti-type II collagen T-cell dependent immunity and stimulates polyclonal B-cell activity

Immunization of castrated female DBA/1 mice with rat type II collagen (CII) induces severe polyarthritis with an onset 3-5 weeks after immunization and with 80-100% incidence. Estrogen treatment, inducing physiological 17 beta-estradiol (E2) levels, during a limited period before and after the immunization, or during another period before the expected onset of arthritis, delayed the arthritic onset by approximately 10 days but did not affect the incidence of severity of arthritis. Treatment with physiological doses of E2 after onset of arthritis decreased severity and duration of disease. The T-cell dependent anti-CII autoantibody response was suppressed if the E2 treatment was given immediately before and after CII immunization and was not significantly affected if E2 treatment was given after CII immunization. Neither the total anti-CII Ig levels nor the anti-CII IgG2a levels correlated with development of arthritis. We also titrated the serum levels of estrogen and recorded the vaginal smear response after injections of various doses of E2. This enabled us to work in a physiological range of estrogen levels, spanning the levels found at the end of pregnancy and those found during the normal estrous cycle. These levels were found to suppress antigen-specific T-cell functions but enhance certain B-cell activities since the delayed type hypersensitivity (DTH) reaction against CII was suppressed while the total number of splenic Ig-secreting cells increased. These findings suggest that estrogen in physiological doses is therapeutic for the development of collagen-induced arthritis and that estrogen exerts dualistic effects on the immune system by suppressing T-cell functions and stimulating certain B-cell activities. The suppressive effect on arthritis could not be explained by suppression of anti-CII autoantibody response and must therefore depend on other T-cell-mediated functions.

Immunoglobulin-Secreting Cells of Maternal Origin Can Be Detected in B Cell-Deficient Mice

Abstract It is well known that the transfer of immunoglobulins (Igs) from mother to young via milk contributes to the offsprings immune defense. The present study suggests that not only is IgG transmitted to progeny, but that functional maternal Ig-secreting cells (or B cells) can also be transferred to the neonate. We have used B cell-deficient (μ−/−) mice and found that a high proportion of them obtain long-lasting, partial reconstitution of their serum Ig levels if born to μ+/− mothers. In some of these serum IgG-positive μ−/− mice, Ig-secreting cells were detected in spleen and bone marrow. To ensure that cells of maternal origin were present in the progeny, μ−/− offspring born to μ+/− dams transgenic for green fluorescent protein (GFP) were used. In spleens and bone marrow from some of these μ−/−GFP−/− offspring, GFP-positive cells were detected, which demonstrated that cells of maternal origin could infiltrate the progeny. In addition, splenic Ig-secreting cells were detected in μ−/− mice that were born to μ−/− dams and transferred to a lactating μ+/+ foster dam at birth. This indicates that maternal Ig-secreting cells can be transferred postnatally via milk.

Placental MHC class I antigen expression is induced in mice following in vivo treatment with recombinant interferon-gamma

Allogeneically pregnant mice (NFR/N (Swiss-derived) H2q x 57/B1 H2b) were injected daily from day 11 to 18 of gestation with recombinant rat interferon-gamma (IFN-gamma) and the effects on placental MHC antigen expression were evaluated by immunohistochemistry. The results demonstrated that a daily dose of 200,000 U/mouse induces a significant increase in placental MHC class I expression in the decidua and the basal zone spongiotrophoblast as well as the fetal mesenchyme, while the labyrinthine trophoblast was almost completely MHC negative. No significant induction of MHC class II antigen expression was observed in the placental tissue, with the exception of a few scattered cells. The MHC class II-inducing efficiency of the treatment protocol used was ascertained by staining maternal skin (ear), which proved to contain considerably elevated numbers of MHC class II-positive cells (mainly keratinocytes). The IFN-gamma treated group showed a higher incidence of fetal resorptions than the corresponding controls, but this was probably due to a non-specific toxic effect of the treatment as none of the sera analysed contained detectable amounts of anti-paternal antibodies, no infiltrating lymphocytes were detectable on histological examination of placental sections, and most of the IFN-gamma treated animals successfully completed their pregnancy.

Constant darkness enhances autoimmunity to type II collagen and exaggerates development of collagen-induced arthritis in DBA/1 mice

The humoral function of the pineal gland is known to be strongly dependent on environmental lighting. Melatonin, the best characterized of the photo-dependent pineal hormones, has been reported to affect immune responses in mice. It has been hypothesized that the development of some types of psychosomatic and autoimmune diseases could be due to a disturbed release of this hormone. The present investigation was performed in order to evaluate effects of constant darkness (physiological stimulation of pineal melatonin synthesis) and constant light (physiological suppression of pineal melatonin synthesis) on the course of an experimental autoimmune model, the type II collagen-induced arthritis (CIA) in DBA/1 female mice. Mice kept in darkness develop more severe arthritis than those kept in constant light or in a normal dark/light rhythm (12 h light/12 h dark). Levels of anti-type II collagen antibodies were higher in mice kept in darkness, and the spleens of these animals were enlarged. Since castration of female DBA/1 mice enhances the severity of CIA, and since melatonin is known to exert effects on gonadal function, the experiment was repeated using oophorectomized mice. The same difference in arthritis severity between darkness- and light-exposed mice was obtained in this second experiment. We conclude that the exacerbation of arthritis in darkness is due to a darkness-induced change in levels of critical neurohumoral compound(s), that via gonadal independent mechanisms affect the autoimmune response. The exaggerated severity and chronicity of arthritis may be due to higher levels of melatonin in these animals.