Anders Bucht

Peripheral nerve injury induces endoneurial expression of IFN-γ, IL-10 and TNF-α mRNA

Abstract Axotomy of a peripheral nerve leads to interruption of axon continuity with Wallerian degeneration in the distal segment and regenerative events in the proximal remaining neuron. Local inflammation is a consequence of trauma in general and signal molecules regulating inflammation, such as cytokines, participate in the outcome of nerve trauma. We studied a broad set of potent immunoregulatory cytokines after transection of rat sciatic nerve. The endoneurium of the transected rat sciatic nerve was taken from both proximal and distal stumps. The pooled endoneurium of 6 rats was studied using reverse transcription polymerase chain reaction (RT–PCR) after 14 h; 1, 3, 5, 7 days; 2 and 4 weeks after transection. A new observation was that TNF-α mRNA showed phasic expression pattern; three distinct peaks were seen, immediately (14 h), after 5 days and in the distal part also after 2 weeks. This phenomenon may be related to the breakdown of the blood–nerve barrier and to the recruitment of circulating macrophages. We further noticed that IFN-γ mRNA was expressed between 5 days and 2 weeks. This suggests that T-cells may also take part in the regenerative processes. Furthermore, we observed that IL-10 mRNA is expressed continuously during Wallerian degeneration. The continuous expression of IL-10 mRNA may attenuate the production of inflammatory cytokines by macrophages and other cells.

The endogenous adjuvant squalene can induce a chronic T-cell-mediated arthritis in rats.

Squalene is a cholesterol precursor, which stimulates the immune system nonspecifically. We demonstrate that one intradermal injection of this adjuvant lipid can induce joint-specific inflammation in arthritis-prone DA rats. Histopathological and immunohistochemical analyses revealed erosion of bone and cartilage, and that development of polyarthritis coincided with infiltration of alphabeta(+) T cells. Depletion of these cells with anti-alphabeta TcR monoclonal antibody (R73) resulted in complete recovery, whereas anti-CD8 and anti-gammadelta TcR injections were ineffective. The apparent dependence on CD4(+) T cells suggested a role for genes within the major histocompatibility complex (MHC), and this was concluded from comparative studies of MHC congenic rat strains, in which DA.1H rats were less susceptible than DA rats. Furthermore, LEW.1AV1 and PVG.1AV1 rats with MHC identical to DA rats were arthritis-resistant, demonstrating that non-MHC genes also determine susceptibility. Some of these genetic influences could be linked to previously described arthritis susceptibility loci in an F2 intercross between DA and LEW.1AV1 rats (ie, Cia3, Oia2 and Cia5). Interestingly, some F2 hybrid rats developed chronic arthritis, a phenotype not apparent in the parental inbred strains. Our demonstration that an autoadjuvant can trigger chronic, immune-mediated joint-specific inflammation may give clues to the pathogenesis of rheumatoid arthritis, and it raises new questions concerning the role of endogenous molecules with adjuvant properties in chronic inflammatory diseases.

Differential anti-inflammatory and anti-oxidative effects of dexamethasone and N-acetylcysteine in endotoxin-induced lung inflammation

Inhalation of bacterial endotoxin induces an acute inflammation in the lower respiratory tract. In this study, the anti‐inflammatory effects of the anti‐oxidant N‐acetylcysteine (NAC) and the glucocorticoid dexamethasone were investigated in mice exposed to aerosolized endotoxin (lipopolysaccharide (LPS)). Powerful reduction of neutrophils in bronchoalveolar lavage fluid (BALF) was obtained by a single i.p. injection of dexamethasone (10 mg/kg), whereas treatment with NAC only resulted in reduction of neutrophils when administered at a high dose (500 mg/kg). Measurement of cytokine and chemokine expression in lung tissue revealed a significant decrease of tumour necrosis factor‐alpha, IL‐1α, IL‐1β, IL‐6, IL‐12p40, and MIP‐1α mRNA when mice where treated with dexamethasone but not when treated with NAC. Analysis of oxidative burst demonstrated a remarkable reduction of oxygen radicals in BALF neutrophils after treatment with dexamethasone, whereas the effect of NAC was not significantly different from that in untreated animals. In conclusion, dexamethasone exerted both anti‐inflammatory and anti‐oxidative effects in acute airway inflammation, probably by blocking early events in the inflammatory cascade. In contrast, treatment with NAC resulted in a weak reduction of the inflammatory response but no inhibition of proinflammatory cytokines or reduction of oxidative burst in neutrophils. These results demonstrate dramatic differences in efficiency and also indicate that the two drugs have different actions. Combined treatment with NAC and dexamethasone revealed an additive action but no synergy was observed.

Down‐regulation of the T cell receptor CD3ζ chain in rheumatoid arthritis (RA) and its influence on T cell responsiveness

T cells implicated in chronic inflammatory diseases such as RA respond weakly when stimulated in vitro with mitogen or antigen. The mechanism behind this hyporesponsiveness is unclear, but a depressed expression of the T cell receptor (TCR)‐associated CD3ζ chain has been suggested. In the present work we describe a low expression of CD3ζ in synovial fluid (SF) T cells from RA patients compared with peripheral blood (PB) T cells, but no difference in CD3ζ expression between RA and healthy control PB T cells. In vitro studies demonstrated that granulocytes but not SF macrophages are able to down‐regulate the expression of CD3ζ. Through stimulation with anti‐CD3 antibodies we demonstrated that the TCR‐dependent proliferative response was decreased in SF T cells compared with PB T cells. Stimulation with phorbol ester and ionomycin also resulted in a low proliferative response of SF T cells, indicating that both signal transduction through the TCR (stimulation with anti‐CD3) and events further downstream in the signalling pathways (stimulation with phorbol ester and ionomycin) are affected. A similar depression of T cell activity was observed when induction of IL‐2 and IL‐4 was measured. However, SF T cells were not defective in the induction of interferon‐gamma (IFN‐γ) when stimulated with phorbol myristate acetate (PMA)/ionomycin, in contrast to the diminished IFN‐γ response observed after stimulation with anti‐CD3. This indicates that the hyporesponsiveness of SF T cells can not be generalized to all T cell functions. The differential response to external stimuli is likely to be of importance for the capacity of SF T cells to influence inflammatory reactions.

Non-MHC gene regulation of nerve root injury induced spinal cord inflammation and neuron death

Spinal ventral root avulsion leads to an inflammatory response around lesioned motoneurons and the subsequent degeneration of a large proportion of the neurons. We demonstrate here differences in the regulation of cytokine mRNAs, microglia/macrophage activation, MHC expression and nerve cell survival in the two inbred rat strains DA and ACI. These strains have similar major MHC haplotypes, but differ in their non-MHC background genes. T cells were rare in the lesioned segments and depletion of T cells did not affect the response. Thus, non-MHC gene(s) regulate the inflammation and neuron death after nerve trauma by mechanisms not involving antigen-specific immune responses.

Immunization with Alum–Collagen II Complex Suppresses the Development of Collagen-Induced Arthritis in Rats by Deviating the Immune Response

Collagen‐induced arthritis (CIA) is an experimental rat model sharing a number of features with human rheumatoid arthritis (RA). The model is associated with a proinflammatory (TH1) type of immune response and treatments with cytokines associated with TH2 immune responses are beneficial. Since agents with TH1‐inducing properties, such as Freunds incomplete adjuvant (FIA), are necessary for disease induction, it is of interest to investigate whether an adjuvant with TH2‐inducing properties affects CIA in a different way than does FIA. The authors studied arthritis development in DA rats after immunization with the TH2 stimulatory adjuvant alum adsorbed to rat collagen type II (CII) or collagen II fragments. Such treatments suppressed disease development both prophylactically and therapeutically. This beneficial effect of alum–CII immunization was associated with an increase in the IgG1 anti‐CII antibody response as compared to untreated rats or rats pretreated with alum alone. Treatment with alum without the addition of collagen did not have any clinical effect. In addition, alum–CII treated rats had a significantly higher expression of IL‐4 mRNA than untreated rats in the lymph nodes, 7 days after CIA induction. The authors suggest that alum–CII induces a TH2 immune response against rat CII which counteracts the development of CIA.

The arthritogenic adjuvant squalene does not accumulate in joints, but gives rise to pathogenic cells in both draining and non-draining lymph nodes.

A single intradermal injection of the adjuvant‐oil squalene induces T cell‐mediated arthritis in DA rats. The chain of events leading from non‐specific provocation of the immune system to arthritis, with clinical similarities to rheumatoid arthritis, is largely undetermined. Here, we combined in vivo tracking of tritium‐labelled squalene with lymph node (LN) cell transfer experiments to determine where critical activation events may take place. The majority of squalene remained at the injection site (79%). The amounts recovered in peripheral joints (<1%) were equal to that recovered in other organs that can be targets in autoimmune diseases. This argues that arthritis does not develop as a consequence of adjuvant accumulation in joints. In contrast, substantial amounts of squalene were recovered in hyperplastic LN draining the injection site (1–13%). The adjuvant was deposited to a larger extent in cells than in extracellular matrix. The draining LN cells could transfer arthritis to naïve irradiated DA rats following in vitro stimulation with conA. Interestingly, non‐draining LN were also hyperplastic and harboured arthritogenic cells, although they contained low amounts of squalene (<1%). Consequently, the amount of arthritogenic adjuvant in a particular LN is not closely linked to the development of pathogenic cells. The distribution pattern of squalene was similar in MHC‐identical but arthritis‐resistant PVG.1AV1 and LEW.1AV1 rats, and it was unaffected by T cell depletion with a monoclonal antibody (R73). Thus, T cells and non‐MHC genes do not regulate dissemination of squalene, but rather determine arthritis development at the level of adjuvant response.

Responses of the rat immune system to arthritogenic adjuvant oil.

T‐cell mediated inflammatory joint diseases with similarities to rheumatoid arthritis (RA) can be triggered in arthritis‐prone rat strains by intradermal injection of adjuvant oils. The pathogenesis of oil‐induced arthritis (OIA) remains elusive, and a largely unresolved question is how the rat immune system responds to arthritogenic oils such as incomplete Freunds adjuvant (IFA). Here we report that IFA already induces increased plasma levels of the acute‐phase reactants (APR) fibrinogen and α1‐acid glycoprotein at day 4 postinjection (p.i). In contrast, no early responses were detected in the joints before infiltration of the T cells, which coincided with arthritis onset at 11–14 days post injection (d.p.i.) The infiltrating cells were possibly derived from draining lymph nodes (LN), which were hyperplastic and contained increased cell numbers from 4 days p.i. and onwards. The magnitude of the early increase in cell numbers and APR was regulated by non‐major histocompatibility complex (MHC) genes, as determined by comparison between arthritis‐susceptible DA rats and arthritis‐resistant but MHC‐identical LEW.1AV1 and PVG.1AV1 rats. Arthritis‐prone DA rats developed a weak acute‐phase response, suggesting that this systemic response may be counteracting disease. The DA rats also had the largest early increase in LN‐cell numbers, suggesting that the LN hyperplasia is part of a disease pathway. The analysis of hyperplastic LN after in vivo labelling with bromodeoxyuridine (BrdU) revealed increased numbers and proportions of proliferating lymphocytes, including T cells. Furthermore, polymerase chain reaction (PCR)‐analysis of LN cytokine mRNA revealed upregulation of interleukin (IL)‐1β at 4 d.p.i. We conclude that adjuvant oil exposure triggers both systemic acute phase reactions and local activation of the peripheral lymphoid system. These responses are genetically regulated and may determine arthritis development and susceptibility.

CD8+ cells suppress oil-induced arthritis.

Oil‐induced arthritis is a genetically restricted polyarthritis that develops in the DA rat after injection of the mineral oil Freunds incomplete adjuvant. Here, we investigated the role of the potentially disease‐limiting cell populations CD8+ T cells, γδ T cells, natural killer (NK) cells and NK T cells in inguinal lymph nodes for the development of this adjuvant‐induced arthritis. Flow cytometry analysis before and at disease onset revealed a higher proportion of lymph node T cells expressing NKR‐P1 in the disease‐resistant LEW.1AV1 compared with the disease‐susceptible DA strain, suggesting that NK T cells might be disease protective. However, prophylactic in vivo administration of an anti‐NKR‐P1 MoAb (clone 10/78) did not consistently affect the disease course. The proportion of CD8+ T cells and the ratio CD4+/CD8+ T cells in inguinal lymph nodes did not differ significantly between DA and LEW.1AV1 rats before or at disease onset. Nevertheless, prophylactic in vivo depletion of CD8+ cells by the OX8 MoAb in the DA strain resulted in an earlier disease onset compared with the control group, demonstrating that CD8+ cells regulate arthritis development. In vivo depletion of γδ T cells by the V65 MoAb did not alter the disease course, indicating that the disease‐suppressive CD8+ cells are αβ T cells or NK cells.

Responses of the rat immune system to arthritogenic adjuvant-oil

T-cell mediated inflammatory joint diseases with similarities to rheumatoid arthritis (RA) can be triggered in arthritis-prone rat strains by intradermal injection of adjuvant oils. The pathogenesis of oil-induced arthritis (OIA) remains elusive, and a largely unresolved question is how the rat immune system responds to arthritogenic oils such as incomplete Freunds adjuvant (IFA). Here we report that IFA already induces increased plasma levels of the acute-phase reactants (APR) fibrinogen and alpha1-acid glycoprotein at day 4 postinjection (p.i). In contrast, no early responses were detected in the joints before infiltration of the T cells, which coincided with arthritis onset at 11-14 days post injection (d.p.i.) The infiltrating cells were possibly derived from draining lymph nodes (LN), which were hyperplastic and contained increased cell numbers from 4 days p.i. and onwards. The magnitude of the early increase in cell numbers and APR was regulated by non-major histocompatibility complex (MHC) genes, as determined by comparison between arthritis-susceptible DA rats and arthritis-resistant but MHC-identical LEW.lAV1 and PVG.1AV1 rats. Arthritisprone DA rats developed a weak acute-phase response, suggesting that this systemic response may be counteracting disease. The DA rats also had the largest early increase in LN-cell numbers, suggesting that the LN hyperplasia is part of a disease pathway. The analysis of hyperplastic LN after in vivo labelling with bromodeoxyuridine (BrdU) revealed increased numbers and proportions of proliferating lymphocytes, including T cells. Furthermore, polymerase chain reaction (PCR)-analysis of LN cytokine mRNA revealed upregulation of interleukin (IL)-1beta at 4 d.p.i. We conclude that adjuvant oil exposure triggers both systemic acute phase reactions and local activation of the peripheral lymphoid system. These responses are genetically regulated and may determine arthritis development and susceptibility.