Mechanisms dependent on tryptophan catabolism regulate immune responses in primary Sjögren's syndrome

To investigate the possible role of tryptophan metabolism in immune regulation of primary Sjögren's syndrome (pSS) the serum concentrations of tryptophan and its metabolite kynurenine were measured by reverse‐phase high‐performance liquid chromatography (HPLC) in 103 patients with pSS, 56 patients with sicca symptoms and 309 healthy blood donors. The kynurenine per tryptophan ratio (kyn/trp), which reflects the activity of the indoleamine‐pyrrole 2,3‐dioxygenase (IDO) enzyme involved in tryptophan catabolism, was calculated. Both female and male patients with pSS had significantly higher serum kynurenine concentrations and kyn/trp than subjects with sicca symptoms or healthy blood donors. The median (quartile range) concentration of kynurenine in female patients with pSS was 2·41 µmol/l (1·86–3·26) compared with 1·85 µmol/l (1·58–2·38, P < 0·0001) in subjects with sicca symptoms and 1·96 µmol/l (1·65–2·27, P < 0·0001) in healthy blood donors. Their kyn/trp × 1000 was 34·0 (25·1–44·3) compared with 25·3 (21·1–31·5, P < 0·0001) in subjects with sicca symptoms and 24·3 (21·0–28·9, P < 0·0001) in healthy blood donors. Female pSS patients with high IDO activity (kyn/trp × 1000 ≥ 34·0) had significantly higher ESR, serum C‐reactive protein, serum IgA and serum beta‐2 microglobulin concentrations as well as higher serum creatinine levels, and they had positive antinuclear antibodies more frequently and presented with more American‐European consensus group criteria than those with low IDO activity (kyn/trp × 1000 < 34·0). These data suggest that mechanisms dependent on tryptophan catabolism regulate immune responses in pSS. Tryptophan degradation is enhanced in patients with pSS, and high IDO activity is associated with severity of pSS.     

Interleukin-1 beta gene polymorphism related with allergic pathogenesis in Iris constitution

Iridological constitution has a strong familial aggregation and is implicated in heredity. The aetiology of inflammatory bowel disease is still unknown. However, from genetic epidemiological studies there is considerable evidence that genetic factors are associated with both Crohn's disease and ulcerative colitis. We investigated the relationships between Iridological constitution and interleukin 1 beta (IL-1β) gene polymorphism. IL-1β is a major proinflammatiry cytokine, and the polymorphisms of this gene have been shown to be of importance in a number of diseases. Especially, IL-1 has been suspected of involvement in allergic pathogenesis. Also, IL-1β genotype is one of the genetic markers of gastric cancer. Therefore, we classified 166 individuals according to Iris constitution, and determined IL-1β genotype. The frequencies of Iris constitutions as follows: neurogenic type, 41 (24.7%); abdominal connective tissue weakness type, 53 (31.9%); cardio-renal connective tissue weakness type, 50 (30.1%); the others type, 22 (13.3%). Especially, the frequency of abdominal connective tissue weakness type was higher in C/T genotype than in the remaining constitutions although the statistical power was very weak. Furthermore, we first attempted to explore possible involvement of the IL-1β polymorphism and the Iris constitution.     

Interferon-gamma (IFN-gamma) and IL-4 expressed during mercury-induced membranous nephropathy are toxic for cultured podocytes.

The subepithelial immune deposits of Dorus Zadel Black (DZB) rats with mercury-induced membranous nephropathy consist of autoantibodies directed to laminin P1 and of complement. The animals develop massive proteinuria within 10-14 days which is associated with obliteration of foot processes of glomerular visceral epithelial cells (GVEC), or podocytes. Previous studies indicate that these autoantibodies are probably not the sole mediator of proteinuria and GVEC damage. In this study we investigated whether circulating or macrophage-derived cytokines can contribute to the GVEC changes as detected in vivo. In vivo at the height of the proteinuria, increased intraglomerular IFN-gamma immunoreactivity was found. In diseased rats a five-fold increase in intraglomerular macrophages was found, but we could not detect intraglomerular IFN-alpha, IFN-beta, IL-1 beta or tumour necrosis factor-alpha (TNF-alpha) by using immunohistology. Subsequently, we exposed cultured GVEC to these cytokines to investigate their cytotoxic effects on several physiological and structural parameters. IFN-gamma and IL-4 were the only cytokines that exerted toxic effects, resulting in a rapidly decreased transepithelial resistance of confluent monolayers, which was closely associated with altered immunoreactivity of the tight junction protein ZO-1. IL-4 also affected vimentin and laminin immunoreactivity. IFN-gamma and IL-4 only interfered with monolayer integrity when added to the basolateral side of the GVEC, indicating specific (receptor-mediated) effects. Only IL-4 decreased the viability of the cells, and treated monolayers demonstrated an increased passage of the 44-kD protein horseradish peroxidase. From our experiments we concluded that IFN-gamma subtly affected monolayer integrity at the level of the tight junctions, and that IL-4 additionally induced cell death. We hypothesize that the toxic effects of the cytokines IFN-gamma and IL-4 as seen with cultured podocytes are necessary together with the autoantibodies, for the ultimate induction of proteinuria in mercury nephropathy in the DZB rat.     

Inhibition of CD4+ T lymphocyte binding to fibronectin and immune-cell accumulation in inflammatory sites by non-peptidic mimetics of Arg-Gly-Asp.

The Arg-Gly-Asp (RGD) cell adhesion motif has been demonstrated in various studies to play a pivotal role in leucocyte and platelet interactions with plasma and extracellular matrix (ECM) glycoproteins. The recognition of the RGD sequence is mediated by heterodimeric receptors designated integrins of the beta 1 subfamily, expressed on distinct cell types, including T lymphocytes. We have recently shown that flexible non-peptidic mimetics of RGD, in which the two ionic side groups were separated by a linear spacer of 11 atoms, bound specifically to the platelet integrin alpha 11b beta 3, and inhibited T cell-mediated immune responses. The present study was designed to (i) further characterize the structural requirements for RGD interactions with CD4+ T cells, and (ii) examine the mechanisms by which the RGD mimetics interfere with immune cell reactivity in vivo. We now report that freezing the conformational degrees of freedom in the spacer chain, which fixes the relative orientation of the guanidinium and carboxylate side groups in a favourable manner, results in a higher level of inhibition of T cell binding to immobilized fibronectin, an RGD-containing ECM glycoprotein. In vivo, treatment of mice with relatively low doses of the RGD mimetics, but not the RGD peptide, inhibited the elicitation of an adoptively transferred DTH reaction. This inhibition was achieved by direct impairment of the ability of antigen-primed lymph node cells to migrate and accumulate in inflammatory sites. Hence, we suggest that the design and production of non-peptidic mimetics of RGD offers a novel approach to study defined parameters related to the structure-function requirements of small adhesion epitopes. Furthermore, this approach could be used therapeutically to inhibit pathological processes which depend on RGD recognition.     

Potassium and sodium shifts during in vitro isometric muscle contraction,and the time course of the ion-gradient recovery

Intracellular potassium ([K⁺]_i), interstitial potassium ([K⁺]_inter), intracellular sodium ([Na⁺]_i), and resting membrane potential (RMP) were measured before and after repetitive stimulation of mouse soleus and EDL (extensor digitorum longus) muscles. At rest, RMP was –69.8 mV for soleus and –74.9 mV for EDL (37°C). [K⁺]_i was 168 mM and 182 mM, respectively. In soleus, free [Na⁺]_i was 12.7 mM. After repetitive stimulation (960 stimuli) RMP had decreased by 11.9 mV for soleus and by 18.2 mV for EDL. [K⁺]_i was reduced by 32 mM and 48 mM, respectively, whereas [K⁺]_inter was doubled. In soleus [Na⁺]_i had increased by 10.6 mM, demonstrating that the [K⁺]_i-decrease is three times higher than the [Na⁺]_i-increase. It is concluded that this difference reflects different activity induced movements of Na and K, and that the difference is not due to the Na/K pumping ratio. The possible involvement of the potassium loss in muscle fatigue is discussed. After stimulation RMP recovered with a time constant of 0.9 min for soleus and 1.5 min for EDL. Within the first minutes after stimulation the intracellular potassium concentration increased by 20.4 mM/min for soleus and 21.7 mM/min for EDL. Free [Na⁺]_i decreased with less than 10 mM/min. The mechanisms underlying the different rate of changes are discussed.Parts of this work have been published in preliminary form (Juel and Sjøgaard 1984)     

A functionally active complement system is present in uterine secretion of the mouse prior to implantation.

Sephadex beads were placed carefully in the uterus on days 2 and 3 and left for 6 to 8 h to absorb uterine secretion. The beads were then removed with volatile silicon oil and mounted on small pieces of nitrocellulose paper. Immuno-staining of these bead blots showed they contained the complement components C1q, C3, C4, and C5. We demonstrated that complement component C3 in the uterine secretion could be activated and deposited on model immune complexes, and also that antibody-coated erythrocytes were lysed in utero, that is, a membrane attack complex was produced. Thus, the mouse uterine secretion at the preimplantation stage contains a functionally active complement system.     

Cyclooxygenase-2, Bcl-2, and chromosome 1p analysis in protoplasmic astrocytomas

Protoplasmic astrocytomas are rare gliomas whose nosology remains enigmatic. This study retrospectively reviews the clinicopathologic features of eight tumors, including evaluation of these neoplasms for chromosome 1p loss, Bcl-2 immunoreactivity, and cyclooxygenase-2 immunoreactivity. Patients ranged in age from 3 to 49 years (median 25.5 years) and included six males and two females. All patients presented with a period of seizures (median duration of period, 54 months) before surgery. Five tumors were either totally or partially based in the temporal lobe. In the six patients for whom follow-up information was available, there was no evidence of recurrence at last known follow-up (range 5 to 171 months; median 134 months). Histologically, all tumors were marked by a proliferation of cells with rounded to oval nuclear contours and a paucity of cytoplasmic processes, arranged against a microcystic background. A rare mitotic figure was observed in only one tumor. Vascular proliferative changes and necrosis were not seen in any of the tumors. None of the tumors showed allelic loss on chromosome 1p by fluorescent in situ hybridization (FISH) analysis. Cyclooxygenase-2 (an enzyme involved in the conversion of arachidonate to prostaglandin H2 and G2) immunoreactivity was observed in two tumors. Bcl-2 (an anti-apoptotic protein) immunoreactivity was also confined to two tumors. In conclusion, protoplasmic astrocytomas appear to be low-grade neoplasms, as evidenced by their relatively benign clinical course. Although they histologically resemble microcystic oligodendrogliomas, none of the tumors showed allelic loss on chromosome 1p, a finding that has been described in the majority of low-grade oligodendrogliomas. This suggests that the protoplasmic astrocytoma is a distinct entity from low-grade oligodendroglioma. Similar to other low-grade astrocytomas, only a minority of tumors show evidence of cyclooxygenase-2 and Bcl-2 immunoreactivity.     

Immunoregulatory defects of V alpha 24V+ beta 11+ NKT cells in development of Wegener's granulomatosis and relapsing polychondritis

The frequency of either CD4(-)8(-) (double negative; DN) or CD4(+) V alpha 24(+)V beta 11(+) NKT cells, the expression of CD1d and the binding of CD1d-tetramer loaded with alpha-galactosylceramide (alpha-GalCer) to NKT cells were analysed in peripheral blood mononuclear cells (PBMCs) of patients with Wegener's granulomatosis (WG), relapsing polychondritis (RP) and healthy subjects (HS). DN and CD4(+) V alpha 24(+)V beta 11(+) NKT cells as well as CD1d-alpha-GalCer tetramer-positive NKT cells, were significantly decreased in number in both WG and RP patients compared to those from HS. When cytokine profiles were analysed in these PBMCs upon stimulation with phorbol ester and calcium ionophore, CD4(+) T cells from patients with WG and RP exhibited a Th1 bias, whereas CD4(+) NKT cells from WG patients in remission showed a Th2 bias. These findings suggest that NKT cells (especially CD4(+) NKT cells) play a regulatory role in Th1 autoimmunity in patients with WG and RP. The reduction in NKT cell counts appears to be associated with the low responsiveness to alpha-GalCer. The dysfunction of NKT cells to recognize ligands such as alpha-GalCer may also contribute to the defects observed in NKT cells from WG and RP patients.