Novel path to IL-6 trans-signaling through thrombin-induced soluble IL-6 receptor release by platelets.

Interleukin (IL)-6 is a multifunctional cytokine with a critical role in inflammatory, immunoregulatory and haemopoietic responses. Its receptor consists of an ubiquitously expressed membrane transducing element (gp130) and of the specific element IL-6R-alpha (gp80), present only on hepatocytes and some leukocyte subsets. IL-6R-alpha also exists as soluble protein (sIL-6R) that, in the presence of IL-6, forms a complex able to bind gp130 and, thanks to the mechanism called trans-signaling, transduces IL-6 effect through tyrosine phosphorylation and activation of the signal transducer and transcription activator (STAT)-3. The aim of this study was to analyze the bidirectional relationships between platelet aggregation and IL-6-dependent effects. While platelets do not produce IL-6, we found that resting platelets express gp130, but not gp80, on their membranes. Upon activation by thrombin or calcium ionophore A23187, but not by ADP, the IL-6R-alpha is released in soluble form, while cangrelor, the specific inhibitor of P2Y12 receptor, can partially inhibit sIL-6R release. This sIL-6R is biologically active and, in the presence of IL-6, can trigger IL-6 trans-signaling, inducing an autocrine activation loop (as measured by an increase in gp80 and gp130 content) and STAT3 phosphorylation. On the other hand, IL-6 trans-signaling has no effect on platelet degranulation or aggregation by itself, nor on thrombin-induced platelet aggregation. Our data add an important piece to the puzzle of thrombosis and inflammation: in the presence of IL-6, which can be produced by stressed endothelial cells, the platelet-derived IL-6 trans-signaling could be crucial for the evolution of inflammation within a damaged vessel.

Skeletal muscle cells: from local inflammatory response to active immunity.

The skeletal muscles are the major living component of the human body. They are constituted by stable cells, the myofibres, and by adult multipotent stem cells, the satellite cells, which can multiply to regenerate and repair the damaged tissues. Injections of DNA in muscle cells have been used to produce recombinant proteins with opposite goals: somatic reparation of genetic defects, which needs to elicit no inflammatory or immune response, and DNA vaccination, which needs a robust immune response. Because of possible therapeutical interventions, a growing body of information is being produced dealing with every aspect of the myofibres during inflammatory and autoimmune responses: skeletal muscle-antigen presenting cell (APC) interaction and intrinsic APC capabilities of myoblasts and myocytes, the response to released cytokines and their endogenous production, the regulation of Toll-like receptors and major histocompatibility complex expression. According to these data, the muscle tissue is now emerging no longer as a passive bystander, but more as an active player that, when correctly manipulated, can drive tolerance or immunization to these de novo produced proteins. In the present review, we summarize the recent developments on the control of muscle immune function.

Neurophysiological and mitochondrial abnormalities in MuSK antibody seropositive myasthenia gravis compared to other immunological subtypes.

OBJECTIVE: To compare the electrophysiological and histopathological features of immunological myasthenia gravis (MG) subtypes. METHODS: Fifty MG patients underwent clinical examination, MuSK-Ab and AChR-Ab analysis. The majority underwent quantitative and single-fiber electromyography (QEMG, SFEMG), repetitive nerve stimulation and deltoid muscle biopsy. From muscle specimens with histological mitochondrial dysfunction, we amplified mitochondrial DNA (mtDNA). In specimens with mtDNA deletions, the nuclear gene POLG1 was sequenced. RESULTS: Five AChR-Ab seropositive [AChR(+)] and 5 seronegative [AChR(-)] patients were MuSK-Ab seropositive [MuSK(+)]. Five of 7 neurophysiologically examined MuSK(+) patients (71%) had proximal myopathic pattern, compared to 7 of 31 MuSK(-)/AChR(+) patients (23%) (P=0.012). SFEMG was abnormal in all examined MuSK(+) patients. All 7 biopsied MuSK(+) and 32 MuSK(-) patients (89%) had cytochrome c oxidase (COX) negative fibers. Three of five MuSK(+) and 13 of 20 MuSK(-) patients analyzed had multiple mtDNA deletions but no POLG1 mutations. CONCLUSIONS: Similar degree of SFEMG abnormalities was present in proximal muscles among MuSK(+) and AChR(+) patients. Proximal myopathy was over-represented in MuSK(+) patients; however, both MuSK(+) and MuSK(-) patients had mild myopathy with frequent mitochondrial abnormalities. SIGNIFICANCE: The weakness in MuSK(+) patients is most likely due to disturbed neuromuscular transmission. The frequently encountered mitochondrial dysfunction in MG warrants further study.

Juvenile myasthenia gravis with prepubertal onset.

Juvenile myasthenia gravis (JMG) with prepubertal onset is an uncommon disease. We studied 19 patients with age at onset ranging from 1.5 to 9.2 years and compared their clinical characteristics and response to therapy with 114 cases with MG onset after the prepubertal age, up to 20 years. Neither sex prevalence nor autoimmune diseases other than MG were found in younger patients. Although ocular myasthenia was more frequent than in later-onset JMG, children with generalized symptoms were often severely affected and respiratory involvement was present in 8/19 patients. Anti-acetylcholine receptor antibodies were detected at a lower rate and, in contrast with results in older patients, seronegativity was more frequent among children with generalized disease. Three out of six patients with onset before the age of five showed spontaneous remission. Nine prepubertal patients underwent thymectomy and, as most of them also received immunosuppressive therapy, the influence of surgery on disease outcome remains unclear; in no case was thymoma found. This is in contrast to the good results after thymectomy and the presence of thymoma in the later-onset group. Eleven patients in the prepubertal series were treated with immunosuppressive therapy. At the end of follow-up, most patients were in good condition. The frequency of immunosuppressive therapy and the rate of good therapeutic results did not differ from those observed in older patients.

Effect of transforming growth factor-beta1 on interleukin-6 secretion in human myoblasts.

Transforming growth factor-beta (TGF-beta) is involved in several autoimmune neurological diseases. It is still unclear whether its local action can be pro-inflammatory or anti-inflammatory in the muscle tissue, because of the few reports on this subject. We have previously shown that human myoblasts secrete interleukin-6 (IL-6) when stimulated with inflammatory cytokine such as interleukin-1beta (IL-1beta) or tumor necrosis factor alpha. In the present report, we show that TGF-beta1 can induce IL-6 production; moreover, costimulation or short term pre-incubation with TGF-beta1 increases IL-1beta effect, while a longer incubation inhibits its action.

Neonatal myasthenia gravis: clinical and immunological study of seven mothers and their newborn infants.

We studied 7 mothers with myasthenia gravis (MG) and their infants. We confirmed that the development of neonatal MG was not related to the serum titer of maternal anti-acetylcholine receptor antibody (anti-AChR ab). To investigate the possibility that specific immunization of the newborn infant had occurred, serial serum determinations of total and 'specific' anti-AChR IgG and IgM were performed. We found that: the decay in total IgG was within the normal range in all the babies; there was a shorter half-life of 'specific' IgG, compared to total IgG, in 3 of the cases, 2 of which did have neonatal MG; no difference was found between the decay of anti-AChR ab in the babies who had neonatal MG and those who did not; there was no anti-AChR IgM-associated activity. Our data suggest that neonatal MG is due to maternal anti-AChR abs and that affected infants do not produce specific antibodies.

Constitutive and cytokine-induced expression of MHC and intercellular adhesion molecule-1 (ICAM-1) on human myoblasts.

We studied the expression of MHC-I and MHC-II molecules and ICAM-1 in cultured human myoblasts in response to IL-1beta, IL-4, IL-6, IFN-gamma and LPS. IFN-gamma, LPS and IL-4 greatly increase MHC-I molecule expression. MHC-II molecule expression is induced only by IFN-gamma. Membrane ICAM-1 and mRNA expression are absent under basal conditions, but can be induced by IFN-gamma, IL-1beta, IL-4, LPS and IL-6 with different efficiencies and time-courses. Soluble ICAM-1 secretion can be induced to a different extent by all cytokines. Our study shows that the expression of adhesion-related molecules in muscle is finely regulated by these cytokines.

Constitutive and cytokine-induced production of interleukin-6 by human myoblasts.

Several recent studies have shown that some inflammatory myopathies are autoimmune diseases. It is possible that certain alterations in the muscle-immune cell microenvironment and in the local production of cytokines could take part in the pathogenesis of inflammatory myopathies. In the present study we investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the secretion of interleukin-6 (IL-6) by myoblasts. Purified human myoblasts from normal subjects and from patients with polymositis were cultured in the presence of TNF-alpha and IFN-gamma at two concentrations (100 and 200 U/ml), alone or in combination, for 12, 24 and 48 h. The supernatants were collected and the IL-6 concentrations tested by ELISA (Genzyme). We found that myoblasts secrete IL-6 constitutively. The secretion of IL-6 was greatly increased by TNF-alpha; the increase was both time- and dose-dependent. IFN-gamma caused a moderate increase in IL-6 secretion, but this effect was not significant, despite a slight positive trend over time. There was no synergism in the effect of IFN-gamma and TNF-alpha. It is known that inflammatory myopathies are characterized by mononuclear cell infiltration and muscle regeneration: myoblasts are present in infiltrated tissues. Thus, the local production of cytokines that characterizes the inflammatory reaction, could stimulate myoblasts to secrete IL-6, which might add to the pro-inflammatory effects of IL-6 produced by activated macrophages and T cells.

Steroid-induced proteins in human endometrium.

The synthesis of soluble proteins in human endometria at various phases of menstrual cycle was evaluated by polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Densitometric analysis of the gels revealed alterations in the rate of synthesis of single protein bands throughout the cycle. Administration of conjugated estrogens (Premarin) to women undergoing hysterectomy, or exposure, in vitro, of the endometrial tissue to 17 beta-estradiol produced an increased incorporation of [35S]methionine into a specific protein which migrated on SDS-polyacrylamide gels at a molecular weight of about 55 000. Induction of this protein was observed only in those endometria showing a secretory histological appearance. The protein was resolved into at least 2 different spots in two-dimensional gel electrophoresis. An increase in the rate of synthesis of another endometrial protein with an apparent molecular weight of 51 000 was observed in tissues exposed in vitro to medroxyprogesterone acetate. These steroid-induced proteins may be a useful marker for studying hormone action in both normal and neoplastic endometria.

Effect of pro-inflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus.

We studied a well-selected population of patients with active rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) without immunosuppressive therapy. Control and patient peripheral blood mononuclear cells (PBMC) were incubated with IL-1beta, IL-10, TGF-beta or LPS for 20 h and the in vitro basal and stimulated secretions of IL-6, TNF-alpha, IL-1beta and IL-1ra were measured by ELISA. We found that in the SLE patients the basal secretion of IL-6 was significantly lower and that of IL-1ra significantly higher than in control subjects, while in the RA group the basal IL-1ra secretion was higher than in healthy subjects. SLE and RA PBMC responded to LPS and IL-1beta reaching higher cytokine secretion values than controls. The in vitro response of SLE and RA PBMC to TGFbeta was normal, while that to IL-10 was defective: IL-10 was able to stimulate the production of IL-6 and IL-1ra in PBMC from normal subjects, but it was unable to enhance IL-6 secretion in RA cells and it was also completely ineffective in inducing IL-1ra secretion in both SLE and RA PBMC. Our work add new data useful for the evaluation of IL-10 and IL-1ra as therapeutic agents in rheumatic diseases.

Myasthenia gravis, thymectomy, and antiacetycholine receptor antibody.

The antiacetylcholine receptor antibody was titered in the serum of 63 patients with myasthenia gravis (MG) and 20 control healthy subjects. The titer was significantly high in 92% of MG patients in contrast with none of the controls and no correlation was found with the thymus pathology and the severity of the disease. The titer decreased after thymectomy almost steadily with the improvement of the myasthenic signs. The role of the antibody in the pathogenesis of the disease is discussed.

When is there a full recovery for a myasthenia gravis patient?

A myasthenia gravis (MG) patient who seems to have recovered can later have recurrence of myasthenic signs. Clearly clinical remission does not always correspond to the normalization of all the factors involved in the pathogenesis of the disease. In ten patients who had apparently recovered from MG, electromyographic tests of repetitive supramaximal stimulation were performed and the anti-acetylcholine receptor (anti-AChR) antibody was assessed. In two of the ten patients all these tests were normal, thus showing lack of electromyographic myasthenic fatigability and the absence of circulating anti-AChR antibodies. Our hypothesis is that for these two subjects the risk of a recurrence of MG is lower than for the others.

Anti-MuSK antibodies: correlation with myasthenia gravis severity.

The authors measured anti-muscle-specific tyrosine kinase (anti-MuSK) antibodies (Abs) in 83 serum samples from 40 patients and evaluated their correlation with myasthenia gravis severity and treatment response. Ab concentrations were often reduced by immunosuppression but not after thymectomy. Both in individual cases and in the whole population, a correlation between Ab levels and disease severity was found.

Seronegative myasthenia gravis: comparison of neurophysiological picture in MuSK+ and MuSK- patients.

The aim of this study was to compare the neurophysiological and clinical pictures of a large sample of seronegative myasthenia gravis (SNMG) patients with and without anti-MuSK antibodies. Fifty-two consecutive SNMG patients were retrospectively evaluated. They had undergone an extended neurophysiological evaluation: repetitive nerve stimulation (RNS), single fiber EMG (SFEMG), and electromyography (EMG) with nerve conduction study. A muscle biopsy was performed in 11 of 52 patients, the edrophonium test in 44 of 52 patients and anti-AChR antibodies and anti-MuSK antibodies were tested in all patients. Anti-MuSK antibodies were detected in 25 SNMG patients (48.1%). The number of women in the MuSK+ group was significantly higher (P = 0.01) than in the MuSK- group. Seronegative MuSK+ patients are more severely affected and the deficit often involves the bulbar and the respiratory muscles. No statistically significant differences were observed in the edrophonium test between MuSK+ and MuSK- groups. The RNS test was abnormal in a significantly higher number of MUSK- patients than MUSK+ patients (P < 0.00001). With regard to SFEMG data, MuSK- patients were characterized to have more severe neurophysiological pattern. Our observations showed several differences between the clinical and neurophysiological pictures of MUSK+ and MUSK- patients.

Thymus changes in anti-MuSK-positive and -negative myasthenia gravis.

Morphologic findings of thymuses from 32 anti-acetylcholine receptor (AChR)-negative myasthenia gravis patients, 12 with and 20 without antibodies against the muscle-specific kinase (MuSK), were compared with those from 30 AChR-positive subjects. In contrast with the high frequency of thymic hyperplastic changes in AChR-positive patients, in MuSK-positive subjects histologic alterations were minimal, arguing against an intrathymic disease pathogenesis. Since hyperplastic changes were seen in 35% of MuSK-negative patients, the thymus could be involved in some of these cases.

Ocular myasthenia: diagnostic and therapeutic problems.

Forty-eight patients with purely ocular myasthenia were studied. Tensilon test was positive in 46 patients (95%); decremental response from limb muscles was present in 24 patients (50%); anti-AChR antibodies were detected in 20 patients of 44 (45.5%). Twenty-two patients underwent thymectomy, 18 were given corticosteroids, 42 received AChE drugs. At the end of the observation period, 8% of the patients were in remission, 67% were improved, 25% were unchanged. In our experience, the diagnosis of ocular myasthenia relies mainly on clinical data; AChE drugs are not very effective in extrinsic ocular muscles; indications for thymectomy should be restricted to thymoma cases and, perhaps, to patients in the early stages of the disease, within the first year of onset; corticosteroids are effective in most cases, but relapses after withdrawal are not uncommon.

Thymic hormone containing cells--IX. Steroids in vitro modulate thymulin secretion by human and murine thymic epithelial cells.

We investigated the in vitro effects (kinetics and dose-response) of adrenal and sexual steroid hormones on the secretion of thymulin, a thymic hormone, by human thymic epithelial cells in primary cultures as well as in a rat epithelial cell line. We demonstrated that all steroids tested, in a range of physiological doses, stimulated thymulin production to various extents. Progesterone and estradiol, however, were revealed to be the most efficient. Specific steroid antagonists abrogated the steroid-induced stimulation of thymulin production. These findings confirm our previous in vivo results and demonstrate that steroid hormones can act directly on thymic epithelial cells to modulate their endocrine production.

High-dose intravenous immunoglobulin in myasthenia gravis.

The effects of high-dose intravenous immunoglobulin (i.v.Ig) in 12 MG patients were studied. All patients had severe symptoms. In two cases anti-acetylcholine receptor antibodies (anti-AChR abs) were not detectable. I.v.Ig was administered to 9 patients already on long-term immunosuppressive therapy and to 3 patients at the beginning of azathioprine treatment. 10 patients (83%) improved; the duration of improvement was longer in immunosuppressed patients. Anti-AChR abs generally decreased after infusion but we did not find a constant correlation between reduction in ab titers and clinical improvement. Side effects included one case of severe hemolysis. In our experience i.v.Ig therapy is effective in MG. The chief indication for its use appears to be the treatment of deterioration of the disease in patients already on immunosuppressive therapy.