Anti-ribosomal P antibodies are associated with elevated circulating IFNα and IL-10 levels in systemic lupus erythematosus patients.

OBJECTIVE: The objective of this paper is to analyze the relationship of anti-protein ribosomal P (RibP) antibodies with circulating levels of IFNα, TNFα, IFNγ, IL-17 and IL-10 in SLE. Disease activity and other systemic lupus erythematosus (SLE) features were also analyzed. METHODS: Anti-RibP and other SLE-related antinuclear antibodies (ANA) were determined by fluoro-enzyme immunoassay in the sera of 107 SLE patients. Circulating cytokines were quantified by flow cytometry (IFNα, IL-10 and IL-17) or ELISA (TNFα and IFNγ). RESULTS: Anti-RibP-positive patients (14.9%) displayed significantly higher serum levels of IFNα (p = 0.023) and IL-10 (p = 0.016) than their negative counterparts. This cytokine upregulation was independent of the presence of other ANA even though, in our patient cohort, anti-dsDNA was found to be associated with anti-RibP (OR, CI 95%: 6.03, 1.32-27.93, p = 0.021) and to correlate with IL-10 levels (r = 0.204, p = 0.036). In fact, patients positive for anti-RibP but negative for anti-dsDNA exhibited the highest amounts of both IL-10 and IFN-α that were not related to disease activity since these patients showed lower SLEDAI than patients also positive for anti-dsDNA (p = 0.018). Anti-RibP positivity was also associated with early diagnosis, hypocomplementemia and leukopenia. CONCLUSIONS: Presence of anti-RibP was found to be related to increased serum IFNα and IL-10 levels independently of both antibody status and disease activity.

Interferon beta-1b treatment modulates TNFalpha and IFNgamma spontaneous gene expression in MS.

BACKGROUND: Interferon beta (IFNbeta) lessens the overall frequency of acute attacks in patients with the relapsing-remitting form of multiple sclerosis (RRMS). IFNbeta may act by decreasing the synthesis of inflammatory cytokines. OBJECTIVES: To determine whether IFNbeta-1b treatment had an initial and sustained effect on the in vivo synthesis and secretion of tumor necrosis factor alpha (TNFalpha) and IFNgamma. METHODS: A highly sensitive reverse-transcriptase PCR technique was used to measure baseline levels of mRNA in freshly isolated cells from patients before therapy and at 3, 6, and 12 months of treatment. Also, protein concentration was measured in serum and in culture supernatants from mitogen-stimulated cells. The authors studied 16 patients, of whom 11 did not have clinical exacerbations, whereas 5 had one clinical relapse each during the study. RESULTS: Mean values of TNFalpha mRNA levels in the 11 stable patients decreased significantly at 3 and 6 months of treatment in comparison with initial data. After 6 months of therapy, IFNbeta-1b downmodulated TNFalpha transcripts in the 5 patients who experienced relapse. In this group of patients, TNFalpha levels rose sharply to reach pretreated values at 1 year of IFNbeta-1b treatment. At the beginning of therapy, 6 patients had high concentrations of serum TNFalpha, which decreased to normal values following IFNbeta-1b therapy. IFNgamma mRNA expression also diminished after 6 and 12 months of IFNbeta-1b therapy in the group of stable patients, whereas nonrelevant variations were observed in patients who had one relapse. Initially, patients' peripheral mononuclear cells secreted diminished amounts of TNFalpha and IFNgamma on PHA + PMA mitogen stimulation in comparison with normal control subjects. After 1 year of therapy, IFNbeta-1b restored the normal production of TNFalpha, whereas therapy did not restore IFNgamma secretion to control values. CONCLUSION: IFNbeta-1b decreases the spontaneous expression of two proinflammatory cytokines.

Glucocorticoids increase IL-10 expression in multiple sclerosis patients with acute relapse.

High doses of glucocorticoids (GCs) are widely employed to treat acute attacks in relapsing-remitting multiple sclerosis (MS) patients. Their beneficial effects are partially due to their capacity to regulate the cytokine network. In the present work, we have examined the effect of GCs on the production of the immunosuppressor cytokine IL-10. Blood samples from MS patients suffering an acute relapse were obtained immediately before initiating therapy and after receiving a daily dose of 1 g intravenous methylprednisolone (MP) for four days. Levels of IL-10 mRNA in PBMC were semiquantified by RT-PCR, whereas protein concentration in serum and in cell culture supernatant was measured by ELISA. Our results show that 7 out of the 9 patients studied displayed increased IL-10 mRNA expression as well as higher serum IL-10 concentration following steroid treatment. In contrast, mRNA expression of two inflammatory cytokines, TNFalpha and IFNgamma, decreased following steroid therapy. In vitro experiments employing normal PBMC showed that methylprednisolone (MP) upregulated IL-10 expression as determined by measuring mRNA levels, flow cytometry of intracytoplasmic protein concentration, and the amount of secreted protein. Peak responses of secreted IL-10 by PBMC cultured cells treated with MP were obtained at 48 h. The effect was steroid-specific as IL-10 expression reversed to baseline levels in the presence of the glucocorticoid receptor antagonist RU486. Contrary to the effect of MP on the spontaneous expression of IL-10, this drug downregulated LPS-induced IL-10 synthesis. In fact, the concentration of IL-10 in LPS-induced IL-10 secretion from normal PBMC decreased upon addition of MP to cell cultures. Thus, it seems that MP exerts an opposite effect on the spontaneous and LPS-induced IL-10 production. Our studies indicate that GCs may control inflammatory responses by upregulating production of the immunosuppressor cytokine IL-10.

Long-term effect of IFNbeta1b treatment on the spontaneous and induced expression of IL-10 and TGFbeta1 in MS patients.

Interferon-beta (IFNbeta) is an effective treatment that lessens the frequency and severity of exacerbations in relapsing-remitting multiple sclerosis (RRMS). The mechanism of action of IFNbeta1b may be by upregulating antiinflammatory cytokines levels. We studied the effect of IFNbeta1b treatment on the in vivo gene expression and protein synthesis of two immunosuppressive cytokines, IL-10 and TGFbeta1, and its persistence with chronic therapy. Peripheral blood samples were obtained from 16 patients before and after 3, 6 and 12 months of IFNbeta1b treatment. Eleven patients did not have any clinical relapse, whereas the other five each had one clinical exacerbation during the study. We employed a highly sensitive RT-PCR technique to study the spontaneous gene expression of IL-10 and TGFbeta1. Protein concentration in serum and in culture supernatants from mitogen-stimulated cells were measured by ELISA. In the group of patients who remained clinically stable during the study, IL-10 mRNA levels decreased significantly after 6 months of treatment to normalize at 1 year of therapy as compare with the initial values. In the five patients who relapsed, mRNA IL-10 levels were significantly diminished at 3, 6, and 12 months of therapy. IL-10 serum levels did not vary significantly in any group of patients during the study. Treatment did not modulate mRNA or serum levels of TGFbeta1 at any time period in the group of stable patients. However, in the five patients who relapsed, TGFbeta1 mRNA significantly decreased at 6 and 12 months of therapy. IFNbeta1b treatment was unable to restore the initial low mitogen-induced production of IL-10; only after 1 year of therapy was a slight increase observed. Cytokine therapy did not affect the mitogen-induced production of TGFbeta1. We can conclude that chronic administration of IFNbeta1b does not result in an upregulation of IL-10 and TGFbeta1.

Differential effect of IL10 and TNF{alpha} genotypes on determining susceptibility to discoid and systemic lupus erythematosus.

OBJECTIVE: To ascertain the possible involvement of functional interleukin 10 (IL10) and tumour necrosis alpha (TNFalpha) cytokine promoter polymorphisms on the susceptibility to discoid and systemic lupus erythematosus (DLE, SLE), and their associations with immunological features. METHODS: Single nucleotide polymorphisms of the IL10 (-1082, -819, and -592) and TNFalpha (-308) genes were determined using allele specific probes in 248 lupus patients and 343 matched controls. To assess functional significance of genotypes, basal mRNA cytokine levels were quantified in 106 genotyped healthy controls by real time RT-PCR. Specific autoantibodies and cutaneous manifestations were analysed in SLE patients and associated with functional genotypes. RESULTS: After analysing the distribution of IL10 and TNFalpha transcript levels according to promoter genotypes in healthy individuals, patients and controls were classified into functional single and combined genotypes according to the expected high or low constitutive cytokine production. High TNFalpha genotypes (-308AA or AG) were associated with SLE independently of IL10 alleles, whereas the risk of developing DLE and the prevalence of discoid lesion in SLE were higher in the high IL10/low TNFalpha producer group (-1082GG/-308GG). Cytokine interaction also influences the appearance of autoantibodies. Antibodies against Sm are prevalent among low producer patients for both cytokines, a genotype not associated with lupus incidence, whereas low IL10/high TNFalpha patients have the highest frequency of antibodies to SSa and SSb. CONCLUSIONS: IL10/TNFalpha interaction influences susceptibility to DLE and the appearance of specific autoantibodies in SLE patients, whereas high TNFalpha producer genotypes represent a significant risk factor for SLE.

Glucocorticoids up-regulate constitutive interleukin-10 production by human monocytes.

BACKGROUND: IL-10 plays an immunosuppressive role in inflammatory responses. Increased plasma levels of IL-10 have been detected in patients under glucocorticoid (GC) therapy, indicating that steroids may exert their suppressive effect, in part, by increasing IL-10 production. OBJECTIVES: The aim was to define possible mechanisms by which steroids up-regulate IL-10 production. To this end, we have analysed ex vivo the effect of GCs on the constitutive production of IL-10 by lymphocytes and cells of myeloid origin. METHODS: Monocytes and T cells were isolated by a Percoll gradient and B cells were purified by rosetting. Protein and mRNA IL-10 levels were determined by ELISA and by Northern blot, respectively. RESULTS: Monocytes, but not T or B cells, up-regulated the constitutive production of IL-10 following pre-treatment for at least 12 h with physiological doses of dexamethasone (Dex). Up-regulation of IL-10 occurred at both protein and mRNA levels, probably indicating that the effect of Dex was by incrementing gene transcription. Other steroids had similar outcomes, their effects being dose-related, proportional to the steroid potency and totally reversed by the steroid antagonist RU486. Thus, transcript levels of IL-10 were up-regulated by GCs probably through binding of the GC receptor to its specific glucocorticoid response element sequence in the IL-10 promoter. In contrast to monocytes, differentiated immature macrophages and dendritic cells did not vary their constitutive IL-10 production after pre-treatment with Dex. CONCLUSION: Our results support the fact that steroids up-regulate constitutive IL-10 production by selectively triggering activation signals on monocytes.

Differential expression of IL-4 receptors in human T and B lymphocytes.

IL-4R have been described in unstimulated human T and B lymphocytes. However, a precise comparative study on the expression and regulation of IL-4R in isolated human T and B cell populations has not yet been fully assessed. We examined the mRNA levels and the cell membrane expression of IL-4R in freshly isolated T and B lymphocytes as well as in in vivo- and in vitro-stimulated cells. IL-4R protein expression and transcript levels were higher in tonsillar unstimulated B cells than in T cells. Splenic and peripheral blood B lymphocytes also expressed higher surface IL-4R in their membranes than T cells did. Large B lymphocytes from tonsils (in vivo-activated cells) obtained by Percoll gradient centrifugation displayed higher IL-4R levels than resting cells. On activation in vitro of T lymphocytes with IL-2 or PHA, slight increments on the IL-4R mRNA and protein levels were achieved. However, maximal levels of IL-4R expression were obtained on T cell incubation with IL-4 at a concentration of 100 U/ml. Similarly, the same concentration of this lymphokine up-regulated the surface IL-4R molecules and the IL-4R mRNA levels in purified B lymphocytes. Cross-linkage of surface Ig by insolubilized anti-IgM potentiated the effect of IL-4 in up-regulating IL-4R expression in B cells, probably by inducing outgrowth of IL-4R positive subpopulations. The B cell mitogen, Staphylococcus Aureus Cowan I, although inducing cell proliferation, was ineffective in promoting new receptor synthesis. Cell proliferation was not required for IL-4-dependent IL-4R up-regulation on both T and B lymphocytes.

Epidemiology of systemic lupus erythematosus in a northern Spanish population: gender and age influence on immunological features.

The present work was planned to research epidemiological and immunological features of systemic lupus erythematosus (SLE) in a Caucasian population from the north of Spain (Asturias). There is only one specialized immunology laboratory in this region where samples from all patients with a plausible or a firm diagnosis of SLE are referred for immunological analysis. Since 1992 we have reviewed registered data from samples submitted to the immunology laboratory with a firm, definitive diagnosis of SLE, based on the fulfillment of the American College of Rheumatology (ACR) criteria. We have constructed a database, which included 367 SLE patients. The point prevalence was 34.12/ 100 000 (95% CI: 30.63-37.61/100 000), whereas the incidence rate calculated during the last five years was 2.15/100 000/year (95% CI: 1.76-2.54/100 000/year). The female/male ratio varied according to the age at diagnosis, being maximum (50 : 1) between 22 and 28 years. The median age at diagnosis was significantly lower in females than in males. Immunological features also yielded sex and age peculiarities. The percentage of patients with anti-SSa antibodies yielded significant differences between males (18.6%) and females (34.6%). Anti-RNP and anti-Sm antibodies were more frequently present in childhood-onset patients, the difference with the oldest-onset group being statistically significant. Other analyses did not show significant differences, although, as a whole, we observed a trend towards a higher presence of autoantibodies related to an early disease onset.

Induction of functional CD154 (CD40 ligand) in neonatal T cells by cAMP-elevating agents.

A deficiency of neonatal T lymphocytes to express CD154 antigen in response to ionomycin and phorbol 12-myrsistate 13-acetate (PMA) stimulation or after CD3 cross-linking has been described. In the present report we describe that CD45RA+ newborn cells are able to synthesize and express CD154 at similar or even higher levels than adult cells in response to ionomycin and cAMP-elevating agents which trigger the protein kinase A (PKA) -mediated metabolic pathway. Peak CD154 protein concentrations in newborn cells were found between 4 and 8 hr after stimulation with ionomycin and dibutyryl cAMP. These agents, however, did not induce expression of the early activation antigen CD69. Surface levels of CD154 did not correlate with specific mRNA concentration, indicating that dibutyryl cAMP up-regulates CD154 by acting at a post-transcriptional stage. The CD154 antigen induced by PKA activation of newborn cells was functional, since upon binding to CD40 on B lymphocytes in the presence of interleukin-4 (IL-4), it promoted immunoglobulin heavy-class switching to IgE. We also found a different pattern of cytokine production between neonatal and adult CD4+ T cells. In response to ionomycin and dibutyryl cAMP, cord blood cells were more prone than adult lymphocytes to secrete the T helper type 2-derived immunosuppressive cytokines IL-4 and IL-10. Taking into account that the feto-maternal environment is rich in cAMP-elevating agents, the reduced risk of graft versus host disease associated with cord blood trasplantation, as compared with the risk with adult bone marrow cell transplants, may be due to the bias of neonatal cells to differentiate towards the T helper type 2 functional cell subset.

Soluble, but not immobilized, anti-IgM antibody inhibits post-activation events leading to T-cell-dependent B-cell differentiation.

The potential for surface immunoglobulin-binding ligands to modify B-cell differentiation responses induced by activated T cells has been investigated. Activated T cells in human splenic mononuclear cells cultured on anti-CD3-coated plates induced B cells to produce large amounts of IgM and IgG. In this experimental system, cross-linking of B-cell antigen receptors by soluble, bivalent monoclonal or polyclonal anti-IgM antibodies completely inhibited IgM production, and greatly diminished IgG production, in a dose-dependent manner. Similar results were obtained using a F(ab')2 fragment of a goat anti-IgM antibody. Inhibition of B-cell differentiation by bivalent cross-linking reagents did not require the presence of antigen-presenting cells (APC), as comparable results were obtained in co-cultures of purified T and B cells. In contrast, enhanced immunoglobulin secretion was seen when surface IgM was cross-linked using anti-IgM antibody immobilized on the culture plate. Interestingly, activated T cells induced similar levels of expression on B cells of the activation antigens CD23, CD25 and CD71, and of class II molecules, irrespective of any treatment with soluble or immobilized anti-IgM antibody. This indicates that soluble anti-IgM specifically inhibits B-cell differentiation without altering initial events of T-cell-dependent B-cell activation.

Requirement of a second signal via protein kinase C or protein kinase A for maximal expression of CD40 ligand. Involvement of transcriptional and posttranscriptional mechanisms.

High levels of CD40 ligand (CD40L) protein expression are induced on native T cells by increasing the intracellular Ca2+ concentration. In the present study we have shown that ionomycin induces CD40L gene transcription leading to mRNA accumulation which translates to high levels of protein expression. Conversely, agents which increase the intracellular levels of cyclic AMP (cAMP), such as prostaglandin E2 (PGE2) or dibutyryl cyclic AMP (dbcAMP), were unable to induce CD40L expression on T lymphocytes. Cell activation by phorbol 12-myristate 13-acetate (PMA) treatment had a slight effect on increasing CD40L mRNA and protein levels. However, PMA and dbcAMP synergized with ionomycin to significantly increase and to prolong the CD40L expression. Nuclear run-on assays revealed that PMA, but not dbcAMP, increased threefold the CD40L gene transcription rate induced by ionomycin. This effect was independent of de novo protein synthesis. In addition, at a posttranscriptional level, both reagents synergized with the Ca2+ ionophore to prolong the CD40L mRNA half-life by a mechanism which was also independent of de novo protein synthesis. Moreover, when transcription was blocked with actinomycin D, an increment of the CD40L transcript levels induced by PMA or dbcAMP on ionomycin-treated cells was observed in the presence of cycloheximide. This probably means that newly synthesized protein may contribute to the CD40L mRNA destabilization. In summary, these data show that PMA and dbcAMP synergized with ionomycin to increase the CD40L mRNA and protein levels. The up-regulatory effect of PMA was accomplished at a transcriptional and posttranscriptional level, whereas dbcAMP exerted its synergistic effect exclusively at a posttranscriptional level.

Glucocorticoids inhibit IL-4 and mitogen-induced IL-4R alpha chain expression by different posttranscriptional mechanisms.

BACKGROUND: A high level of expression of IL-4Ralpha chain on the surface of lymphocytes has been described in certain allergic and inflammatory autoimmune diseases. Progression of these diseases are usually controlled by steroid treatment. One mechanism by which these drugs exert their antiinflammatory and immunosuppressive effects is by widely repressing or enhancing the production of cytokines and their receptors. OBJECTIVES: The effect of glucocorticoids on IL-4Ralpha chain expression has not been previously studied, and this is the aim of the present report. For this purpose, human lymphocytes were induced to express IL-4Ralpha chain by means of protein kinase C (PKC) activation with phorbol myristate acetate (PMA) or by triggering the Janus kinase-Stat pathway with IL-4 in the presence or absence of pharmacologic doses of dexamethasone. METHODS: IL-4Ralpha cell surface expression was studied by flow cytofluorometry. The levels and stability of mRNA were assessed by Northern blot analysis. The effect of dexamethasone on the IL-4Ralpha rate of transcription was determined by nuclear run-on experiments. RESULTS: Dexamethasone significantly downregulated PMA-induced IL-4Ralpha mRNA and protein levels in total peripheral blood mononuclear cells and in isolated T cells. The mechanism involved a posttranscriptional regulation of IL-4Ralpha expression because dexamethasone decreased the PMA-induced IL-4Ralpha mRNA half-life. However, we found that PMA did not influence the transcription rate of IL-4Ralpha gene, irrespective of the presence or absence of dexamethasone. This immunosuppressor also diminished the IL-4-induced IL-4Ralpha expression on the surface of isolated T and B lymphocytes but, interestingly, without modifying mRNA levels that indicates that dexamethasone downregulated IL-4-dependent IL-4Ralpha expression by acting at a translational or posttranslational level. In fact, we observed that the drug did not affect IL-4-induced IL-4Ralpha gene transcription rate nor did it shorten mRNA half-life. The effect of dexamethasone on the IL-4Ralpha was steroid specific because it was totally reversed by the glucocorticoid receptor antagonist RU486. CONCLUSION: Our results support that dexamethasone may influence the course of allergic and inflammatory diseases by downregulating the expression of IL-4Ralpha.

Upregulated expression of IL-4 receptors and increased levels of IL-4 in rheumatoid arthritis patients.

The level of IL-4R expression on peripheral lymphocyte subsets from rheumatoid arthritis (RA) patients and controls was studied by flow cytometric analysis of the binding of phycoerythrin-labelled IL-4 (PE-IL-4). In normal lymphocytes, IL-4R is mainly expressed on CD19+ cells, although it was also seen, at lower levels, on CD3+, CD4+ and CD8+ cells. In RA patients, a significantly increased spontaneous expression of IL-4R was observed, compared with controls, in the CD3+, CD4+ and CD19+ cell subsets. No significant differences in IL-4R expression were found between patients receiving steroids and those who were not, suggesting that steroids are not involved in upregulating IL-4R levels in vivo. Because IL-4 is a potent upregulator of IL-4R, we considered the possibility that incremented levels of circulating IL-4 in RA accounted for the high surface expression of IL-4R. By ELISA, we found abnormally high levels of immunoreactive IL-4 in 35.13% of patient serum samples, while it was undetectable in control sera. In addition, we examined IL-4 mRNA expression by polymerase chain reaction (PCR) in the PBMC of patients and controls. IL-4 PCR products were observed in four out of 10 patients studied but in none of the controls. No correlation was observed between the seric concentrations of IL-4 and IL-4R, indicating that activator factors other than IL-4 contribute to the upregulation of IL-4R expression in RA. Since the patients' sedimentation rate and CRP values did not correlate with the concentration of circulating IL-4, we conclude that this lymphokine does not contribute to the deleterious effect of the disease. Rather, due to its antiinflammatory properties, the overproduction of IL-4 in RA may be a compensatory mechanism neutralizing the harmful effect of activated macrophages.