Erik Åhlin

Increased IL‐17A secretion in response to Candida albicans in autoimmune polyendocrine syndrome type 1 and its animal model

Autoimmune polyendocrine syndrome type 1 (APS‐1) is a multiorgan autoimmune disease caused by mutations in the autoimmune regulator (AIRE) gene. Chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal failure are hallmarks of the disease. The critical mechanisms causing chronic mucocutaneous candidiasis in APS‐1 patients have not been identified although autoantibodies to cytokines are implicated in the pathogenesis. To investigate whether the Th reactivity to Candida albicans (C. albicans) and other stimuli was altered, we isolated PBMC from APS‐1 patients and matched healthy controls. The Th17 pathway was upregulated in response to C. albicans in APS‐1 patients, whereas the IL‐22 secretion was reduced. Autoantibodies against IL‐22, IL‐17A and IL‐17F were detected in sera from APS‐1 patients by immunoprecipitation. In addition, Aire‐deficient (Aire0/0) mice were much more susceptible than Aire+/+ mice to mucosal candidiasis and C. albicans‐induced Th17‐ and Th1‐cell responses were increased in Aire0/0 mice. Thus an excessive IL‐17A reactivity towards C. albicans was observed in APS‐1 patients and Aire0/0 mice.

Cytokine induction by circulating immune complexes and signs of in-vivo complement activation in systemic lupus erythematosus are associated with the occurrence of anti-Sjögren's syndrome A antibodies

Circulating immune complexes (IC) and levels of IC‐induced cytokines have been correlated with complement activation and autoantibody profiles in systemic lupus erythematosus (SLE). SLE sera were analysed concerning levels of immune complexes (IC), classical complement function and different antinuclear and anti‐C‐reactive protein (CRP) autoantibodies. Blood mononuclear cells from healthy donors were stimulated with isolated IC and production of interleukin (IL)‐10, IL‐6 and IL‐12p40 was measured. Functional experiments revealed that increased levels of IC‐induced cytokines were associated with both increased classical complement activation and the occurrence of anti‐Sjögrens syndrome A (SSA) and anti‐SSB but not other autoantibodies. Biochemical measurement of circulating IC showed that the degree of complement activation and the occurrence of anti‐SSA were synergistically associated with levels of circulating IC in SLE sera, as complement activation was a prerequisite for the enhancing effect of anti‐SSA. Anti‐CRP was associated with complement activation, but not with other autoantibodies. Our results indicate that anti‐SSA and possibly anti‐SSB antibodies influence IC formation and subsequent IC‐induced cytokine induction, and that they thereby participate in the inflammatory process in active SLE.

Circulating Immune Complexes (IC) and IC-Induced Levels of GM-CSF Are Increased in Sudanese Patients with Acute Visceral Leishmania donovani Infection Undergoing Sodium Stibogluconate Treatment: Implications for Disease Pathogenesis

Infection with Leishmania donovani is associated with IL-10 as well as with GM-CSF. Immune complexes (IC) exert important functions by stimulation of monocytes/macrophage-mediated production of pro- and anti-inflammatory cytokines in rheumatic diseases. In this investigation, we have explored IC-induced cytokine production during Leishmania infection. Sera from 43 patients with visceral leishmaniasis (VL), 17 patients with post-kala-azar dermal leishmaniasis, and 20 healthy Sudanese controls were precipitated with polyethylene glycol (PEG). The PEG precipitates were added to serum-free PBMC for 20 h,whereupon supernatant levels of IL-1β, IL-6, IL-10, IL-1 receptor antagonist protein, TNF-α, TNF receptor p75, and GM-CSF were investigated using ELISA. Circulating levels of C1q-binding IC were also measured in the serum samples. PEG precipitates from Leishmania-infected patients induced significantly higher levels of GM-CSF (p = 0.0037) and IL-10 (p < 0.0001), as well as of IL-6 (p < 0.0001) and IL-1 receptor antagonist (p = 0.0238) as compared with PEG precipitates from controls. Patients with acute VL as well as VL patients receiving sodium stibogluconate treatment displayed significantly increased levels of PEG precipitate-induced GM-CSF. The induction of GM-CSF by circulating IC was especially prominent in acute VL patients receiving sodium stibogluconate treatment; ANOVA revealed significant interaction between disease activity and treatment for PEG precipitate-induced levels of GM-CSF (disease activity, p = 0.0006; treatment, p = 0.0005; interaction, p = 0.0046). Parallel associations were determined for C1q-binding immune complexes, but not for any cytokine other than GM-CSF. The importance of IC-induced GM-CSF in leishmaniasis warrants further study.

Autoantibodies associated with RNA are more enriched than anti-dsDNA antibodies in circulating immune complexes in SLE.

To what extent different autoantibodies accumulate in systemic lupus erythematosus (SLE) immune complexes (ICs), and whether such accumulation is associated with disease activity has been investigated. ICs were isolated from SLE sera by both polyethylene glycol (PEG) precipitation and C1q-binding. Autoantibody specificities were determined using a lineblot assay quantified by densitometry. To compare the relative levels of autoantibodies, levels were normalized to the total levels of IgG measured by ELISA in sera and parallel ICs. Samples were investigated both in a cross-sectional design as well as in a paired design with samples obtained during both active and inactive SLE. All investigated autoantibody specificities except anti-dsDNA were enriched in circulating ICs as compared with parallel sera. The group of antibodies against RNA-associated antigens (anti-RNP/Sm, anti-Sm, anti-SSA/Ro60, anti-SSA/Ro52, anti-SSB/La) all exhibited higher median enrichment than the DNA-associated (anti-dsDNA, anti-histones, anti-nucleosomes) or cytoplasmic (anti-ribosomal P) antigens. In particular autoantibodies against RNP/Sm and SSA/Ro52 had the highest degree of enrichment in SLE PEG precipitates. These findings were corroborated by analysis of autoantibody content in C1q-bound ICs. There was no difference in degree of IC accumulation of the investigated autoantibodies during active and inactive SLE. Our findings demonstrate a difference in enrichment between autoantibodies against RNA- and DNA-associated autoantigens in isolated SLE IC, suggesting that the RNA-associated autoantibodies are more prone to form circulating ICs in SLE, in contrast to antibodies against DNA-associated autoantigens such as dsDNA. These finding have implications in understanding mechanisms of differential autoantibody accumulation in target organs in SLE.

Activity and turnover of eosinophil and neutrophil granulocytes are altered in visceral leishmaniasis

Visceral leishmaniasis (VL) is a health issue in Sudan. Our aim was to investigate the involvement of eosinophils and neutrophils in VL by serum and plasma measurements of eosinophil cationic protein (ECP) and myeloperoxidase (MPO) and some key cytokines and chemokines. Blood was collected from 125 VL patients and 181 healthy Sudanese controls from the same rural area. Results showed reduced eosinophil and neutrophil counts in the VL group (P=0.0001 and P=0.002, respectively). Serum-ECP levels were higher in the controls (P<0.0001), while plasma MPO levels were higher in the VL group (P<0.0001). Levels of IL-5, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-17 were increased among the VL group (P<0.0001, P=0.017 and P=0.03, respectively), whereas eotaxin and IL-8 levels were reduced (P<0.0001 and P=0.002, respectively). Positive correlations were found between IL-8 and ECP/MPO (P<0.0001). We conclude that eosinophil and neutrophil turnover and activity are increased in subjects in rural areas of Sudan. In VL the turnover was further increased, but the relatively low secretory activity of eosinophils and neutrophils in VL may relate to the reduced production and availability of the chemokines eotaxin and IL-8. The combined assay of ECP and MPO in serum and plasma provides further insight into the mechanisms of eosinophil and neutrophil involvement in disease and constitutes a novel approach to the study of disease processes.

Immune complex-mediated cytokine production is regulated by classical complement activation both in vivo and in vitro

Immune complexes (IC) induce a number of cellular functions, including the enhancement of cytokine production from monocytes, macrophages and plasmacytoid dendritic cells. The range and the composition of cytokines induced by IC in vitro is influenced by the availability of an intact classical complement cascade during cell culture, as we have showed in our studies on artificial IC and on cryoglobulins purified from patients with lymphoproliferative diseases. When IC purified from systemic lupus erythematosus sera were used to stimulate in vitro cytokine production, the amount of circulating IC and IC-induced cytokine levels depended both on in vivo classical complement function as well as on the occurrence of anti-SSA, but not on anti-dsDNA or any other autoantibodies. Collectively these findings illustrate that studies on IC-induced cytokine production in vitro requires stringent cell culture conditions with complete control and definition of access to an intact classical complement pathway in the cell cultures. If IC are formed in vivo, the results have to be interpreted in the context of classical complement activation in vivo as well as the occurrence of IC-associated autoantibodies at the time of serum sampling.

Anti-Citrullinated Peptide Antibodies in Sudanese Patients with Leishmania donovani Infection Exhibit Reactivity not Dependent on Citrullination

African patients with Leishmania donovani infections have signs of strong systemic inflammation and high levels of circulating immune complexes (IC) and rheumatoid factor (RF), all serologic markers of rheumatic disease. As inflammation in general is associated with citrullination, we sought to investigate ACPA responses in Sudanese Leishmania patients. Serum samples were collected from Sudanese patients with visceral leishmaniasis (VL) and post‐kala‐azar dermal leishmaniasis (PKDL) as well as from ACPA‐positive Sudanese rheumatoid arthritis patients and compared to healthy Sudanese controls. Levels of circulating C1q‐binding IC and anticyclic citrullinated peptide 2(CCP2) were investigated using ELISA, and RF was measured with nephelometry. C1q adsorption was carried out to investigate anti‐CCP2 content in IC. Citrulline specificity was evaluated with control plates with cyclic arginine‐containing control peptides. Leishmania‐infected patients had elevated levels of RF and circulating IC but also a significant increase in anti‐CCP2 (12%) as compared to healthy controls. Anti‐CCP2‐positive Leishmania patients displayed lower anti‐CCP2 levels than Sudanese patients with rheumatoid arthritis (RA), and anti‐CCP2 levels in Leishmania patients showed a continuum not resembling the dichotomous pattern seen in patients with RA. Whereas the anti‐CCP reactivity of Sudanese RA sera was strictly citrulline dependent, anti‐CCP2‐positive Leishmania sera reacted equally well with ELISA plates containing arginine control peptides. There was a strong correlation between anti‐CCP2 and circulating IC among the Leishmania patients, but IC depletion only marginally diminished anti‐CCP2 levels. Our findings stress the importance to interpret a positive CCP test carefully when evaluated in non‐rheumatic conditions associated with macrophage activation.

Anticorpos antipeptídeos citrulinados e fator reumatoide em pacientes sudaneses com infecção por Leishmania donovani

[online]. 2011, vol.51, n.6, pp. 579-586. ISSN 0482-5004. http://dx.doi.org/10.1590/S0482-50042011000600005. RESUMO: A Revista Brasileira de Reumatologia decidiu retratar o artigo entitulado Ahlin E, Elshafei A, Nur M, El Safi SH, Johan R, Elghazali G. Anticorpos antipeptideos citrulinados e fator reumatoide em pacientes sudaneses com infeccao por

Autoantibodies associated with RNA are more enriched than anti-dsDNA antibodies in circulating immune complexes in SLE

Background and objective We have earlier shown that polyethylene glycol (PEG)-precipitated circulating immune complexes (CIC) from systemic lupus erythematosus (SLE) patients induce cytokines in vitro, and that levels of both CIC and CIC-induced cytokines are dependent on (1) SLE disease activity and (2) the occurrence of anti-SSA but not anti-dsDNA autoantibodies. These findings implicated anti-SSA in immune complex formation, but the formal proof that anti-SSA participates in the formation of SLE CIC to a greater degree than did other antinuclear antibody-associated autoantibodies was lacking. The aim of this study was to investigate to what extent different autoantibodies accumulate in SLE immune complexes, and whether such accumulation is dependent on SLE disease activity. Methods Circulating immune complexes were isolated from SLE sera by both PEG precipitation (n=19) and C1q binding (n=8). Autoantibody specificities were determined with a semiquantitative lineblot assay quantified by densitometry. To compare the relative levels of autoantibodies they were normalised against total IgG measured by ELISA in sera and CIC in parallel. Samples were investigated both in a cross-sectional design (n=19), as well as in a paired design with samples obtained during active and inactive SLE (n=10). Results All autoantibodies were enriched in CIC compared to in serum. The group of antibodies against RNA-associated antigens showed higher median enrichment than anti-dsDNA (anti-RNP/Sm p=0.0228, anti-Sm p=0.0392, anti-SSA/Ro60 p=0.0539, anti-SSA/Ro52 p=0.0122, anti-SSB/La p=0.0342). Similarly, the other DNA-associated antigens (antihistones and antinucleosomes) were also less enriched as compared with the RNA-associated specificities. These findings were corroborated by analysis of autoantibody content in C1q-bound CIC. There was no difference in degree of accumulation of the investigated autoantibodies in CIC during active and inactive SLE. Conclusion Our findings demonstrate a difference in enrichment between autoantibodies against RNA- and DNA-associated autoantigens in isolated SLE CIC, suggesting that the RNA-associated autoantibodies are more prone to form complexes in the circulation in SLE, in contrast to antibodies against DNA-associated autoantigens including dsDNA. These finding have implications on by what mechanisms different autoantibodies accumulate in target organs in SLE.

Anti-cyclic citrullinated peptide antibodies, other common autoantibodies, and smoking as risk factors for lymphoma in patients with rheumatoid arthritis

Objectives: Patients with rheumatoid arthritis (RA) are at increased risk of lymphoma. There is no biomarker to indicate future lymphoma risk in RA and it is not known whether factors associated with an increased risk of RA also confer an increased risk of lymphoma. We investigated whether anti-cyclic citrullinated peptide (CCP) antibodies, other autoantibodies, and smoking, are associated with lymphoma development in RA. Method: From two population-based case–control studies, the Scandinavian Lymphoma Etiology (SCALE) study and the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) I study, we identified lymphoma cases with a validated RA diagnosis (n = 50), to whom we matched study participants with RA but no lymphoma (n = 261), lymphoma but no RA (n = 257), and neither RA nor lymphoma (n = 233). Lymphomas were classified according to the WHO classification. Blood samples were analysed for immunoglobulin G (IgG), IgM, and IgA isotypes and IgG1–4 subclasses of anti-CCP antibodies and for 15 antinuclear antibody (ANA)-associated specific autoantibodies. Relative risks were estimated as crude and adjusted odds ratios (adjOR) with 95% confidence intervals (CIs) using logistic regression. Results: We found no association between anti-CCP IgG ≥ 25 units/mL (adjOR 1.4, 95% CI 0.7–2.7), anti-CCP IgG ≥ 500 units/mL (adjOR 1.4, 95% CI 0.7–3.0), anti-CCP Ig of other isotypes, other autoantibodies (adjOR any vs none 0.6, 95% CI 0.3–1.2), or cigarette smoking (adjOR ever vs never 1.1, 95% CI 0.5–2.2) and lymphoma risk among patients with RA. Conclusion: In this study, neither anti-CCP antibodies (IgG, IgG1–4, IgM, or IgA), nor other common autoantibodies, nor smoking predicted lymphoma risk in RA.