Amir I. Elshafie

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.

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.

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.

General false positive ELISA reactions in visceral leishmaniasis. Implications for the use of enzyme immunoassay analyses in tropical Africa.

Leishmaniasis is a neglected disease in tropical countries. Clinical and laboratory features may mimic autoimmune diseases and this can complicate the Leishmania diagnosis. Due to our previous investigation for false anti-CCP2 reactivity in Leishmania-infected subjects and our interest in immunity against the joint-specific collagen type II (CII) in rheumatoid arthritis (RA) we investigated the same cohort for anti-CII antibodies. We found elevated anti-CII reactivity in Leishmania-infected patients as compared to controls. When anti-CII OD values were compared with BSA-blocked control plates we found higher reactivity against BSA than in CII-coated plates in many Leishmania-infected patients. The percentage of such false positive anti-CII reactions increased with inflammatory activity, and was found in almost all Leishmania patients with highly active inflammatory disease, but was as low in Sudanese healthy controls as well as among Swedish RA patients. The correlation coefficients between false positive anti-CII and anti-CCP2 measured with a commercial ELISA were highest for patients with the most inflammatory disease but non-significant for Sudanese controls and Swedish RA patients, arguing that our findings may have general implications for ELISA measurements in leishmaniasis. ELISA investigations in areas endemic for leishmaniasis might benefit from individual-specific control wells for each serum sample. This approach might also be applicable to other geographical areas or patient groups with high incidence of inflammatory and infectious diseases.

The genetically determined production of the alarmin eosinophil-derived neurotoxin is reduced in visceral leishmaniasis

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis. Recent findings indicate that dendritic cells have a key role in the defense against the Leishmania parasite and that the activity of this cell may be modified by the eosinophil secretory protein eosinophil‐derived neurotoxin (EDN). We hypothesized that the interactions between dendritic cells and EDN might be of importance in the disease development. Cellular content of EDN was analyzed by ELISA. The single‐nucleotide polymorphisms at positions 405, 416, and 1122 in the EDN gene were analyzed by real‐time PCR with TaqMan® reagents. The study cohorts comprised 239 Sudanese subjects (65 healthy controls and 174 with VL) and 300 healthy Swedish controls. The eosinophil content of EDN was lower in VL as compared with controls (p < 0.0001). The EDN405 (G>C) genotype distribution was similar among Swedish and Sudanese controls, whereas VL subjects had a higher prevalence of the EDN405‐GG genotype (p < 0.0001). The content of EDN in the eosinophils was closely linked to the EDN405 polymorphism (p = 0.0002). Our findings suggest that the predisposition to acquire VL is related to the genetic polymorphism of the EDN gene and the reduced production by the eosinophil of this gene product.

Active Rheumatoid Arthritis in Central Africa: A Comparative Study Between Sudan and Sweden.

Objective. To compare clinical characteristics and treatment between simultaneously investigated Sudanese and Swedish outpatients with rheumatoid arthritis (RA). Methods. Outpatients with RA from Sudan (n = 281) and Sweden (n = 542) diagnosed according to the 1987 American College of Rheumatology criteria were recruited between December 2008 and September 2010 and compared concerning clinical presentation, treatment, and laboratory findings, including immunoglobulin M with rheumatoid factor (IgM-RF). Results. Sudanese patients had lower inclusion age (median 49 vs 68 yrs), disease duration (48 vs 107 mos), and disease onset age (43 vs 56 yrs) as compared with Swedish patients (p < 0.0001 for all). When stratified concerning the age of inclusion, Swedish patients between 41–50 years had, however, a significantly lower age of onset, with a similar trend for all age groups above 30 years. The female preponderance was higher among Sudanese patients (89.3% vs 72.5%, p < 0.0001), and smoking was nonexistent among Sudanese female patients (p < 0.0001). Erythrocyte sedimentation rate levels and number of tender joints were significantly higher among Sudanese patients. The proportion of IgM-RF positivity was lower among Sudanese patients with RA (52.4% vs 75.5%, p < 0.0001). Higher proportions of Sudanese patients with RA were treated with methotrexate (MTX) and disease-modifying antirheumatic drug combinations, but none of them used biologics. Sudanese patients used lower doses of MTX and sulfasalazine (p < 0.0001) and higher doses of prednisolone (p < 0.0001) than Swedish patients. Conclusion. Sudanese patients with RA have significantly higher disease activity and are often IgM-RF–seronegative. Together with reports from Uganda and Cameroon, our data indicate a cluster of highly active and often seronegative RA in central Africa.

Anti-type II collagen immune complex-induced granulocyte reactivity is associated with joint erosions in ra patients with anti-collagen antibodies

Objective RA patients with anti-collagen antibodies (anti-CII) are characterised by acute RA onset (Mullazehi et al, ARD 2007) and early joint erosions (Mullazehi et al ART 2012), as well as increased production of TNF by peripheral blood mononuclear cells from anti-CII immune complexes (IC) in vitro (Mullazehi et al A&R 2006). We have previously (abstract EWRR 2012) shown that polymorphonuclear neutrophil granulocytes (PMN) react towards anti-CII IC. The aim was to investigate whether functional PMN responses are associated with the clinical acute onset RA phenotype. Methods A set of 72 baseline patient sera (24 anti-CII positive, 24 anti-CII negative/RF positive and 24 anti-CII negative/RF negative) was chosen from a clinically well-characterised RA cohort with 2-year radiological follow-up (Larsen score). PMN and PBMC isolated from healthy donors were stimulated with anti-CII IC prepared with sera from the patients. PMN expression of CD16 and CD66b were measured by flow cytometry, and PMN production of myeloperoxidase (MPO) and IL-17, and PBMC production of TNF was measured with ELISA. Results CD66b, MPO, and TNF were significantly up-regulated and CD16 was significantly down-regulated by IC made with anti-CII positive sera. Even anti-CII low positive sera were able to impact the expression of CD16, CD66b and TNF. There was linear correlation to CD16, CD66b, MPO and TNF production in relation to anti-CII levels (r = -0.3152, 0.6755, 0.2532 and 0.5846, respectively). CD16 correlated with early joint erosion (p = 0.024, 0.034, 0.046 at baseline, one and two years) and CD66b was associated with changes in joint erosion (p = 0.017 and 0.016, at one and two years compared to baseline, respectively). CD66b was associated with baseline CRP and PBMC production of TNF was associated with baseline ESR, in accordance to our earlier findings. No correlations were observed with IL-17. Conclusion We have earlier shown that PBMC anti-CII IC-induced production of TNF was associated with CRP and ESR in newly diagnosed RA patients. Here we show that PMN reactivity against anti-CII IC is uniquely associated with joint destruction. PMN reactivity towards anti-CII IC in cartilage of patients can contribute to joint destruction in newly diagnosed RA patients.

A1.04 Type II collagen immune complex induce granulocyte dependent augmentation of chemokines via TLR4; a possible therapeutic target in early ra

Background RA patients with anti-collagen type II (CII) antibodies have a distinct acute onset phenotype, associated with cytokine induction by surface-bound anti-CII immune complexes (IC). Mononuclear cells and PMN are accumulated both in the synovial fluid and tissue, in close proximity to hyaline cartilage where anti-CII IC might form. Therefore we wanted to investigate to what extent these two cell types cooperate in anti-CII IC induced responses. Methods Healthy donor granulocytes (PMN) and PBMC were stimulated individually or together (cocultures) with surface-bound IC (anti-CII IC, tetanus toxoid (TT)/anti-TT IC, directly bound IgG), GM-CSF or LPS. Blocking and neutralising studies were performed with antibodies against TLR4, FcgγRIIa, FcgγRIII and GM-CSF. Supernatant cytokine and chemokine levels were analysed with ELISA or ALBIA. Results Anti-CII IC induced cytokine production by PBMC, whereas PMN alone produced negligible cytokine levels. TNF-aα production was downregulated in all coculture systems compared to PBMC cultures, whereas levels of CXCL8, RANTES and MCP-1 were specifically upregulated in anti-CII IC-stimulated cocultures. The CXCL8 upregulation was dependent on anti-CII IC, as CXCL8 production was downregulated in cocultures stimulated with other IC or LPS. The increase of CXCL8 in anti-CII IC stimulated cocultures was totally dependent on TLR4, partly on PMN enzymes, FcgγRIIa, FcgγRIII, and density of anti-CII in IC. Like anti-CII IC, GM-CSF induced coculture-dependent CXCL8 enhancement, and GM-CSF neutralisation diminished the anti-CII IC-dependent CXCL8 enhancement. Conclusion In anti-CII-positive RA patients, PMN amplify accumulation of inflammatory cells by inducing chemokines. This mechanism is dependent on TLR4, PMN enzymes, GM-CSF and the joint-specific autoantigen CII. Local TLR4 blockade, PMN enzyme inhibition or GM-CSF neutralisation might be used to suppress acute joint inflammation in early RA.

A5.24 Neutrophil Granulocytes Respond to Surface-Bound Immune Complexes Containing Anti-Type II Collagen Antibodies from RA Patients

Background and Objectives We have earlier shown that surface-bound immune complexes containing anti-type II collagen antibodies (anti-CII IC) from rheumatoid arthritis (RA) patients and anti-CII IC stimulate monocyte proinflammatory cytokine production, associated with an acute onset RA phenotype. Anti-CII IC in joint cartilage are exposed to cells in the synovial fluid (SF). Neutrophil granulocytes are the major cell type in SF, where they co-localise with mononuclear cells (MNC). The objective was to investigate whether also granulocytes respond to anti-CII IC, and whether such a response was dependent on interaction with other cells in SF. Materials and Methods An anti-CII RA serum together with human native collagen (CII) was used to create surface-bound anti-CII IC. Heparinised blood from 8 healthy donors was separated into neutrophil granulocytes (>95% purity) and MNC. For each donor, the granulocyte cell fractions as well as co-cultures (granulocytes + MNC) (0.5 × 10E6/ml of each cell fraction) was cultured on anti-CII IC as well as on negative control IC prepared with normal human serum on CII and in a positive control IC system with purified IgG coated onto plastic. After 18 hours, cells were harvested for the measurement of CD11b, CD66b, CD16 and CD32 on granulocytes by flow cytometry, and supernatant levels of TNF and IL-8 was measured by ELISA. Results In granulocyte cultures both anti-CII IC and control IC induced significant up-regulation of CD11b and CD66b, and significant down-regulation of CD16 and CD32. When the granulocytes were co-cultured with MNC, there was a significant increase in CD11b up-regulation and CD16 down-regulation than granulocytes, with no effect on CD32 and CD66b. In the co-culture system, the anti-CII IC-induced production of IL-8 was significantly increased, but no such difference was noted for TNF. Isolated granulocyte fractions produced very low levels of TNF and IL-8 after IC stimulation. Conclusions Isolated granulocytes respond to RA anti-CII IC in a model system mimicking IC in RA cartilage. The granulocyte responses depend on interaction with MNC. Our anti-CII dependent RA phenotype is a human counterpart to collagen antibody-induced arthritis. Strong granulocyte reactivity to anti-CII IC might therefore be related to the Ncf1 gene involved in NADPH activity important in collagen-induced arthritis models.

A10.15 IgA Rheumatoid Factor is more Predominant than anti-CCP in Sudanese Rheumatoid Arthritis Patients, whereas IgG RF is a Strong Prognostic Marker and Associated with Early Onset

Background and Objectives The aim was to investigate the diagnostic and prognostic impact of the conventionally used autoantibodies (IgG) anti-CCP and IgM rheumatoid factor (RF) as well as IgA and IgG RF in the first ever collected cohort of Sudanese rheumatoid arthritis (RA) patients. Materials and Methods 264 consecutive RA patients (87% females) diagnosed according to the 1987 ACR criteria attending two rheumatology centres in Khartoum between December 2008 and September 2010 were included, together with 168 healthy Sudanese blood donor controls. Autoantibody levels were investigated in Uppsala, and RF specificity levels aligned to the anti-CCP specificity (97.6%). Results Anti-CCP was elevated in 52% (131/252) of the patients, a figure not different from what has been found in Sweden (57%, Rönnelid ARD 2005; p = 0.2). Among the Sudanese RA patient, 57.2%, 51% and 49.8% had IgA, IgM and IgG RF, respectively. The areas under the Receiver Operator Characteristics (ROC) curves were 0.94 for anti-CCP, and 0.95, 0.82 and 0.85 for IgA, IgG and IgM RF, respectively. IgG RF was associated with young age (p = 0.0005) and lower age of disease onset (p < 0.0001), as well as with higher total number of affected joints (p = 0.03). Hand deformities like swan neck deformity (p = 0.0001) and boutonnière deformity (p = 0.02) were also primarily associated with IgG RF. Association with the other investigated autoantibodies were weaker or absent. The prognostic impact of IgG RF was not secondarily dependent on anti-CCP, as the correlation between anti-CCP was stronger for IgM RF (r = 0.49) and IgA RF (r = 0.31) than for IgG RF (r = 0.23). Conclusions The occurrence of anti-CCP in Sudanese RA patients does not differ from Sweden. In contrary to what has been found in Caucasian RA populations, IgA RF is a diagnostically more sensitive marker than anti-CCP IgG RF is the strongest marker for bad prognosis, and associated with early disease onset.