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The paradox of high availability and low recognition of soluble HLA-G by LILRB1 receptor in rheumatoid arthritis patients.

Veit TD, Chies JA, Switala M, Wagner B, Horn PA, Busatto M, Brenol CV, Tavares Brenol JC, Machado Xavier R, Rebmann V - PLoS ONE (2015)

Bottom Line: Remarkably, the proportion of patients presenting specific binding of sHLA-G to LILRB1 was significantly decreased as compared to controls (56% vs. 81%, p=0.027).Patients without rheumatoid factor (RF-) were significantly overrepresented in the group of patients positive for LILRB1 binding as compared to patients without LILRB1 binding (31% vs 10%, p=0.033).Unlike in controls, no significant differences in sHLA-G levels were observed among patients grouped by 14 pb genotype.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunogenética, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

ABSTRACT
HLA-G is a regulatory molecule involved in immunologic tolerance. Growing evidence indicates that HLA-G plays a role in the regulation of inflammatory processes and autoimmune diseases. This study aimed at a systematic evaluation of soluble HLA-G (sHLA-G) in plasma of rheumatoid arthritis (RA) patients with long-lasting chronic inflammation. RA patients (n=68) and healthy controls (n=26) had their plasmatic sHLA-G measured by ELISA whereas the binding capability of sHLA-G to its cognate LILRB1 receptor was measured by a Luminex-based assay. All subjects were PCR-genotyped for HLA-G 14 bp polymorphism (rs66554220). Significantly higher sHLA-G levels were observed in patients (p<0.001), however no significant differences were observed in LILRB1 binding capacity between RA patients and controls. Remarkably, the proportion of patients presenting specific binding of sHLA-G to LILRB1 was significantly decreased as compared to controls (56% vs. 81%, p=0.027). Patients without rheumatoid factor (RF-) were significantly overrepresented in the group of patients positive for LILRB1 binding as compared to patients without LILRB1 binding (31% vs 10%, p=0.033). Furthermore, methotrexate treated patients (n=58) revealed significantly lower LILRB1 binding to sHLA-G molecules than non-treated patients (medians: 12.2 vs. 67.7 units/ml, p=0.031). Unlike in controls, no significant differences in sHLA-G levels were observed among patients grouped by 14 pb genotype. Thus, in a substantial number of late RA patients, the circulating sHLA-G molecules are impaired regarding LILRB1 recognition, meaning that although increased levels are observed; these molecules are not qualified to exert their protective functions against inflammation. Our findings offer new insights into the immunopathology of RA patients with long-lasting anti-RA-treatment and highlight the importance to also measure the binding capability of sHLA-G to LILRB1.

No MeSH data available.


Related in: MedlinePlus

Correlation of sHLA-G levels and its recognition by LILRB1 receptor in RA patients and healthy controls.Straight line indicates the linear regression and dotted line indicates the 95% confidence interval of regression. Closed cycles indicate plasma samples of RA patients with HLA-G molecules with an impaired LILRB1 recognition.
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pone.0123838.g003: Correlation of sHLA-G levels and its recognition by LILRB1 receptor in RA patients and healthy controls.Straight line indicates the linear regression and dotted line indicates the 95% confidence interval of regression. Closed cycles indicate plasma samples of RA patients with HLA-G molecules with an impaired LILRB1 recognition.

Mentions: In order to evaluate the potential recognition of sHLA-G molecules present in the RA patients plasma we analyzed the binding capacity of sHLA-G to its cognate LILRB1 receptor [28]. Importantly, in the recognition assay we used the same antibody (mAb G233) to capture sHLA-G as for the quantitative assessment, but this time bound molecules were exposed to recombinant human LILRB1 followed by anti-human LILRB1 antibody instead of polyclonal rabbit anti-human β2-microglobulin antiserum. Results of these assay showed that, despite the high sHLA-G levels in patients, no quantitative differences in recognition by LILRB1 were observed between patients and controls (Fig 2). The median in LILRB1 recognition was 12.9 FU/ml in the patients’ group and 15.3 FU/ml in the controls’ group (p = 0.632, Mann-Whitney test). However, the proportion of patients presenting specific recognition of sHLA-G molecules by LILRB1 (above the calculated detection limit of the test) was significantly decreased as compared to healthy controls (56% vs. 81%, p = 0.027, Chi-square test). Of note, among individuals with no detectable LILRB1 recognition, significantly higher median values of sHLA-G were identified in patients as compared to controls (5.42 vs. 0.83 FU/mL, p < 0.001, Mann-Whitney test, Table 2). This suggests that lack of LILRB1 recognition is mainly due to low amounts (not detectable) of sHLA-G in healthy individuals, whereas in RA patients it is rather a consequence of a large amount of non-recognized sHLA-G molecules. This assumption is further supported by the correlation analysis between sHLA-G levels and LILRB1 recognition: Although a significant positive correlation between sHLA-G level and LILRB1 recognition was found in both healthy controls (r = 0.57, p = 0.003) and RA patients (r = 0.52, p < 0.001), the correlation did differ with respect to the slope of their linear regression line (Fig 3): For healthy controls the slope of regression line was with 4.43 ± 2.2 steeply rising, whereas for RA patients the slope of regression line was with 0.96 ± 0.18, clearly decreased. In addition to 30 RA patients without detectable LILRB1 recognition, despite substantial levels of circulating sHLA-G molecules, two RA patients revealed sHLA-G molecules in a concentration above 45 ng/ml with a very weak recognition by the LILRB1 receptor. Thus, in a substantial number of late RA patients the circulating sHLA-G molecules in the blood were not or only hardly recognized by the LILRB1 receptor suggesting that these sHLA-G molecules are functionally inactive with regard to this receptor.


The paradox of high availability and low recognition of soluble HLA-G by LILRB1 receptor in rheumatoid arthritis patients.

Veit TD, Chies JA, Switala M, Wagner B, Horn PA, Busatto M, Brenol CV, Tavares Brenol JC, Machado Xavier R, Rebmann V - PLoS ONE (2015)

Correlation of sHLA-G levels and its recognition by LILRB1 receptor in RA patients and healthy controls.Straight line indicates the linear regression and dotted line indicates the 95% confidence interval of regression. Closed cycles indicate plasma samples of RA patients with HLA-G molecules with an impaired LILRB1 recognition.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390237&req=5

pone.0123838.g003: Correlation of sHLA-G levels and its recognition by LILRB1 receptor in RA patients and healthy controls.Straight line indicates the linear regression and dotted line indicates the 95% confidence interval of regression. Closed cycles indicate plasma samples of RA patients with HLA-G molecules with an impaired LILRB1 recognition.
Mentions: In order to evaluate the potential recognition of sHLA-G molecules present in the RA patients plasma we analyzed the binding capacity of sHLA-G to its cognate LILRB1 receptor [28]. Importantly, in the recognition assay we used the same antibody (mAb G233) to capture sHLA-G as for the quantitative assessment, but this time bound molecules were exposed to recombinant human LILRB1 followed by anti-human LILRB1 antibody instead of polyclonal rabbit anti-human β2-microglobulin antiserum. Results of these assay showed that, despite the high sHLA-G levels in patients, no quantitative differences in recognition by LILRB1 were observed between patients and controls (Fig 2). The median in LILRB1 recognition was 12.9 FU/ml in the patients’ group and 15.3 FU/ml in the controls’ group (p = 0.632, Mann-Whitney test). However, the proportion of patients presenting specific recognition of sHLA-G molecules by LILRB1 (above the calculated detection limit of the test) was significantly decreased as compared to healthy controls (56% vs. 81%, p = 0.027, Chi-square test). Of note, among individuals with no detectable LILRB1 recognition, significantly higher median values of sHLA-G were identified in patients as compared to controls (5.42 vs. 0.83 FU/mL, p < 0.001, Mann-Whitney test, Table 2). This suggests that lack of LILRB1 recognition is mainly due to low amounts (not detectable) of sHLA-G in healthy individuals, whereas in RA patients it is rather a consequence of a large amount of non-recognized sHLA-G molecules. This assumption is further supported by the correlation analysis between sHLA-G levels and LILRB1 recognition: Although a significant positive correlation between sHLA-G level and LILRB1 recognition was found in both healthy controls (r = 0.57, p = 0.003) and RA patients (r = 0.52, p < 0.001), the correlation did differ with respect to the slope of their linear regression line (Fig 3): For healthy controls the slope of regression line was with 4.43 ± 2.2 steeply rising, whereas for RA patients the slope of regression line was with 0.96 ± 0.18, clearly decreased. In addition to 30 RA patients without detectable LILRB1 recognition, despite substantial levels of circulating sHLA-G molecules, two RA patients revealed sHLA-G molecules in a concentration above 45 ng/ml with a very weak recognition by the LILRB1 receptor. Thus, in a substantial number of late RA patients the circulating sHLA-G molecules in the blood were not or only hardly recognized by the LILRB1 receptor suggesting that these sHLA-G molecules are functionally inactive with regard to this receptor.

Bottom Line: Remarkably, the proportion of patients presenting specific binding of sHLA-G to LILRB1 was significantly decreased as compared to controls (56% vs. 81%, p=0.027).Patients without rheumatoid factor (RF-) were significantly overrepresented in the group of patients positive for LILRB1 binding as compared to patients without LILRB1 binding (31% vs 10%, p=0.033).Unlike in controls, no significant differences in sHLA-G levels were observed among patients grouped by 14 pb genotype.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunogenética, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

ABSTRACT
HLA-G is a regulatory molecule involved in immunologic tolerance. Growing evidence indicates that HLA-G plays a role in the regulation of inflammatory processes and autoimmune diseases. This study aimed at a systematic evaluation of soluble HLA-G (sHLA-G) in plasma of rheumatoid arthritis (RA) patients with long-lasting chronic inflammation. RA patients (n=68) and healthy controls (n=26) had their plasmatic sHLA-G measured by ELISA whereas the binding capability of sHLA-G to its cognate LILRB1 receptor was measured by a Luminex-based assay. All subjects were PCR-genotyped for HLA-G 14 bp polymorphism (rs66554220). Significantly higher sHLA-G levels were observed in patients (p<0.001), however no significant differences were observed in LILRB1 binding capacity between RA patients and controls. Remarkably, the proportion of patients presenting specific binding of sHLA-G to LILRB1 was significantly decreased as compared to controls (56% vs. 81%, p=0.027). Patients without rheumatoid factor (RF-) were significantly overrepresented in the group of patients positive for LILRB1 binding as compared to patients without LILRB1 binding (31% vs 10%, p=0.033). Furthermore, methotrexate treated patients (n=58) revealed significantly lower LILRB1 binding to sHLA-G molecules than non-treated patients (medians: 12.2 vs. 67.7 units/ml, p=0.031). Unlike in controls, no significant differences in sHLA-G levels were observed among patients grouped by 14 pb genotype. Thus, in a substantial number of late RA patients, the circulating sHLA-G molecules are impaired regarding LILRB1 recognition, meaning that although increased levels are observed; these molecules are not qualified to exert their protective functions against inflammation. Our findings offer new insights into the immunopathology of RA patients with long-lasting anti-RA-treatment and highlight the importance to also measure the binding capability of sHLA-G to LILRB1.

No MeSH data available.


Related in: MedlinePlus