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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

sHLA-G recognition by LILRB1 receptor.Straight line indicates the linear regression and dotted line indicates the 95% confidence interval of regression. MFI = mean fluorescence intensity. One unit/ml sHLA-G5 corresponds to 1ng/ml of purified sHLA-G5.
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pone.0123838.g001: sHLA-G recognition by LILRB1 receptor.Straight line indicates the linear regression and dotted line indicates the 95% confidence interval of regression. MFI = mean fluorescence intensity. One unit/ml sHLA-G5 corresponds to 1ng/ml of purified sHLA-G5.

Mentions: For the measurement of LILRB1 receptor recognition to sHLA-G molecules in plasma the Luminex-x-MAP technology and instruments were used (Luminex). Microspheres with color code 36 were covalently coupled with the G233 mAb [28]. Recognition of the mAb to the micropheres was performed as recently described [29]: G233 coupled microspheres (1250 per sample) were incubated in a total volume of 50μl with plasma diluted 1:4 in Luminex buffer (Cayman). Thereafter the bound HLA-G molecules were exposed to recombinant human LILRB1 receptor protein fused to the Fc region of human IgG1 (R&D Systems). Then, the bound LILRB1 receptor was detected by the anti-human LILRB1 mAb (BD Biosciences), PE conjugated. Measurement of the microspheres was carried out by the Luminex 100 IS System (Luminex). In total, the median fluorescence intensity from 100 microspheres was calculated in each sample. For the determination of HLA-G molecules recognized by the LILRB1 receptor, purified HLA-G5 was used in concentrations ranging from 0–224 ng/ml (Fig 1). The LILRB1 recognition of sHLA-G is given in fluorescence units (FU)/ml. One unit corresponds to 1 ng purified HLA-G5. The detection limit was 11.9 units/ml. Luminex buffer was used as a negative control.


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)

sHLA-G recognition by LILRB1 receptor.Straight line indicates the linear regression and dotted line indicates the 95% confidence interval of regression. MFI = mean fluorescence intensity. One unit/ml sHLA-G5 corresponds to 1ng/ml of purified sHLA-G5.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123838.g001: sHLA-G recognition by LILRB1 receptor.Straight line indicates the linear regression and dotted line indicates the 95% confidence interval of regression. MFI = mean fluorescence intensity. One unit/ml sHLA-G5 corresponds to 1ng/ml of purified sHLA-G5.
Mentions: For the measurement of LILRB1 receptor recognition to sHLA-G molecules in plasma the Luminex-x-MAP technology and instruments were used (Luminex). Microspheres with color code 36 were covalently coupled with the G233 mAb [28]. Recognition of the mAb to the micropheres was performed as recently described [29]: G233 coupled microspheres (1250 per sample) were incubated in a total volume of 50μl with plasma diluted 1:4 in Luminex buffer (Cayman). Thereafter the bound HLA-G molecules were exposed to recombinant human LILRB1 receptor protein fused to the Fc region of human IgG1 (R&D Systems). Then, the bound LILRB1 receptor was detected by the anti-human LILRB1 mAb (BD Biosciences), PE conjugated. Measurement of the microspheres was carried out by the Luminex 100 IS System (Luminex). In total, the median fluorescence intensity from 100 microspheres was calculated in each sample. For the determination of HLA-G molecules recognized by the LILRB1 receptor, purified HLA-G5 was used in concentrations ranging from 0–224 ng/ml (Fig 1). The LILRB1 recognition of sHLA-G is given in fluorescence units (FU)/ml. One unit corresponds to 1 ng purified HLA-G5. The detection limit was 11.9 units/ml. Luminex buffer was used as a negative control.

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