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Cross-linking of IgGs bound on circulating neutrophils leads to an activation of endothelial cells: possible role of rheumatoid factors in rheumatoid arthritis-associated vascular dysfunction.

Rollet-Labelle E, Vaillancourt M, Marois L, Newkirk MM, Poubelle PE, Naccache PH - J Inflamm (Lond) (2013)

Bottom Line: Cross-linking of IgGs bound on neutrophils leads to FcγRs-dependent tyrosine phosphorylation, mobilisation of intracellular calcium and the extracellular release of superoxide anions and lysozyme.Incubation of endothelial cells with the supernatant of activated neutrophils increases ICAM-1 expression and IL-8 production by endothelial cells.Our results show that activation of neutrophils' FcγRs by rheumatoid factors could participate in rheumatoid arthritis-associated vascular damage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Départements de Microbiologie-Infectiologie et Immunologie et de Médecine, Centre de recherche en rhumatologie et immunologie, Centre de recherche du CHU de Québec, Université Laval, Québec, QC, Canada. emmanuelle.rollet@crchul.ulaval.ca.

ABSTRACT

Background: Rheumatoid arthritis is characterized by the presence of circulating auto-antibodies, including rheumatoid factors, which recognize the Fc portion of IgGs. The neutrophil is the most abundant circulating leukocyte and it expresses high levels of FcγRs on its surface. The aim of the present study was to examine the capacity of circulating human neutrophils to be activated by rheumatoid factors and the consequences of these events on endothelium.

Methods: Neutrophil-bound IgGs were cross-linked with anti-human IgGs to mimick the presence of circulating rheumatoid factors and FcγRs-dependent signalling events and functions were examined. The IgG and IgM composition of rheumatoid factors isolated from the serum of RA patients was characterized. Adhesion of neutrophils to endothelial cells was quantified in response to the addition of rheumatoid factors.

Results: Cross-linking of IgGs bound on neutrophils leads to FcγRs-dependent tyrosine phosphorylation, mobilisation of intracellular calcium and the extracellular release of superoxide anions and lysozyme. Incubation of endothelial cells with the supernatant of activated neutrophils increases ICAM-1 expression and IL-8 production by endothelial cells. Finally, rheumatoid factors enhance neutrophil adhesion to endothelial cells.

Conclusions: Our results show that activation of neutrophils' FcγRs by rheumatoid factors could participate in rheumatoid arthritis-associated vascular damage.

No MeSH data available.


Related in: MedlinePlus

Rheumatoid factors induce FcγRs-dependent adhesion of neutrophils to HUVECs. A: An aliquot of the preparation of rheumatoid factors (100 ng and 10 ng) was analyzed by SDS-PAGE followed by immunoblotting with horseradish peroxidase-labelled anti-human IgG or anti-human IgM antibodies. The same amounts of commercial human IgG and IgM were analyzed on the same gel. The IgM band was used for quantification (arrow). B: Neutrophils were incubated with calcein and with or without PPP, as described in Methods. Neutrophils were then plated on a confluent monolayer of endothelial cells and RFs (500 μg/ml) were added for 2 hours. Non-adherent neutrophils were discarded and the amount of adherent cells was quantified as the amount of fluorescence bound to endothelial cells. Results are expressed as mean +/− SEM of three independent experiments. ** = P < 0.005, *** = P < 0.0005 (one-sample t test). C: Heat aggregated IgGs (HA-IgGs) were prepared as previously described [41]. Neutrophils were incubated with calcein, plated on endothelial cells and 100 μg/ml HA-IgG (HA) were added for 2 hours. Results are expressed as mean +/− SEM of three independent experiments, each carried out in triplicate. ** = P < 0.005, *** = P < 0.0005 (Mann–Whitney test).
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Figure 6: Rheumatoid factors induce FcγRs-dependent adhesion of neutrophils to HUVECs. A: An aliquot of the preparation of rheumatoid factors (100 ng and 10 ng) was analyzed by SDS-PAGE followed by immunoblotting with horseradish peroxidase-labelled anti-human IgG or anti-human IgM antibodies. The same amounts of commercial human IgG and IgM were analyzed on the same gel. The IgM band was used for quantification (arrow). B: Neutrophils were incubated with calcein and with or without PPP, as described in Methods. Neutrophils were then plated on a confluent monolayer of endothelial cells and RFs (500 μg/ml) were added for 2 hours. Non-adherent neutrophils were discarded and the amount of adherent cells was quantified as the amount of fluorescence bound to endothelial cells. Results are expressed as mean +/− SEM of three independent experiments. ** = P < 0.005, *** = P < 0.0005 (one-sample t test). C: Heat aggregated IgGs (HA-IgGs) were prepared as previously described [41]. Neutrophils were incubated with calcein, plated on endothelial cells and 100 μg/ml HA-IgG (HA) were added for 2 hours. Results are expressed as mean +/− SEM of three independent experiments, each carried out in triplicate. ** = P < 0.005, *** = P < 0.0005 (Mann–Whitney test).

Mentions: Although the presence of RFs is associated with the severity of RA symptoms, very few studies have defined a biologic or pathologic function of these antibodies [40]. The results presented above suggest that RFs either as ligands of FcγRs or as antibodies recognizing membrane-bound IgGs might activate neutrophils. To verify this hypothesis, we decided to test the capacity of human RFs to induce neutrophil adhesion to HUVECs as this experiment is performed in 96-well plates and consequently requires low amounts of RF. We first characterized the immunoglobulin composition of the RF preparation. An aliquot was submitted to SDS-PAGE followed by immunoblotting with anti-human IgG or anti-human IgM. We observed that this RF preparation is an IgM/IgG complex containing 5% IgM and 95% IgG, as estimated by densitometry (Figure 6A). Thus, the complexes collected using 5% PEG are the IgM-RFs bound to their cognate IgG antigen. Being a pentamer, IgM-RFs preferentially form immune complexes in the presence of IgGs. As the methods used to dissociate IgMs from IgGs (acid, high salt) can cause denaturation of the RF with resulting loss of binding activity, we used in the subsequent experiments the native RF preparation as so. We incubated neutrophils with the RF complexes and monitored their adhesion to HUVECs as in Figure 4. To assess that RF can bind membrane-bound IgGs, we performed these experiments without or with a pre-incubation of neutrophils with PPP on the adhesion capacity (see Figure 1). We observed an increase of adhesion to HUVECs when neutrophils were incubated with 500 μg/ml of purified RF and this adhesion was enhanced when neutrophils were pre-incubated with PPP (Figure 6B). This observation confirms the hypothesis that RFs can bind IgGs on neutrophils leading to cell activation. Adhesion was not modified in the presence of commercial non-specific IgM alone (data not shown). The relatively high concentration of RFs required for observing a functional effect when compared with the commercial anti-human cross-linking antibodies (20 μg/ml) is explained by the low concentration of IgM-RFs present in the preparation. Furthermore, as the affinity of most IgM-RFs for IgG is relatively low (of the order of 105 to 107) there is a constant on/off binding of the IgG-RF to IgGs or FcγR-bound IgGs. As we showed that the RF preparation was an IgG-containing complex, we tested the capacity of IgG complexes to enhance adhesion. Incubation of neutrophils with heat-agregated IgGs (HA-IgGs, 100 μg/ml) enhances their adhesion to HUVECs in an FcγRs-dependent manner (Figure 6C). Taken together, these results indicate that RFs from RA patients can activate neutrophils by recognizing either IgGs or FcγRs on neutrophils leading to an increased adhesion to endothelium.


Cross-linking of IgGs bound on circulating neutrophils leads to an activation of endothelial cells: possible role of rheumatoid factors in rheumatoid arthritis-associated vascular dysfunction.

Rollet-Labelle E, Vaillancourt M, Marois L, Newkirk MM, Poubelle PE, Naccache PH - J Inflamm (Lond) (2013)

Rheumatoid factors induce FcγRs-dependent adhesion of neutrophils to HUVECs. A: An aliquot of the preparation of rheumatoid factors (100 ng and 10 ng) was analyzed by SDS-PAGE followed by immunoblotting with horseradish peroxidase-labelled anti-human IgG or anti-human IgM antibodies. The same amounts of commercial human IgG and IgM were analyzed on the same gel. The IgM band was used for quantification (arrow). B: Neutrophils were incubated with calcein and with or without PPP, as described in Methods. Neutrophils were then plated on a confluent monolayer of endothelial cells and RFs (500 μg/ml) were added for 2 hours. Non-adherent neutrophils were discarded and the amount of adherent cells was quantified as the amount of fluorescence bound to endothelial cells. Results are expressed as mean +/− SEM of three independent experiments. ** = P < 0.005, *** = P < 0.0005 (one-sample t test). C: Heat aggregated IgGs (HA-IgGs) were prepared as previously described [41]. Neutrophils were incubated with calcein, plated on endothelial cells and 100 μg/ml HA-IgG (HA) were added for 2 hours. Results are expressed as mean +/− SEM of three independent experiments, each carried out in triplicate. ** = P < 0.005, *** = P < 0.0005 (Mann–Whitney test).
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Figure 6: Rheumatoid factors induce FcγRs-dependent adhesion of neutrophils to HUVECs. A: An aliquot of the preparation of rheumatoid factors (100 ng and 10 ng) was analyzed by SDS-PAGE followed by immunoblotting with horseradish peroxidase-labelled anti-human IgG or anti-human IgM antibodies. The same amounts of commercial human IgG and IgM were analyzed on the same gel. The IgM band was used for quantification (arrow). B: Neutrophils were incubated with calcein and with or without PPP, as described in Methods. Neutrophils were then plated on a confluent monolayer of endothelial cells and RFs (500 μg/ml) were added for 2 hours. Non-adherent neutrophils were discarded and the amount of adherent cells was quantified as the amount of fluorescence bound to endothelial cells. Results are expressed as mean +/− SEM of three independent experiments. ** = P < 0.005, *** = P < 0.0005 (one-sample t test). C: Heat aggregated IgGs (HA-IgGs) were prepared as previously described [41]. Neutrophils were incubated with calcein, plated on endothelial cells and 100 μg/ml HA-IgG (HA) were added for 2 hours. Results are expressed as mean +/− SEM of three independent experiments, each carried out in triplicate. ** = P < 0.005, *** = P < 0.0005 (Mann–Whitney test).
Mentions: Although the presence of RFs is associated with the severity of RA symptoms, very few studies have defined a biologic or pathologic function of these antibodies [40]. The results presented above suggest that RFs either as ligands of FcγRs or as antibodies recognizing membrane-bound IgGs might activate neutrophils. To verify this hypothesis, we decided to test the capacity of human RFs to induce neutrophil adhesion to HUVECs as this experiment is performed in 96-well plates and consequently requires low amounts of RF. We first characterized the immunoglobulin composition of the RF preparation. An aliquot was submitted to SDS-PAGE followed by immunoblotting with anti-human IgG or anti-human IgM. We observed that this RF preparation is an IgM/IgG complex containing 5% IgM and 95% IgG, as estimated by densitometry (Figure 6A). Thus, the complexes collected using 5% PEG are the IgM-RFs bound to their cognate IgG antigen. Being a pentamer, IgM-RFs preferentially form immune complexes in the presence of IgGs. As the methods used to dissociate IgMs from IgGs (acid, high salt) can cause denaturation of the RF with resulting loss of binding activity, we used in the subsequent experiments the native RF preparation as so. We incubated neutrophils with the RF complexes and monitored their adhesion to HUVECs as in Figure 4. To assess that RF can bind membrane-bound IgGs, we performed these experiments without or with a pre-incubation of neutrophils with PPP on the adhesion capacity (see Figure 1). We observed an increase of adhesion to HUVECs when neutrophils were incubated with 500 μg/ml of purified RF and this adhesion was enhanced when neutrophils were pre-incubated with PPP (Figure 6B). This observation confirms the hypothesis that RFs can bind IgGs on neutrophils leading to cell activation. Adhesion was not modified in the presence of commercial non-specific IgM alone (data not shown). The relatively high concentration of RFs required for observing a functional effect when compared with the commercial anti-human cross-linking antibodies (20 μg/ml) is explained by the low concentration of IgM-RFs present in the preparation. Furthermore, as the affinity of most IgM-RFs for IgG is relatively low (of the order of 105 to 107) there is a constant on/off binding of the IgG-RF to IgGs or FcγR-bound IgGs. As we showed that the RF preparation was an IgG-containing complex, we tested the capacity of IgG complexes to enhance adhesion. Incubation of neutrophils with heat-agregated IgGs (HA-IgGs, 100 μg/ml) enhances their adhesion to HUVECs in an FcγRs-dependent manner (Figure 6C). Taken together, these results indicate that RFs from RA patients can activate neutrophils by recognizing either IgGs or FcγRs on neutrophils leading to an increased adhesion to endothelium.

Bottom Line: Cross-linking of IgGs bound on neutrophils leads to FcγRs-dependent tyrosine phosphorylation, mobilisation of intracellular calcium and the extracellular release of superoxide anions and lysozyme.Incubation of endothelial cells with the supernatant of activated neutrophils increases ICAM-1 expression and IL-8 production by endothelial cells.Our results show that activation of neutrophils' FcγRs by rheumatoid factors could participate in rheumatoid arthritis-associated vascular damage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Départements de Microbiologie-Infectiologie et Immunologie et de Médecine, Centre de recherche en rhumatologie et immunologie, Centre de recherche du CHU de Québec, Université Laval, Québec, QC, Canada. emmanuelle.rollet@crchul.ulaval.ca.

ABSTRACT

Background: Rheumatoid arthritis is characterized by the presence of circulating auto-antibodies, including rheumatoid factors, which recognize the Fc portion of IgGs. The neutrophil is the most abundant circulating leukocyte and it expresses high levels of FcγRs on its surface. The aim of the present study was to examine the capacity of circulating human neutrophils to be activated by rheumatoid factors and the consequences of these events on endothelium.

Methods: Neutrophil-bound IgGs were cross-linked with anti-human IgGs to mimick the presence of circulating rheumatoid factors and FcγRs-dependent signalling events and functions were examined. The IgG and IgM composition of rheumatoid factors isolated from the serum of RA patients was characterized. Adhesion of neutrophils to endothelial cells was quantified in response to the addition of rheumatoid factors.

Results: Cross-linking of IgGs bound on neutrophils leads to FcγRs-dependent tyrosine phosphorylation, mobilisation of intracellular calcium and the extracellular release of superoxide anions and lysozyme. Incubation of endothelial cells with the supernatant of activated neutrophils increases ICAM-1 expression and IL-8 production by endothelial cells. Finally, rheumatoid factors enhance neutrophil adhesion to endothelial cells.

Conclusions: Our results show that activation of neutrophils' FcγRs by rheumatoid factors could participate in rheumatoid arthritis-associated vascular damage.

No MeSH data available.


Related in: MedlinePlus