Background/Purpose: It has been reported that the expression levels of β1 integrins and the ligands such as VCAM-1 are elevated in the affected joints of RA patients.  Cas-L is a cytoplasmic docking protein downstream of β1 integrin-mediated signaling pathway and is essential for β1 integrin-mediated cell migration and costimulation in T cells.  We found that the levels of Cas-L were markedly elevated in various tissues from HTLV-I tax transgenic mice, a murine model of RA.  Interestingly, a large amount of Cas-L positive lymphocytes and leukocytes were shown to infiltrate into the inflamed joints, suggesting its roles for the pathophysiology of RA.  To further evaluate the involvement of Cas-L in the development of RA, we analyzed collagen-induced arthritis using Cas-L knockout mice.

Methods: We immunized Cas-L knockout mice (KO) and wild-type mice (WT) on a C57BL6 background with the emulsion of chicken type II collagen (IIC) and complete Freund’s adjuvant on day 0 and day 21.  The animals were assessed for the severity and occurrence of arthritis.  In addition, limb joints were assessed by HE staining, immunohistochemistry, and x-ray.  Serum samples were collected on the day 21 and the levels of multiple cytokines and chemokines were measured by Luminex.  In vitro, IIC-specific proliferative response of lymphocytes from the draining lymph nodes (LN) was analyzed by ³H-thymidine incorporation assay.  CD4⁺T cells were purified from splenocytes by MACS, and costimulatory response (CD3 plus fibronectin and CD3 plus CD28) were analyzed by the same assay.  Furthermore, we made bone marrow chimera with WT (graft)/KO (host) and KO (graft)/WT (host), then challenged with collagen-induced arthritis.

Results: Although the overall incidence of arthritis was similar between KO and WT, the onset of the disease was retarded in KO, and the severity of arthritis was significantly reduced in KO compared to WT.  By immunohistochemical analysis, the hindlimbs from WT showed more severe infiltration of inflammatory cells, bone destruction, inflammatory changes, and synovial thickening compared to KO.  In WT, inflammatory cytokines such as TNF-α, IL- 17, IL-6 showed higher levels compared to KO.  On the contrary, the level of anti-inflammatory cytokine IL-10 was lower than KO mice.  Lymphocytes proliferative response against IIC was reduced in KO.  Furthermore, costimulatory response of CD4+ T cells to CD3 plus fibronectin was reduced in KO, in contrast, the response to CD3 plus CD28 was higher in KO compared to WT.  Finally, bone marrow chimera revealed that the severity of CIA was lower in KO (graft)/WT (host) than WT (graft)/KO (host). 

 Conclusion: Gene-targeting of Cas-L conferred the resistance against CIA with altered balance of the pro-inflammatory and anti-inflammatory cytokines.  The costimulatory response of the T cells and IIc-specific response of lymphocytes were also altered.  It seems that hematopoietic cells were more responsible for the gene-targeting effect of Cas-L based on the bone marrow chimera experiment.  Taken together, Cas-L may play a pivotal role in the pathophysiology of collagen-induced arthritis.  It is thus suggested that Cas-L may be a potential molecular target for the treatment of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/gene-targeting-of-an-integrin-mediated-signaling-adaptor-molecule-crk-associated-substrate-lymphocyte-type-reduced-the-severity-of-collagen-induced-arthritis-its-possible-involvement-in-the-pathophy/