PMC6875445_01
Histopathological and immunohistochemical analyses of renal tissues from the present patient with refractory progressive proliferative lupus nephritis. (A) Hematoxylin and Eosin staining. Bottom: magnified view of a section of the top image. (B) Immunostaining for CD4+ lymphocytes using anti-CD4 monoclonal antibody (mAb) with 3,3'-diaminobenzidine (DAB) (brown). (C) Immunostaining for P-gp on lymphocytes using JSB-1 anti-P-gp mAb (P-gp) or isotype-matched negative control antibody immunoglobulin G1 (IgG1 control) with Vulcan Fast Red (FR) (red). (B, C) Nuclear counterstaining with hematoxylin.
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At her sixth admission in November 2010 due to severe flare with erythema, massive edema, elevated levels of anti-double-stranded DNA antibody, and hypocomplementemia, an examination of a renal biopsy specimen showed both features of active proliferative LN classified as ISN/RPS IVG (A/C) and the marked accumulation of inflammatory cells in the renal interstitial tissue (Fig. 1A). Immunohistochemical staining of serial 5-mum-thick sections from the same specimens showed that the majority of these inflammatory cells were CD4+ cells (Fig. 1B) and P-gp-positive cells (Fig. 1C), suggesting that P-gp+CD4+ cells had accumulated in the renal interstitial tissue.
The reduction in P-gp+CD4+ lymphocytes following IVCY-MTX therapy was associated with an improvement in refractory proliferative lupus nephritis. A flow cytometric analysis showed P-gp expression on target molecule positive CD4+ cells at 6 weeks before treatment (-6W), just at the start of treatment (0W), at 4 weeks after the commencement of treatment (4W), and at 12 weeks after the commencement of treatment (+12W). Percentages represent the proportion of P-gp and target molecule double-positive CD4+ lymphocytes. Clinical course; PSL eq: prednisolone equivalent, IVCY: intravenous cyclophosphamide pulse therapy, MZ: mizoribine, MTX: methotrexate, Cre: creatine, Alb: albumin, SLEDAI: systemic lupus erythematosus disease activity index, ds DNA: anti-double-stranded (ds) DNA antibody, CH50: 50% hemolytic complement activity
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A further analysis showed that the CD69+CD4+ cells included CD69+P-gp+CD4+cells (Fig. 2, -6W). In addition, CXCR3+CD4+ cells were detected in peripheral blood, and the majority of P-gp+CD4+ cells expressed CXCR3 (Fig. 2, -6W). A further analysis of the cytokine production by peripheral P-gp+CD4+ cells showed the production of interleukin (IL)-2 and IL-6 but not IL-17 (Fig. 2, 0W). This resulted in a significant decrease in peripheral CD69+P-gp+CD4+ cells within 4 weeks (Fig. 2, 4W), along with improvement in proliferative LN and recovery of the serum albumin level to >2.5 g/dL. MTX combined with IVCY therapy eliminated CD69+P-gp+CD4+ cells, accompanied by the disappearance of CXCR3+P-gp+CD4+ cells and IL-2- and IL-6-producing P-gp+CD4+ cells within 12 weeks (Fig. 2, 12W).