Humoral factors in one-kidney, one-clip (1K,1C) hypertension in rats increase growth of smooth muscle cells cultured from rat arteries. To further characterize the plasma factor or factors involved, we prepared male rats with early, benign 1K,1C hypertension and paired them with one-kidney (1K) normotensive controls. In the presence of growth stimulated by background levels (1%) of fetal calf serum (FCS), plasma-derived serum (PDS), fresh or frozen, from 99 1K,1C rats differentially increased [3H]thymidine incorporation of growth-arrested rat aortic cells; increases were up to 93% more than those evoked by the paired 1K PDS and were concentration related (P < 0.01). However, there was no evidence for a differential effect of 1K,1C PDS in the absence of FCS nor of PDS after boiling. On the other hand, neither treatment of PDS with proteolytic enzymes nor charcoal absorption ablated this differential trophic effect. Thus this study provides evidence that the humoral factor(s) in hypertension are resistant to freezing, proteolysis, and charcoal absorption, but sensitive to boiling, and they require background levels of growth for expression.
Background We previously reported that IL-6 inhibition by anti-IL6R antibody (mMR16-1) attenuates HLA-A2 recall antibody responses in a mouse model of allo-sensitization. Follicular T heper (Tfh) cell development with subsequent antigen-specific B-cell and plasma cell generation are dependent on IL-6 and IL-21. Here, we examine the effects of anti-IL6R treatment on follicular T-helper cells during memory alloantibody response to skin allograft. Methods C57BL/6 mice were pre-sensitized with skin allografts (SG) from a HLA.A2 transgenic mouse. At Day 70 the pre-sensitized mice were re-stimulated with a second HLA.A2+ skin graft. Recall alloantibody responses were monitored by measurement of serum anti-HLA.A2 antibodies. Splenic lymphocytes isolated from SG recipients were subjected to a two-step intracellular staining procedure with antibody marker, and analyzed in FACS for intracellular cytokine production by CD4+ cells of IFNg, IL-17a and IL-21, and cell surface CD4, CD185 (CXCR5) expression. Results All the pre-sensitized mice had moderate levels (113.9+143.9 MFI) of DSA IgG in the sera before 2nd skin grafting. Re-sensitization in the control group (n=6) resulted in a recall response featured by a surge of anti-HLA.A2 IgG at day 14 (633+52 MFI) and a peak at day 28 (827+110 MFI). mMR16-1 significantly reduced DSA IgG levels ( 336±116 MFI at day 14 and 530±136 MFI at day 28, P=0.003, P=0.015, respectively, as compared to controls). FACS analysis showed that mMR16-1 did not significantly change the relative subpopulation (%) of CD4+ or CD8+ T cells, B220+ B-cells or CD11b+ monocyte/macrophages in the spleens. There was no significant difference in IL17a+ CD4 subset or IFNg+ CD4 subset between treatment and control groups. However, mMR16-1 significantly reduced IL-21 expressing CD4 T-helper cells (18+3.9% vs. 31+7.2% in control, p=0.006). In addition, there was a significant decrease in relative population of CD4+CXCR5+ Tfh cells (14.8+3.3% v. 22.8+7.8%, p=0.04) in anti-IL6R treated group compared to controls. Conclusion The data indicate that IL-6 signal inhibition using anti-IL6R antibody may down-regulate IL-21 production by CD4+CXCR5+ follicular T helper cells. Since IL-21 is a cytokine critical of follicular B cell development, modification of Tfh cells in turn restrains follicular B cell activation and maturation, leading to alloantibody reduction.
