William F. Glass

CD4+ T cells specific to a glomerular basement membrane antigen mediate glomerulonephritis

Ab-mediated mechanisms have been considered the major causes of glomerulonephritis (GN). However, recent studies suggest that T cells may be more important in mediating GN. To investigate the effects of antigen-specific CD4(+) T cells, we generated Th1 cell lines specific for this antigen from rats that had been immunized with a recombinant form of the glomerular basement membrane (GBM) antigen, Col4alpha3NC1. Upon the transfer of in vitro-activated T cell lines to pertussis toxin-primed, naive syngeneic rats, the recipients developed severe proteinuria/albuminuria, which plateaued after approximately 35 days. Although no IgG binding to GBM or C3 deposition could be detected by immunofluorescence, five out of eleven rats exhibited severe GN, as judged by the formation of characteristic crescent-shaped lesions in the glomeruli, whereas the others exhibited modest GN. Thus Col4alpha3NC1-specific T cells directly initiated glomerular injury in the recipients. One notable difference from GN induced by active immunization was a T cell infiltration in the renal interstitium, which affected some tubules. We therefore injected fluorescence-labeled Col4alpha3NC1-specific into naive rats, and we found that they were enriched 4.5-fold in the kidney cortex relative to nonspecific control T cells 24 hours later. Many of the T cells were located in the Bowmans space and had a flattened shape, suggesting that the primary target for the T cells was in or adjacent to the Bowmans capsule.

Regulation of the mesangial cell myofibroblast phenotype by actin polymerization

Mesangial cells in diverse glomerular diseases become myofibroblast‐like, characterized by activation of smooth muscle α‐actin (α‐SMA) expression. In cultured mesangial cells, serum‐deprivation markedly increases α‐SMA expression, cell size, and stress fiber formation. Since stress fibers are assembled from actin monomers, we investigated the hypothesis that alterations in stress fiber formation regulate α‐SMA expression and hypertrophy. Human mesangial cells were treated with agents that disrupt or stabilize actin stress fibers. Depolymerization of actin stress fibers in serum‐deprived cells with actin‐depolymerizing agents, cytochalasin B (CytB) and latrunculin B (LatB), or with inhibitors of Rho‐kinase, Y‐27632 and HA‐1077 decreased α‐SMA mRNA as judged by Northern blot analysis. Western blot analysis showed that CytB also reduced α‐SMA protein levels. In serum‐fed cells, agents that stabilized actin stress fibers, jasplakinolide (Jas) and phalloidin, increased α‐SMA mRNA and protein. Treatment of human or rat mesangial cells with CytB, LatB, or Y‐27632 decreased α‐SMA promoter activity. In contrast, Jas increased promoter activity 5.6‐fold in rat mesangial cells. The presence of an RNA polymerase inhibitor blocked degradation of α‐SMA mRNA in cells treated with CytB suggesting that destabilization of this message is dependent on a newly transcribed or rapidly degraded factor. Inhibition of actin polymerization by CytB, LatB, Y‐27623, and HA‐1077 inhibited incorporation of 3[H]‐leucine into newly synthesized protein. Additionally, CytB and LatB decreased cell volume as determined by flow cytometry. Collectively, these results indicate that the state of polymerization of the actin cytoskeleton regulates α‐SMA expression, hypertrophy, and myofibroblast differentiation in mesangial cells.

Orthotopic treatment model of prostate cancer and metastasis in the immunocompetent mouse: efficacy of flt3 ligand immunotherapy.

We established an orthotopic treatment model of prostate cancer to generate reproducible primary and metastatic carcinoma in immunocompetent C57BL/6 mice. Using an in vivo selection scheme of intraprostatic implantation of TRAMP‐C1 cells, primary prostate tumors were cultured and recycled three times by intraprostatic injection resulting in the selection and establishment of the recycled cell line TRAMP‐C1P3. Prostate tumors were detected ∼30 days post‐implantation with periaortic lymph node metastasis in 19/20 (95%) of mice. Tissue culture amplification, DNA ploidy and PCR amplification of the SV40 transgene were used to detect metastatic TRAMP‐C1P3 in lymph node specimens. Tissue culture amplification and DNA ploidy were as sensitive as SV40 transgene amplification by PCR in detection of early metastatic disease in draining lymph nodes. To establish the use of the orthotopic model of prostate cancer for immunotherapy, mice were injected orthotopically with TRAMP‐C1P3 cells and 7 days post‐implantation treated daily for 28 days with either flt3L or carrier control. Carrier‐treated mice had clinically detectable prostate tumors, lymph node metastasis and were moribund at 29–35 days, whereas flt3L therapy markedly suppressed primary TRAMP‐C1P3 growth and lymph node metastasis, and prolonged survival. In summary, we have established a reproducible and clinically relevant orthotopic treatment model of prostate cancer in immunocompetent mice with application to a variety of therapeutic strategies. We demonstrate that flt3L treatment suppressed orthotopic prostate tumor growth and lymph node metastasis reinforcing a role for flt3L as an immunotherapeutic strategy for prostate cancer.

Prostate Tumor Microenvironment Alters Immune Cells and Prevents Long-Term Survival in an Orthotopic Mouse Model Following flt3-Ligand/CD40-Ligand Immunotherapy

A novel orthotopic metastatic model of mouse prostate cancer was developed using MHC-negative TRAMP-C1P3 (tr ansgenic a denocarcinoma of m ouse p rostate) cells derived by serial passage of the parental TRAMP-C1 line in mouse prostate glands. TRAMP-C1P3 cells grew efficiently in mouse prostate glands and reproducibly metastasized to draining lymph nodes. Using this model, we show that Fms-like tyrosine kinase-3 ligand (flt3-L) dramatically inhibited growth of preexisting orthotopic TRAMP-C1P3 tumors and the development of metastatic disease. Mice remained in remission for several months following termination of flt3-L treatment but eventually relapsed and died of progressive disease. flt3-ligand treatment induced a pronounced mixed inflammatory cell infiltrate that consisted of CD8&agr;-CD4− dendritic cells (CD11c+), macrophages, granulocytes (Gr-1+) and to a lesser extent T cells (CD4+ and CD8+). Dendritic cells isolated from TRAMP-C1P3 tumors were phenotypically immature (CD11c+ B7.2-I-A−CD40−), and this phenotype was also predominant in peripheral organs of mice treated with flt3-L alone or in combination with the DC maturation factor, CD40-L. Diminished expression of TCR-&bgr;, CD3-ε, and CD3-ζ was also observed on intratumoral T cells, although these signaling proteins were reexpressed following in vitro culture with IL-2. The TCR/CD3 complex remained intact on peripheral T cells except in mice treated with flt3-L where CD3-ζ loss was observed. In contrast to &agr;&bgr;-T cells, tumor-infiltrating &ggr;&dgr;-T cells maintained expression of their antigen receptors but not CD3ε. Thus, TRAMP-C1P3 tumors quickly establish a microenvironment that profoundly diminishes expression of molecules critical for normal dendritic cell and T cell function, thus limiting the efficacy of flt3-L and CD40-L immunotherapy. Overall, these data suggest that long-term cures of established MHC-negative tumors may not be achieved until therapeutic interventions are engineered to overcome this immunosuppressive microenvironment.

COX-2 inhibition potentiates the antiproteinuric effect of enalapril in uninephrectomized SHR

PGE(2) and PGI(2) reduce extracellular matrix deposition and their production is altered after ACE inhibitor (ACEi) treatment. We therefore hypothesized that cyclooxygenase (COX)-2 inhibition would exacerbate renal injury and antagonize the effects of ACEi. To test these hypotheses, WKY and SHR were uninephrectomized (UNX) and treated with either vehicle, enalapril, NS398 or enalapril+NS398. NS398 did not affect systolic blood pressure nor antagonize the antihypertensive effect of enalapril. Urinary protein excretion in UNX WKY was significantly decreased after treatment with either enalapril or NS398. In UNX SHR, enalapril reduced proteinuria, but NS398 alone had no effect. Administration of both drugs, however, further reduced proteinuria. In UNX WKY, treatment with either NS398 alone or both drugs reduced glomerular volume and similar results were observed in SHR. Surprisingly, these results disprove our original hypothesis and suggest that inhibition of COX-2 provides additional renoprotection to that of enalapril alone.

Expression of α-Actinin-1 in Human Glomerular Mesangial Cells In Vivo and In Vitro

Recent studies have demonstrated important roles of α-actinins in glomerular disease, while little information is known about the expression profile of α-actinins in human glomerular mesangial cells. Here, immunofluorescence and confocal microscopy showed that α-actinin-1 exclusively distributed along mesangial cells in human glomeruli of IgA nephropathy. RT-PCR and Western blot further confirmed the expression of α-actinin-1 in primary cultured human mesangial cells. We also found that transforming growth factor-β 1 (TGF-β 1) stimulated ACTN1 gene transcription and that transiently transfected α-actinin-1 significantly increased TGF-β 1-induced plasminogen activator inhibitor-1 (PAI-1) promoter activity in human mesangial cells. These findings suggest that α-actinin-1 may play a role in human glomerular disease.