Crescentic glomerulonephritis in CD4- and CD8-deficient mice. Requirement for CD4 but not CD8 cells.

The contribution of CD4 and CD8 cells to crescentic glomerulonephritis (GN) was studied in mice genetically deficient in CD4, CD8, and with combined CD4 and CD8 (CD4/CD8) deficiency. Wild-type (C57BL/6) mice developed GN with mild proliferative changes 7 days after an intravenous dose of sheep anti-mouse glomerular basement membrane globulin. Crescents were observed in 12.5 +/- 6.1% of glomeruli on day 14. On day 21, 51.5 +/- 7.3% of glomeruli were affected by crescents, and mice had marked azotemia and proteinuria. CD4 and combined CD4/CD8-deficient mice developed minimal evidence of GN. On day 21, their glomeruli showed only mild proliferative changes and crescents, azotemia, and proteinuria were absent. In contrast, CD8-deficient mice developed severe crescentic GN with three of five mice dying on day 20 with ascites and edema. The two mice surviving to day 21 had severe azotemia. Crescent development was accelerated (day 14, 51.6 +/- 2.4% of glomeruli; day 20 or 21, 62.0 +/- 4.0% of glomeruli). These studies demonstrate that CD4 cells are crucial for the development of crescentic GN in mice and that genetic absence of CD8 cells accelerates disease. They support the hypothesis that crescent formation is a manifestation of CD4-dependent (and CD8-independent) delayed type hypersensitivity in the glomerulus.

Combined treatment of acute EAE in Lewis rats with TNF-binding protein and interleukin-1 receptor antagonist.

Experimental autoimmune encephalomyelitis (EAE) is a term given to describe a collection of animal models representing the human disease multiple sclerosis (MS). Although not fully understood, the involvement of cytokines and the immune system in either EAE or human MS is well established. Past efforts have shown that inhibition of proinflammatory cytokines tumor necrosis factor (TNF-alpha) or interleukin-1 (IL-1) result in amelioration of acute EAE in Lewis rats. The present study examined this model for the effect of concomitant inhibition of both TNF-alpha and IL-1, which resulted in a modest but significant therapeutic effect that was superior to inhibition of either single agent alone with respect to four of the five variables used to follow the progression of disease in this model, i.e., clinical severity, frequency of disease, loss of body weight, and day of onset. These results are in accordance with the idea that combination treatments are likely to prove superior to single agent therapy in the treatment of autoimmune inflammatory disease.

Chemokine expression in experimental tubulointerstitial nephritis.

Chemokines may be important in the pathogenesis of leukocyte infiltration in tubulointerstitial nephritis associated with glomerular disease. We studied the renal cortical expression of the C-C (macrophage inflammatory protein-1alpha (MIP-1alpha)), monocyte chemotactic protein-1 (MCP-1), and RANTES) and C-X-C (interferon-inducible protein-10 (IP-10), MIP-2, and cytokine-induced neutrophil chemoattractant (CINC)) chemokines 4, 6, 8, 10, 14, and 21 days after the induction of puromycin aminonucleoside (PAN) nephrosis. There was a 7- to 10-fold increase in the steady state mRNA expression of IP-10 and MCP-1 in the renal cortex of rats 6 to 8 days after the administration of PAN that declines thereafter reaching control values by day 21. The site of IP-10 and MCP-1 mRNA production was localized to intrinsic tubulointerstitial cells and not to infiltrating monocytes or macrophages. By comparison, there was a low basal expression of RANTES mRNA in the renal cortex of nephrotic rats that did not differ from those of control rats. In contrast, CINC, MIP-2, and MIP-1alpha mRNAs were not detected. Translation of MCP-1 mRNA into protein was confirmed with an ELISA. These changes in chemokine gene expression were associated with a tubulointerstitial T lymphocyte and macrophage infiltration beginning on day 6 that peaked on day 10. Administration of a neutralizing Ab to rat MCP-1 (n = 5) beginning on day 4 resulted in a 45% decline in tubulointerstitial macrophage infiltration from 8.4 +/- 1.3% to 4.6 +/- 0.4% (p < 0.001) on day 6. These data provide evidence that MCP-1, and possibly IP-10, are important in the pathogenesis of monocyte/macrophage infiltration in the tubulointerstitial nephritis associated with PAN nephrosis.

Myofibroblast and alpha 1 (III) collagen expression in experimental tubulointerstitial nephritis.

Despite the importance of tubulointerstitial fibrosis as a predictor of renal function in patients with primary glomerular disease, the identity of the cell(s) that is the source of interstitial collagen production remains unknown. The present study was performed to identify the site of alpha 1(III) production during the development of tubulointerstitial fibrosis. We studied a model of experimental tubulointerstitial nephritis associated with puromycin aminonucleoside (PAN) nephrosis. There was a twofold increase in renal cortical alpha 1 (III) mRNA expression coincident with the onset of tubulointerstitial myofibroblasts infiltration in rats with PAN nephrosis beginning on day 6, which increased to a fivefold difference by day 10. There were 60.8 +/- 40.3 myofibroblast/mm2 within the renal tubulointerstitium of rats with PAN nephrosis on day 6 that peaked at 240.2 +/- 11.1 myofibroblast/mm2 on day 14, which then declined to 43.7 +/- 9.8 myofibroblast/mm2 by day 21. By combining in situ hybridization with immunohistochemistry, alpha 1(III) mRNA expression was colocalized to cells that labeled for alpha-smooth muscle actin identifying them as myofibroblasts. Interestingly, the major site of alpha 1(III) mRNA expression shifted to tubuloepithelial cells with the waning of myofibroblast infiltration on day 21. To determine if PDGF-BB induced myofibroblasts to synthesize alpha 1(III) mRNA, we examined kidneys from rats that had been treated with PDGF-BB (5 mg/kg/day). alpha 1(III) mRNA expression also localized to cells that labeled for alpha-smooth muscle actin. These data demonstrate the cellular source of alpha 1(III) production within the renal tubulointerstitium following injury, and suggest that PDGF-BB may be mediating this production.

Leukemia inhibitory factor ameliorates experimental anti-GBM Ab glomerulonephritis.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that has been identified in acute and chronic inflammatory conditions such as rheumatoid arthritis, sepsis, and renal allograft rejection. We investigated the glomerular expression of LIF at 30 minutes, and 3, 6, 9, 15 and 24 hours after administration of anti-GBM Ab (N = 3) by the RNase protection assay. Control rats received rabbit sera and were sacrificed at 30 minutes, and 6 and 24 hours. LIF mRNA relative to GAPDH mRNA was detected at low levels within the glomeruli of occasional control rats. However with the induction of anti-GBM Ab GN, there was a marked increase in LIF steady-state mRNA beginning at three hours which persisted through 24 hour. LIF mRNA was also detected in cultured mesangial cells stimulated with IL-1 beta, identifying this cell type as a potential glomerular source for this cytokine. To investigate the in vivo effect of LIF, Lewis rats were continuously infused with recombinant (r) human (h) LIF (approximately 0.5 ng/hr) or saline vehicle i.p. with ALZA osmotic pumps beginning at t = -24 hours (N = 8). All rats were injected with anti-GBM Ab intravenously at t = 0 (N = 16). LIF infusion decreased 24-hour urinary protein excretion by 85% (17 +/- 15 vs. 114 +/- 37 mg/day, P = 0.0001) and was associated with a 60% decrease in glomerular macrophage infiltration (0.8 +/- 0.2 vs. 2.0 +/- 0.6 ED-1 cells/glom, P = 0.0001). The administration of rhLIF did not affect the binding of the anti-GBM Ab to glomeruli. The beneficial effects of LIF were associated with a decrease in glomerular MCP-1 (56%), IL-1 (41%) and TNF (17%) steady state mRNA expression. The latter was associated with a 29% decrease in TNF-alpha protein expression within the glomerular lysate of nephritic rats administered LIF when compared with control rats. These data demonstrate a potential role for LIF in the therapy of anti-GBM Ab GN.

Heparin attenuates bleomycin but not silica-induced pulmonary fibrosis in mice: possible relationship with involvement of myofibroblasts in bleomycin, and fibroblasts in silica-induced fibrosis.

Pulmonary fibrosis was elicited in mice or rats by the intratracheal instillation of bleomycin or silica. Daily injections of heparin significantly reduced the collagen deposition in bleomycin, but not in silica, injected mice, as evaluated by the lung hydroxyproline content on day 15 after instillation. Heparin also reduced the bleomycin-induced morbidity and mortality. Study of the broncho-alveolar lavage fluid (BAL) detected no significant change in the number of leucocytes or the amount of protein in heparin treated mice. Histologies of bleomycin instilled mice suggested that heparin did reduce the alveolar remodelling but not the alveolitis, evidenced by leucocytic infiltration. As detected by electron microscopy (EM), bleomycin increased the number of leucocytes and platelets within the alveolar capillaries but this was not significant ly reduced by heparin. The phenotype of the interstitial cell involved in these two types of pulmonary fibrosis was investigated by immunohistochemistry and EM. While in bleomycin injected animals the interstitial cells had the phenotype of an actin (alpha-actin in the rat) and lipid containing interstitial cell, with a poorly developed RE, in silica injected animals in contrast, the interstitial cells were without cytoplasmic actin or lipid but with a markedly developed endoplasmic reticulum (ER). Thus bleomycin and silica induced the growth of two different types of interstitial cells, the myofibroblast and the regular fibroblast, which might be a reason why heparin selectively inhibits bleomycin but not silica-induced fibrosis.

Fate of cationic liposomes and their complex with oligonucleotide in vivo.

The present studies describe the biodistribution of cationic liposomes and cationic liposome/oligonucleotide complex following intravenous injection into mice via the tail vein. (111)In-diethylenetriaminepentaacetic acid stearylamide ((111)In-DTPA-SA) was used as a lipid-phase radiolabel. Inclusion of up to 5 mol% DTPA-SA in liposomes composed of 3beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) did not influence liposome formation or size, nor the binding/uptake or fusion of the cationic liposomes with CHO cells in vitro. Moreover, nuclear delivery of oligonucleotide to CHO cells was unaffected by the probe. The biodistribution of liposomes with increasing concentration of DC-Chol (1:4-4:1, DC-Chol/DOPE, mol/mol) at 24 h post-injection revealed no dependence on lipid composition. Uptake was primarily by liver, and accumulation in spleen and skin was also observed. Comparatively little accumulation occurred in lung. Clearance of injected liposomes by liver was very rapid (approximately 84.5% of the injected dose by 7.5 h post-injection). Liposome uptake by liver and spleen were equally efficient in the dose range of 3.33 to 33.33 mg/kg body weight, yet possible saturation of liver uptake at a dose of 66.80 mg/kg may have allowed for increased spleen accumulation. Preincubation of cationic liposomes with phosphorothioate oligonucleotide induced a dramatic yet transient accumulation of the lipid in lung which gradually redistributed to liver. Similar results were observed when monitoring iodinated oligonucleotide in the complex. Immuno-histochemical studies revealed large aggregates of oligonucleotide within pulmonary capillaries at 15 min post-injection, suggesting the early accumulation in lung was due to embolism. Immuno-histochemical studies further revealed labeled oligonucleotide to be localized primarily to Kupffer cells at 24 h post-injection. Immuno-electron microscopy revealed localization of oligonucleotide primarily to the lumen of pulmonary capillaries at 15 min post-injection. Immuno-electron microscopy revealed localization of oligonucleotide primarily to the lumen of pulmonary capillaries at 15 min post-injection, and to phagocytic vacuoles of Kupffer cells at 24 h post-injection. By these methods, nuclear delivery of oligonucleotide in vivo was not observed. Increasing concentration of mouse serum inhibited cellular binding/uptake of cationic liposomes in vitro, without or with complexed oligonucleotide. We therefore postulate that interaction with plasma components, including opsonin(s), inhibits cellular uptake of the injected liposomes as well as the liposome/oligonucleotide complex, and mediates rapid uptake by Kupffer cells of the liver. These results are relevant to the design of cationic liposomes for efficient delivery of nucleic acid in vivo.

Expression of keratinocyte growth factor in embryonic liver of transgenic mice causes changes in epithelial growth and differentiation resulting in polycystic kidneys and other organ malformations.

Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to hepatocytes during the later period of mouse gestation using a human apolipoprotein E (ApoE) gene promoter and its associated liver-specific enhancer. Human KGF was detectable in liver extracts and serum prepared from e17.5-e19.5 embryos, concomitant with the appearance of morphological abnormalities in several organs which express KGF receptor. The most striking phenotypic aberration in the ApoE-hKGF transgenic embryos was marked hyperplasia and cystic dilation of the cortical and medullary kidney collecting duct system, a phenotype resembling infantile polycystic kidney disease in humans. Transgenic embryos had enlarged livers, with prominent biliary epithelial hyperplasia, and also exhibited enhanced bronchiolar epithelial and type II pneumocyte proliferation. There was variable hyperplasia of intestinal epithelia, and urothelium of the urinary bladder and ureters. When compared to age-matched littermate controls, marked epidermal papillomatous acanthosis and hyperkeratosis in the skin, with a notable decrease in the number of developing hair follicles was seen in transgenic embryos. The pancreas exhibited significant ductal hyperplasia, with an increase in the number of ductal epithelial cells staining positive for insulin expression. High systemic levels of KGF during the latter stages of embryogenesis causes abnormalities in epithelial growth and differentiation within multiple organ systems and results in perinatal lethality. Correct temporal and spatial expression of KGF during the latter stages of organ development is likely to play a critical role in mesenchymal-epithelial signaling required for normal embryonic growth and development.

Platelet-derived growth factor-BB induces renal tubulointerstitial myofibroblast formation and tubulointerstitial fibrosis.

Tubulointerstitial fibrosis correlates closely with renal function, although the mechanism regulating tubulointerstitial fibrogenesis remains poorly understood. Since platelet-derived growth factor (PDGF) is a growth factor for fibroblasts, we examined the effect of daily (for 7 days) PDGF-AA or PDGF-BB administration on renal tubulointerstitial architecture in rats. PDGF-AA administration at a dose of 5 mg/kg did not affect the renal tubulointerstitium. By comparison, PDGF-BB induced dose-dependent renal tubulointerstitial cell proliferation and fibrosis. At 5 mg/kg, PDGF-BB increased BrdU labeling in tubulointerstitial fibroblasts at 24 hours (19-fold), which peaked at 72 hours (23-fold) with bromodeoxyuridine uptake returning to control values by 7 days. Tubulointerstitial proliferation was associated with the differentiation of these cells into myofibroblasts as evidenced by alpha-smooth muscle actin expression beginning on day 3. The expression of alpha-smooth muscle actin peaked on day 5 and had markedly declined by day 21. In addition, apoptotic cells were identified within the tubulointerstitium on day 3 and progressively increased through day 7, suggesting that the disappearance of myofibroblasts may have occurred through apoptosis. These changes were accompanied by increased expression of alpha 1 (III) collagen mRNA and interstitial accumulation of type III collagen within the renal cortex. By morphometric analysis, an approximately twofold increase in collagen III immunolabeling within the interstitial compartment was evident at 24 hours and peaked on days 5 to 7 (approximately fourfold). These data suggest that PDGF-BB may be an important mediator of tubulointerstitial hyperplasia and fibrosis.

Growth factors in porcine full and partial thickness burn repair. Differing targets and effects of keratinocyte growth factor, platelet-derived growth factor-BB, epidermal growth factor, and neu differentiation factor.

The topical application of recombinant growth factors such as epidermal growth factor, platelet-derived growth factor-BB homodimer (rPDGF-BB), keratinocyte growth factor (rKGF), and neu differentiation factor has resulted in significant acceleration of healing in several animal models of wound repair. In this study, we established highly reproducible and quantifiable full and deep partial thickness porcine burn models in which burns were escharectomized 4 or 5 days postburn and covered with an occlusive dressing to replicate the standard treatment in human burn patients. We then applied these growth factors to assess their efficacy on several parameters of wound repair: extracellular matrix and granulation tissue production, percent reepithelialization, and new epithelial area. In full thickness burns, only rPDGF-BB and the combination of rPDGF-BB and rKGF induced significant changes in burn repair. rPDGF-BB induced marked extracellular matrix and granulation tissue production (P = 0.013) such that the burn defect was filled within several days of escharectomy, but had no effect on new epithelial area or reepithelialization. The combination of rPDGF-BB and rKGF in full thickness burns resulted in a highly significant increase in extracellular matrix and granulation tissue area (P = 0.0009) and a significant increase in new epithelial area (P = 0.007), but had no effect on reepithelialization. In deep partial thickness burns, rKGF induced the most consistent changes. Daily application of rKGF induced a highly significant increase in new epithelial area (P < 0.0001) but induced only a modest increase in reepithelialization (83.7% rKGF-treated versus 70.2% control; P = 0.016) 12 days postburn. rKGF also doubled the number of fully reepithelialized burns (P = 0.02) at 13 days postburn, at least partially because of marked stimulation of both epidermal and follicular proliferation as assessed by proliferating cell nuclear antigen expression. In situ hybridization for KGFR in porcine burns revealed strong expression of KGFR on hair follicles and basal epidermis, confirming direct rKGF action on follicular as well as epidermal keratinocytes. Although the epithelial proliferation induced by rKGF resulted in marked neoepidermal psoriasiform hyperplasia with exaggerated rete ridges and neoepidermal and follicular maturation as assessed by expression of cytokeratin 10, a marker of keratinocyte terminal differentiation was not delayed and appeared to be accelerated in some rKGF-treated burns. Recombinant epidermal growth factor induced a trend toward increased new epithelial area in deep partial thickness burns, but had no effect on reepithelialization. The recombinant neu differentiation factor-alpha 2 isoform had no significant biological effects in either full or deep partial thickness burns.(ABSTRACT TRUNCATED AT 250 WORDS)