Identification of tissue-specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels.

Nonhistone protein antigens resolved by electrophoresis in sodium dodecyl sulfate were identified immunochemically after being transferred to nitrocellulose. Use of antiserum to dehistonized chromatin from Novikoff hepatoma revealed numerous protein antigens specific to the chromatin of Novikoff hepatoma in comparison to that of normal rat liver.

Urokinase-dependent adhesion loss and shape change after cyclic adenosine monophosphate elevation in cultured rat mesangial cells.

Mesangial cells in culture change shape and become less adhesive in response to cAMP elevation (e.g., treatment with isoproterenol plus isobutylmethylxanthine (IM). Inhibitors of serine proteases inhibit cellular shape change in response to IM. To further examine the role of cell surface proteases in shape change, adhesion plaque proteins (i.e., preparations of ventral membranes and extracellular matrix) were separated in SDS-polyacrylamide gels containing gelatin with and without plasminogen. Four discrete zones of lysis were evident in plasminogen gels (indicative of activation of plasminogen) from control adhesion plaques: one inconspicuous zone with a Mr approximately 150 kD, another at approximately 115 kD, and a doublet at approximately 35-32 kD. Another diffuse zone of lysis centered around Mr approximately 70 kD and contained a defined band of approximately 56 kD. Adhesion plaques contained most of the plasminogen activators (PA). 5 min after IM treatment, the Mr approximately 150- and approximately 115-kD PA were increased in activity. Vasopressin (VP), which prevented shape change and adhesion loss when added along with IM, inhibited the increase in these PA. Preincubation with monoclonal or polyclonal antibodies to urokinase-type plasminogen activator (uPA) totally inhibited the IM-inducible shape change and adhesion loss. Activation of plasminogen throughout the gels revealed multiple protease resistant bands that markedly increased with IM treatment (maximal at 45 min). These may represent focal control mechanisms. uPA thus may mediate focal proteolysis, which results in shape change and decreased adhesion.

Flt3-ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer.

We assessed the in vivo efficacy of Flt3-ligand (Flt3-L) treatment in C57BL/6 mice bearing a well-established MHC class I-negative prostate carcinoma TRAMP-C1. Flt3-L immunotherapy was initiated approximately 30 days after tumor inoculation, a time when > or =80% of the mice had palpable TRAMP-C1 tumors. Treatment with Flt3-L at 10 microg/day for 21 consecutive days suppressed TRAMP-C1 tumor growth and induced tumor stabilization (P = 0.0337). Enhanced tumor regression was demonstrated at a higher dose of 30 microg/day (P < 0.0001). Tumors excised from mice treated with Flt3-L were smaller than carrier-treated controls and contained a more pronounced mixed inflammatory cell infiltrate primarily composed of mphi. In regressor nice, tumors reappeared at the site of injection when Flt3-L therapy was terminated. When the experiment was repeated with MHC class I-positive TRAMP-C1 cells, tumor stabilization and/or regression was again observed after treatment (P < 0.0001); however, once again, tumors reappeared after the termination of therapy despite an extended treatment schedule (35 days). MHC class I-negative variants were present in tumors isolated from carrier- and Flt3-L-treated mice, and this phenotype could be reversed by IFN-gamma treatment in vitro. Thus, Flt3-L treatment of mice with preexisting transplantable prostate tumors results in tumor regression that is dose-dependent and accompanied by a pronounced mixed-cell inflammatory tumor infiltrate. However, disease relapse was invariably observed after the termination of therapy, which suggests that Flt3-L treatment of advanced MHC- prostate cancers will require adjuvant modalities to achieve a durable response.

Lactoferrin: nuclear localization in the human neutrophilic granulocyte?

Conditions were established whereby nuclear or cytoplasmic immunocytochemical localization of lactoferrin was observed in the human peripheral blood granulocyte. A positive nuclear staining reaction was obtained when cells were either not fixed or treated with most fixatives. However, treatment of blood smears with formaldehydeacetone prior to the immunocytochemical localization gave a cytoplasmic staining reaction. A method was developed that allowed us to examine proteins obtained from granulocyte nuclei isolated from fixed cells. Only a trace amount of lactoferrin was detected in the electrophoretically separated nuclear proteins obtained from granulocytes treated for 1 min with formaldehyde-acetone. However, lactoferrin was major protein component in nuclei isolated from untreated cells, cells treated with other fixatives, or cells preincubated in buffered saline prior to formaldehyde-acetone fixation. formaldehyde-acetone treatment of nuclear materials containing lactoferrin did not extract lactoferrin or mask it from detection, thus indicating that lactoferrin found in the mature neutrophilic granulocyte nucleus is most likely acquired from other cellular organelles during tissue processing.

Transforming growth factor beta regulates cyclooxygenase-2 in glomerular mesangial cells.

This study examines the hypothesis that transforming growth factor beta (TGFbeta) regulates cyclooxygenase-2 (COX-2) and induces prostaglandin E synthase (mPGES-1) in rat mesangial cells. COX-2 expression was determined by Northern blot analysis after treatment with either TGFbeta1 or the selective COX-2 inhibitor, NS398. mPGES-1 expression was determined by real-time polymerase chain reaction. The effect of TGFbeta1 on COX-2 gene transcription was assessed using a luciferase reporter assay, and mRNA stability was also determined. To determine whether TGFbeta1 activates elements of the COX-2 promoter, we performed gel shift analyses to examine activation of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB). Prostaglandin E(2) (PGE(2)) and thromboxane B2 (TxB2) production was assayed by enzyme immunoassay. Finally, the pathophysiological relevance of COX-2 inhibition on the downstream effects of TGFbeta was assessed by examining collagen type I mRNA and net collagen production. COX-2 mRNA and mPGES-1 were induced after treatment with TGFbeta1 for 4 h, and this rise was accompanied by a three-fold increase in PGE(2) production that could be antagonized by selective inhibition of COX-2 with NS398. TGFbeta1 increased transcription by approximately 50% and activated both AP-1 and NF-kappaB. These effects were antagonized by co-treatment with NS398. Treatment with TGFbeta1 also doubled the half-life of COX-2 mRNA. Neither collagen type I mRNA nor net collagen production were altered by co-treatment with NS398. In conclusion, these results indicate that TGFbeta stimulates COX-2 and mPGES-1, with additional effects on transcription and stability of COX-2 mRNA.

Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP.

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle-like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (alpha V beta 3 integrin) and fibronectin (alpha 5 beta 1 integrin) receptors in these changes. Mesangial cells on vitronectin-rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin-coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin-coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin-coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin--but not on vitronectin-coated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus-type extracellular matrix contacts.

Two-chain urokinase, receptor, and type 1 inhibitor in cultured human mesangial cells.

Urokinase-type plasminogen activator (u-PA), its receptor (u-PAR), and type 1 inhibitor (PAI-1) in cultured human mesangial cells were investigated. Treatment with phospholipase C (PLC) released plasminogen activators [with relative mol wt (M(r)) of 55,000 and 100,000] and u-PAR into the culture medium. By Western blot, both u-PA and PAI-1 were present in the M(r) 100,000 band. Since PAI-1 binds only active, two-chain u-PA (tcu-PA), formation of the M(r) 100,000 band reflects conversion of the single-chain, proenzyme form of u-PA (scu-PA) to tcu-PA. Immunofluorescence staining of whole cells demonstrated the presence of PAI-1, u-PA, and u-PAR. Immunofluorescence staining and Western blot analysis showed enrichment of PAI-1, u-PA, and u-PAR in a preparation of substratum-attached extracellular matrix and membrane proteins termed adhesion plaques. Using a chromogenic assay, we found that PAI-1 expression in adhesion plaques exceeded that of u-PA. We conclude that cultured human mesangial cells produce receptor-bound u-PA/PAI-1 complexes localized to adhesion plaques.

Extracellular matrix distribution and hillock formation in human mesangial cells in culture without serum.

Smooth muscle cell and mesangial cell hillock formation have been proposed as in vitro models of vascular sclerosis and glomerular sclerosis. This growth pattern is characterized by multilayered ridges and nodules, termed hills or hillocks, separated by less populated areas termed valleys. In this study, it was discovered that an extracellular matrix rich in pericellular fibronectin-fibrils was key to hillock formation. Human mesangial cells were plated onto serum-coated or noncoated substrata in serum-free medium. Subconfluent cells on serum-coated substrata migrated together, forming aggregates, but cells on noncoated substrata remained evenly dispersed. When plated at confluent densities, cells in serum-coated dishes formed hillocks, but cells in noncoated dishes did not. In serum-coated dishes, the substratum underlying subconfluent cells was vitronectin-rich but fibronectin-poor, whereas the pericellular matrix contained abundant fibronectin fibrils. In contrast, the substratum of subconfluent cells plated in noncoated dishes lacked vitronectin but was fibronectin-rich, whereas the pericellular matrix contained few fibronectin fibrils. The distributions of integrin receptors for fibronectin (rabbit anti-alpha 5 beta 1) and vitronectin (rabbit anti-alpha V, beta 3, and beta 5) followed the distributions of their ligands, fibronectin and vitronectin, respectively. Antibodies to fibronectin blocked hillock formation by cells on serum-coated substrata and prevented spreading of cells on noncoated substrata. In summary, key steps in hillock formation are: (1) migration, (2) secretion of fibronectin and assembly of pericellular fibrils, (3) fibronectin fibril-mediated cell-cell adhesion, and (4) aggregation of cells with further migration to form multiple layers. A similar mechanism may play a role in vascular and glomerular sclerosis.

Regulation of mesangial cell adhesion and shape by thrombin.

Adenosine 3',5'-cyclic monophosphate (cAMP) elevation in cultured rat mesangial cells causes urokinase-dependent adhesion loss, stress-fiber fragmentation, and shape change. Thrombin cleaves single-chain urokinase (scu-PA), causing its inactivation, but not two-chain u-PA [tcu-plasminogen activator (PA)] or tissue-type PA. We tested the ability of thrombin to inhibit the effects of cAMP elevation in mesangial cells and inactivate cell-associated scu-PA. In an assay of trypsin-sensitive adhesion, 65.9% of control cells and 5.5% of cells treated with isoproterenol + methylisobutylxanthine (IM) remained adherent. In the presence of 0.01, 0.1, 1.0, and 10.0 unit/ml thrombin, 20.9, 46.6, 50.4, and 53.3%, respectively, of IM-treated cells remained attached. Thrombin also inhibited stress-fiber fragmentation and shape change. The effects of thrombin were blocked by hirudin or antithrombin III plus heparin. Direct zymography in gels containing gelatin and plasminogen revealed loss of a closely spaced pair of PA bands with thrombin treatment (1.0 unit/ml). Hirudin blocked the loss. alpha-Thrombin inactivated by diisopropyl fluorophosphate neither inhibited shape change nor caused loss of the PA bands; however, gamma-thrombin was nearly as active as native alpha-thrombin in both regards. Pretreatment of the cells with as little as 1.0 unit/ml thrombin for 1.0 min caused marked inhibition of shape change and near total loss of the slower migrating u-PA band (of the doublet). The faster migrating band was inhibited less. The results indicate that the slower migrating band represents scu-PA; the nature of the faster migrating band is less certain. Thrombin reversed the adhesion loss and shape change caused by 8-(4-chlorophenylthio)-cAMP and MIX. Thus physiological concentrations of thrombin rapidly inactivate mesangial cell scu-PA and inhibit and reverse cAMP-stimulated adhesion loss and shape change.(ABSTRACT TRUNCATED AT 250 WORDS)

A cytokeratin 8-like protein with plasminogen-binding activity is present on the external surfaces of hepatocytes, HepG2 cells and breast carcinoma cell lines.

Plasminogen binding to cell surfaces may be important for tumor invasion and other processes that involve cellular migration. In this investigation, the principal plasminogen-binding protein was identified in the plasma membrane fraction of rat hepatocytes. The protein had an apparent mass of 59 kDa, was insoluble in a spectrum of detergents, and was identical to cytokeratin 8 (CK 8) as determined by sequence analysis of nine amino acids at the N terminus of two cyanogen bromide fragments. The 59 kDa protein bound CK 8-specific antibody in western blot analyses. These studies demonstrate that CK 8 or a CK 8-like protein binds plasminogen. Given this newly determined and potentially important CK 8 function, immunofluorescence and immunoelectron microscopy studies were performed to determine whether CK 8 may be present on the external surfaces of unpermeabilized, viable hepatocytes. All of the cells in each preparation were immunopositive with two separate CK 8-specific antibodies. A punctate pattern of immunofluorescence was detected on the cell surface with approximately even intensity from cell to cell. By immunoelectron microscopy, CK 8 was preferentially associated with microvilli. In order to determine whether other epithelial cells express cell-surface CK 8, immunofluorescence and immunoelectron microscopy studies were performed with HepG2 hepatocellular carcinoma cells and with BT20 and MCF-7 breast carcinoma cells. The pattern of antigen expression was equivalent with each cell type and comparable to that observed with hepatocytes. These studies support the hypothesis that CK 8 is associated with the external cell surface where it may express important proteinase receptor function.

Membranous glomerulonephritis associated with testicular seminoma.

Proteinuria, often nephrotic in range, is a recognized paraneoplastic syndrome of solid tumours, with membranous glomerulonephritis (MGN) the most common histopathological lesion seen on renal biopsy. A 56-year-old male was found to have proteinuria on routine medical examination. History, physical and serological evaluation failed to reveal an aetiology and subsequent renal biopsy showed MGN, presumed to be idiopathic. Prednisone therapy was begun but this proteinuria did not resolve (> 1 g 24 h-1). Eleven months later the patient discovered a testicular mass which was found to be a stage I seminoma upon excision and metastatic evaluation. His proteinuria rapidly normalized after orchectomy and regional lymph node radiotherapy. This is the first known case of MGN associated with testicular seminoma.

Tissue specificity and cellular distribution of Novikoff hepatoma antigenic proteins p39, p49, and p56.

Three Novikoff hepatoma protein antigens (approximately Mr = 39,000, 49,000, and 56,000) were partially purified from Novikoff hepatoma chromatin. Rabbit antiserum to these three proteins was used to examine various rat tissues for the presence of these antigens. Immunological specificity of the antiserum was assessed using quantitative microcomplement fixation assay or by visualization of the immunoreactive complexes with peroxidase-antiperoxidase procedure after transferring the electrophoretically separated proteins to nitrocellulose sheets. The immunoreactivity was localized with the three proteins p39, p49, and p56 in Novikoff hepatoma. The p56 protein was found to be present in normal rat liver, 24-h regenerating rat liver, fetal rat liver, or kidney, albeit in much smaller amounts as found in Novikoff hepatoma. the p49 and p39 antigens were specific for Novikoff hepatoma. Immunoabsorption experiments confirmed the specificity of this antiserum. Assessment of various subcellular fractions of Novikoff hepatoma revealed that the p39, p49, and p56 protein antigens are present in the cytoplasmic fractions as well as in isolated chromatin.

Alpha 2-macroglobulin functions as a cytokine carrier to induce nitric oxide synthesis and cause nitric oxide-dependent cytotoxicity in the RAW 264.7 macrophage cell line.

Nitric oxide (NO) is an important mediator of macrophage activities. We studied the regulation of macrophage NO synthesis by alpha 2-macroglobulin (alpha 2M), a proteinase inhibitor and carrier of certain growth factors, including transforming growth factor-beta (TGF-beta). Native alpha 2M and the alpha 2M receptor-recognized derivative, alpha 2M-methylamine (alpha 2M-MA), increased nitrite generation by the RAW 264.7 murine macrophage cell line. The level of nitrite accumulation, which is an index of NO synthesis, was comparable to that observed with interferon-gamma. Native alpha 2M and alpha 2M-MA also increased inducible nitric oxide synthase (iNOS) mRNA levels and substantially reduced the number of viable cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium/succiny l dehydrogenase assay or trypan blue exclusion. At slightly higher alpha 2M concentrations, [3H]thymidine incorporation was inhibited. All of these activities were counteracted nearly completely when the iNOS competitive inhibitor NG-monomethyl-L-arginine was included. By in situ nick translation, native alpha 2M and alpha 2M-MA increased the percentage of cells with detectable single strand chromatin nicks from 4 to 12 and 17%, respectively. This change suggested apoptosis; however, electron microscopy studies demonstrated variability in the morphology of injured cells. To determine the mechanism by which alpha 2M increases macrophage NO synthesis, we studied proteolytic alpha 2M derivatives that retain partial activity. A 600-kDa derivative that retains growth factor binding activity increased RAW 264.7 cell NO synthesis and iNOS mRNA levels comparable to native alpha 2M and alpha 2M-MA. The purified 18-kDa alpha 2M receptor-binding fragment had no effect on NO synthesis or iNOS expression. Thus, the growth factor-carrier activity of alpha 2M and not its receptor-binding activity is essential for NO synthesis regulation. A TGF-beta-neutralizing antibody mimicked the activity of alpha 2M, increasing RAW 264.7 cell NO synthesis and decreasing cellular viability. These studies demonstrate that alpha 2M can regulate macrophage NO synthesis and profoundly affect cellular function without gaining entry into the cell and without binding specific plasma membrane receptors.

High glucose causes an increase in extracellular matrix proteins in cultured mesangial cells.

Diabetic nephropathy is a major cause of the increased morbidity and mortality in insulin-dependent diabetes mellitus. The most significant renal lesion of diabetic nephropathy is expansion of the glomerular mesangium. Thickening of the glomerular basement membrance is also apparent. Mesangial expansion is largely due to the accumulation of extracellular matrix (ECM) proteins such as fibronectin, laminin, and type IV collagen. To determine whether high glucose is responsible for the observed increase in mesangial cell ECM protein accumulation, mesangial cells were grown in tissue culture medium containing 10 mmol/l (millimolar) glucose (normal) or 30 mmol/l glucose (high). The degree of ECM protein accumulation was determined by immunocytochemistry and a solid-phase enzyme-linked immunosorbent assay (ELISA) developed in the laboratory. Mesangial cells cultured for 1 week contained fibronectin as the most abundant ECM protein, followed by laminin and type IV collagen. Type IV collagen was seen only after the cells had piled up into 'hillocks' (approximately 4 weeks of continuous growth without passaging). After 4 weeks in 30 mmol/l glucose, mesangial cells contained increased amounts of all three matrix proteins. Fibronectin and laminin were increased by approximately 60%, while type IV collagen was increased 50%. Cells subcultured in medium containing 30 mmol/l glucose for 8 months displayed a twofold increase in fibronectin and laminin. Thus, high glucose per se can cause changes in mesangial cell ECM. This cell culture model should be useful in elucidating the mechanisms involved.

Enzymatic modification of Novikoff hepatoma lamins A and C.

A significant increase in the molecular weights of lamin A and more so of lamin C was observed when isolated Novikoff hepatoma chromatin was incubated in the presence of Ca2+. This increase did not occur to any significant degree in similar preparations of normal rat liver nuclei. Although detectable in Coomassie Brilliant Blue stained gels, this increase to a higher molecular weight (by approximately 2000 Mr) was much more visible when the electrophoretically separated lamins were transferred to nitrocellulose sheets and stained (using peroxidase-antiperoxidase) with polyclonal antiserum to the three major lamin proteins. This modification could also be induced when whole Novikoff hepatoma cell lysates were incubated in the presence of calcium. Again, this change did not occur in normal rat liver cells treated in the same manner. Further analysis has provided evidence that this modification is most likely mediated by the transaminating activity of an intrinsic nuclear transglutaminase forming a cross-link between the affected lamins and an unknown low molecular weight (approximately equal to 2000 Mr) moiety.

Regulation of smooth muscle alpha-actin expression and hypertrophy in cultured mesangial cells.

BACKGROUND: Mesangial cells during embryonic development and glomerular disease express smooth muscle alpha-actin (alpha-SMA). We were therefore surprised when cultured mesangial cells deprived of serum markedly increased expression of alpha-SMA. Serum-deprived mesangial cells appeared larger than serum-fed mesangial cells. We hypothesized that alpha-SMA expression may be more reflective of mesangial cell hypertrophy than hyperplasia. METHODS: Human mesangial cells were cultured in medium alone or with fetal bovine serum, thrombin, platelet-derived growth factor-BB (PDGF-BB) and/or transforming growth factor-beta1 (TGF-beta1). Alpha-SMA expression was examined by immunofluorescence, Western blot, and Northern blot analysis. Cell size was analyzed by forward light scatter flow cytometry. RESULTS: Alpha-SMA mRNA was at least tenfold more abundant after three to five days in human mesangial cells plated without serum, but beta-actin mRNA was unchanged. Serum-deprived cells contained 5.3-fold more alpha-SMA after three days and 56-fold more after five days by Western blot. Serum deprivation also increased alpha-SMA in rat and mouse mesangial cells. The effects of serum deprivation on alpha-SMA expression were reversible. Mesangial cell mitogens, thrombin or PDGF-BB, decreased alpha-SMA, but TGF-beta1 increased alpha-SMA expression and slowed mesangial cell proliferation in serum-plus medium. Flow cytometry showed that serum deprivation or TGF-beta1 treatment caused mesangial cell hypertrophy. PDGF-BB, thrombin, or thrombin receptor-activating peptide blocked hypertrophy in response to serum deprivation. CONCLUSIONS: We conclude that increased alpha-SMA expression in mesangial cells reflects cellular hypertrophy rather than hyperplasia.

Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium.

Nodular expansion of glomerular mesangium with increased amounts of extracellular matrix (ECM) material is pathognomic of diabetic nephropathy. The precise mechanisms involved in this accumulation are unknown. Recently, we reported using a solid-phase enzyme-linked immunosorbent assay (ELISA) technique that glomerular mesangial cells, the principal cell type residing in glomerular mesangium, accumulate 50-60% more fibronectin (FN), laminin (LM), and type IV collagen (T-IV) when cultured in medium containing high glucose (30 mM) (S. H. Ayo, R. A. Rodnik, J. Garoni, W. F. Glass II, and J. I. Kreiberg. Am. J. Pathol. 136: 1339-1348, 1990). ECM assembly is controlled by its rate of synthesis and degradation, as well as its binding and rate of incorporation into the ECM. To elucidate the mechanisms involved, pulse-chase experiments were designed to estimate ECM protein synthesis from the incorporation of Trans-35S [( 35S]methionine, [35S]cysteine) into immunoprecipitated FN, LM, and T-IV. mRNA levels were examined, and degradation rates were estimated from the disappearance of radioactivity from matrix proteins in mesangial cells previously incubated with Trans-35S. One week of growth in 30 mM glucose resulted in approximately 40-50% increase in the synthesis of all three matrix proteins compared with 10 mM glucose-grown cells. This was accompanied by a significant increase in the transcripts for all three matrix proteins (approximately twofold). The specific activity of the radiolabel in trichloroacetic acid-precipitable cell protein showed no difference between cells grown in 10 or 30 mM glucose, indicating that total protein synthesis was unchanged. After 1 wk, the rate of FN, LM, and T-IV collagen degradation was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

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 (transgenic adenocarcinoma of mouse prostate) 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 CD8alpha-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-beta, CD3-epsilon, and CD3-zeta 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-zeta loss was observed. In contrast to alphabeta-T cells, tumor-infiltrating gammadelta-T cells maintained expression of their antigen receptors but not CD3epsilon. 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.