Constitutive activation of STAT3 is associated with the acquisition of an interleukin 6-independent phenotype by murine plasmacytomas and hybridomas.

Interleukin 6 (IL-6), the major growth factor for myeloma cells, signals through the activation of signal transducers and activators of transcription (STAT) proteins. An important step in the malignant progression of murine plasmacytomas is the transition from dependence on IL-6 to a state of IL-6 independence. To elucidate the mechanism whereby IL-6 independence occurs, intracellular signaling events elicited by IL-6 in both IL-6-dependent and -independent plasmacytomas and hybridomas were compared. It was found that STAT3, a key molecule involved in IL-6 signaling, was constitutively activated and phosphorylated in IL-6-independent cell lines compared to the IL-6-dependent cells. Further comparison of upstream signaling pathways revealed that JAK-1 was constitutively present in anti-phosphotyrosine immunoprecipitates of IL-6-independent cells; gp130 was constitutively phosphorylated in a subset of IL-6-independent plasmacytomas, whereas other IL-6-independent lines showed no detectable gp130 phosphorylation in the absence of exogenous IL-6. Secretion of a factor capable of supporting the growth of IL-6-dependent cells was observed in one of the IL-6-independent plasmacytomas, but not in others, making an autocrine mechanism an unlikely explanation for IL-6 independence. These findings provide evidence that the constitutive activation of STAT3, either in the absence of detectable receptor-proximal events or associated with the concomitant activation of gp130, can contribute to the process of IL-6 independence.

Evaluation of methods for transient transfection of a murine macrophage cell line, RAW 264.7.

Monocyte/macrophage cell lines are fastidious cells commonly used in transient transfection experiments. In the course of a study of gene regulation by lipopolysaccharide (LPS), we have compared several methods for DNA-mediated cell transfection to determine which would be optimally applicable to the macrophage line, RAW 264.7. Both the response level (LPS inducibility) and the degree of inter-assay variation were evaluated for each transfection technique. The following methods were compared: Lipofectin, LipofectAMINE, LipofectAMINE PLUS, SuperFect, Ca3(PO4)2 DNA co-precipitation, DEAE dextran-mediated transfection and electroporation. The transfected plasmid DNA included a luciferase reporter construct containing the junB minimal promoter under the control of an LPS-inducible 1300-bp regulatory fragment downstream of junB 5'-flanking sequence, as well as a beta-galactosidase reporter construct under the adenovirus promoter and enhancer used as an internal control. Electroporation, followed by a resting period of 16-24 h before stimulation with LPS, had the highest inducibility of all methods. DEAE dextran and Ca3(PO4)2 precipitation showed the least and the greatest inter-assay variation, respectively. For all other methods, inter-assay variability fell within this range. The results presented may serve as both a general reference and a guide for reporter gene studies in this or other macrophage cell lines.

TGF-beta: a balancing act.

Regulation of developmental processes as well as host defense and repair mechanisms requires the maintenance of a delicate balance of positive and negative regulatory signals. TGF-beta, a molecule known for its many diverse activities, can promote or inhibit cell growth and function. Disruption of the balance between these opposing activities can contribute to aberrant development, malignancy, or pathogenic immune and inflammatory responses. TGF-beta transgenic mouse studies highlight the essential function(s) of TGF-beta and its receptors and provide insight to potential therapeutic approaches to manipulate TGF-beta expression.

Lacrimal gland inflammation is responsible for ocular pathology in TGF-beta 1 null mice.

Mice homozygous for a nonfunctional transforming growth factor-beta 1 gene develop rampant inflammation in vital organs that contributes to a shortened life span. The presence of circulating anti-nuclear anti-bodies, immune deposits in tissues, leukocyte infiltration, and increased major histocompatibility complex antigen expression resembles an autoimmune-like syndrome. One of the overt symptoms that appears in these mice lacking transforming growth factor-beta 1 is the development of dry crusty eyes that close persistently as their health declines. Histologically, the eyes appear normal with little or no inflammation. However, inflammatory lesions, predominantly lymphocytic, develop in the lacrimal glands, disrupting their structure and function and severely limiting their ability to generate tears. This histopathology and aberrant function mimic that of Sjögren's syndrome, a human autoimmune disease characterized by dry eyes and dry mouth. Impeding the leukocyte infiltration into the glands with synthetic fibronectin peptides, which block adhesion, not only prevents the inflammatory pathology but also prevents the persistent eye closure characteristic of these mice.

Cytokine regulation of schistosome-induced granuloma and fibrosis.

Schistosomiasis mansoni, a major cause of hepatic fibrosis in many developing countries, triggers a granulomatous inflammatory reaction in response to its eggs that lodge in the liver. The egg antigens are eliminated slowly, and the persistent granulomatous response leads to prolonged matrix synthesis and hepatic fibrosis. In mice, soluble egg antigens (SEA) induce interleukin 4 synthesis, promoting a dominant T helper type 2 lymphocyte accumulation with the release of additional cytokines (IL-5, IL-10), which not only suppress Th1 lymphocyte subset cytokines, but mediate the characteristic pathophysiology. Manipulation of the cytokine profile with antagonists or exogenous cytokine delivery alters the course of the hepatic inflammation and fibrosis. In the evolution of the granulomatous response to the S. mansoni eggs, transforming growth factor beta (TGF-beta) is also produced that may modulate inflammation and regulate fibrogenesis. In TGF-beta 1-gene targeted mutant mice that over-express TGF-beta 1 (TGF-beta 1 transgenics) or in which TGF-beta 1 has been inactivated (TGF-beta 1-/-; null mutation) or partially inactivated (TGF-beta 1+/-; null mutation heterozygotes), the altered production of TGF-beta 1 impacts on S. mansoni granuloma and hepatic fibrosis. In addition to the Th1/Th2 cytokine balance, modulation of TGF-beta 1 may change the outcome of chronic inflammatory fibrotic disease.

Autoimmune Sjögren's-like lesions in salivary glands of TGF-beta1-deficient mice are inhibited by adhesion-blocking peptides.

The targeted disruption of the TGF-beta1 gene in mice (TGF-beta1 -/-) leads to extensive inflammation in vital organs, cachexia, and death within 3 to 4 wk. Significant inflammatory lesions develop initially in the periductal regions of the salivary glands and escalate as the animals become symptomatic. These inflammatory sites, characterized by lymphocytic infiltration and increased proliferation, cytokine mRNA expression, and IgG-positive cells, resemble lesions of Sjögren's syndrome. Moreover, the inflammatory pathology, enhanced MHC expression, and Ab production are consistent with an autoimmune-like etiology. Glandular atrophy and loss of acini with reduced saliva production appear to contribute to the wasting syndrome characteristic of the TGF-beta1 -/- mice. To determine whether the structural and functional defects were developmental due to the absence of TGF-beta1 or secondary to the inflammation, TGF-beta1 -/- mice were treated with synthetic fibronectin peptides, which block leukocyte infiltration. Daily systemic injections of RGD, CS-1, and/or peptides derived from the heparin-binding region of the A chain not only prevented leukocyte infiltration in the salivary glands of the TGF-beta1 -/- mice, but also reversed the acinar and ductal derangements. These data suggested that salivary gland development is not jeopardized in the absence of TGF-beta1, but that the extensive infiltration of inflammatory cells compromises glandular structure and function. The essential nature of TGF-beta1 in controlling inflammatory and immune processes is confirmed by these studies. Moreover, these TGF-beta1 -/- mice provide an important model of autoimmune disease that can be used in the design of therapeutic interventions.

Estrogen regulation of JE/MCP-1 mRNA expression in fibroblasts.

We have recently demonstrated that 17 beta-estradiol (E2) inhibits peritoneal adhesion formation. Because macrophages play a central role in inflammation and wound healing, we chose to investigate whether the E2 could inhibit the expression of JE, the murine monocyte chemoattractant protein-1 (MCP-1). To accomplish this, murine fibroblasts were cultured with physiological concentrations of E2 (3-300 pg/ml) with or without inducers of JE/MCP-1 mRNA expression. Untreated cells failed to express the message, but, following stimulation, a marked increase in JE/MCP-1 mRNA expression was observed. At 10-30 pg/ml, E2 had no effect on JE/MCP-1 mRNA expression in stimulated fibroblasts. In contrast, lower and higher doses of E2 inhibited the expression of JE/MCP-1 mRNA in stimulated fibroblasts. Treatment with tamoxifen reversed the E2-inhibition of expression of the message. These data demonstrate that JE/MCP-1 mRNA expression is controlled, in part, by estrogen and suggest that macrophage recruitment may be affected by circulating levels of E2.

Estrogen suppression of connective tissue deposition in a murine model of peritoneal adhesion formation.

Estrogen's involvement in inflammation and wound healing is poorly understood. To examine the role of estrogen in peritoneal adhesion formation, we gave ovariectomized female C57BL/6 mice time-release pellets containing placebo, 0.05 mg 17 beta-estradiol (low E2), or 5 mg 17 beta-estradiol (high E2) before i.p. injection of talc in saline or saline alone. Analyses of abdominal wall connective tissue thickness and peritoneal cell populations were performed. Talc-treated mice receiving low and high E2 replacement had a decreased amount of abdominal connective tissue deposition (29% and 65% decrease, respectively) as compared with talc-treated mice receiving placebo pellets. At high E2 replacement, the difference in connective tissue deposition was significant statistically (p less than 0.01). Immunohistochemical analysis revealed that the number of macrophages in adhesion tissue was proportionate to the amount of connective tissue present, regardless of the circulating levels of E2. Northern blot analysis of abdominal wall tissue showed that five of six talc-treated animals given placebo expressed mRNA for the murine monocyte chemoattractant protein-1 (MCP-1), JE. Conversely, only one of five talc-treated animals that received E2 replacement expressed JE/MCP-1 mRNA, suggesting that the hormone may inhibit connective tissue deposition by altering the production of chemotactic factors. Furthermore, E2 suppressed talc-induced expression of JE/MCP-1 mRNA in murine macrophages. Since macrophages play a central role in the wound healing process, these studies suggest that E2 inhibition of adhesion formation could be mediated by suppressing macrophage activation and/or recruitment to inflammatory sites.

Bronchoalveolar lavage fluid endotoxin elevation in human single lung transplant recipients during rejection.

Endotoxin has been implicated as an aetiological factor in liver transplant rejection and acute graft-versus-host disease. To investigate the role of endotoxin in human single lung transplant rejection we measured the level of endotoxin in bronchoalveolar lavage (BAL) fluid from six subjects at baseline and during rejection, which was defined histologically from transbronchial biopsy. Differential cell counts of BAL fluid cells, the levels of protein and albumin in BAL fluid, and serum albumin levels were also examined. BAL fluid albumin to serum ratio was calculated to evaluate alveolar-capillary leakage. A significant elevation of BAL fluid endotoxin with rejection compared with baseline was observed. Standardizing endotoxin levels to BAL fluid volume, protein, or albumin were all of similar significance. Examination of BAL fluid cell population revealed a significant elevation in the percentage of lymphocytes with rejection. No significant difference between BAL fluid protein levels, BAL fluid albumin levels. BAL fluid albumin to protein ratio, serum albumin levels, or BAL fluid albumin to serum albumin ratio was seen with rejection. We conclude that BAL fluid endotoxin levels increase during lung rejection, endotoxin levels can be accurately standardized to millilitres of BAL fluid, and abnormal alveolar-capillary leakage does not appear to account for the increased BAL fluid endotoxin.

Estrogen modulation of JE/monocyte chemoattractant protein-1 mRNA expression in murine macrophages.

The chemotactic cytokine, monocyte chemoattractant protein-1 (MCP-1), and its murine homologue, JE, have been detected in atherosclerotic lesions but not in normal arteries, implicating that these proinflammatory cytokines may be involved in the pathogenesis of atherosclerosis. Epidemiologic studies reveal that postmenopausal women receiving estrogen replacement for treatment of osteoporosis have a greatly reduced risk of developing cardiovascular disease. Because JE/MCP-1 and estrogen play regulatory roles in the development of atherosclerotic lesions, we chose to examine the effect of estrogen treatment on JE/MCP-1 mRNA expression in macrophages. 17 beta-estradiol (E2) inhibited LPS-stimulated JE/MCP-1 mRNA expression in ANA-1 and J774A.1 murine macrophage cell lines and in thioglycolate-elicited murine peritoneal macrophages. Inhibition of JE/MCP-1 mRNA ranged from 50 to 90%, with a maximal effect occurring at a concentration of 300 pg/ml E2. Conversely, E2 had little effect on LPS-stimulated TNF-alpha mRNA production. Treatment of LPS-stimulated macrophages with moxestrol, an estrogen agonist, resulted in a similar inhibition, and the addition of the estrogen antagonist, tamoxifen, reversed E2 inhibition of JE/MCP-1 mRNA expression. Progesterone failed to inhibit LPS-induced JE/MCP-1 mRNA expression. Immunohistochemical analysis revealed the presence of estrogen receptors in ANA-1 cells, indicating that E2 inhibition of LPS-induced JE/MCP-1 mRNA expression in murine macrophages may be mediated through the estrogen receptor. Thus, another mechanism whereby estrogen exerts antiatherogenic effects may be through prevention of macrophage accumulation in the atherosclerotic lesion.

Abdominal wall thickness as a means of assessing peritoneal fibrosis in mice.

Herein we describe a method for the quantitative assessment of connective tissue deposition within the peritoneal cavity. Female C57BL/6 mice (8-10 weeks) were given a single intraperitoneal injection of varying concentrations of talc (100 mg, 50 mg, 30 mg, 20 mg) in 1 ml of PBS or PBS alone. After 14 days, animals were killed. Adhesion formation was measured by the standard method of Myllârniemi et al. (1966), namely a gross visual inspection of the peritoneal cavity. This analysis gave a crude assessment of connective tissue deposition in the abdominal cavity but did not allow one to distinguish more subtle differences between intermediate dosage groups. In addition, a histological evaluation was performed. For the latter method, portions of the abdominal wall of mice were fixed and processed for histological analysis using Masson's Trichrome stain which allows for differentiation of connective tissue components. The thickness of connective tissue between the parietal peritoneum and the underlying abdominal wall muscle was measured. A dose-dependent increase in connective tissue deposition was observed in talc-treated animals compared to saline control animals. A differential cell count of the peritoneal exudate cells (PEC) showed that there was no change in cell populations in talc treated animals (compared to control animals). Given the above results, the measurement of connective tissue thickness was found to give the most accurate assessment of peritoneal fibrosis than other previously used methods.