Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages.

Taxol, a microtubule stabilizing agent, exhibits promise in the treatment of breast and ovarian tumors. Recently, this novel drug has been shown to activate murine macrophages to express TNF-alpha and to down-regulate TNF-alpha receptors, activities shared by bacterial LPS. Our study sought to determine if taxol could regulate gene expression in murine macrophages and to examine further the ability of taxol to generate an LPS-like signal. Toward this end, the ability of taxol to induce TNF-alpha mRNA and five other genes (IL-1 beta, IP-10, D3, D7, and D8) associated with LPS-activation of macrophages was examined by Northern blot analysis. Taxol alone (1-30 microM) induced murine C3H/OuJ macrophages to secrete bioactive TNF-alpha and express increased levels of each of the six genes under investigation. The magnitude and the kinetics of induction of each gene closely resembled that seen with Escherichia coli K235 LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, however, failed to induce detectably any of the genes in response to taxol, despite being sensitive to the microtubule stabilizing effects of taxol as determined by immunofluorescence microscopy. The gene induction activity of taxol was in marked contrast to an alternative macrophage activator, heat killed Staphylococcus aureus, which induced a distinct gene profile in C3H/OuJ macrophages and which was equally active in C3H/OuJ and C3H/HeJ macrophages. These data are consistent with an ability of taxol to generate an LPS-like signal, possibly through a common signaling intermediate. As a first step toward identifying signal responses shared by taxol and LPS, we have shown that taxol, as shown previously for LPS, rapidly induces the tyrosine phosphorylation of a 41- and 42-kDa protein.

Autocrine and paracrine regulation of astrocyte function by transforming growth factor-beta.

Recent evidence indicates that astrocytes have a wide range of functions, usually attributed to cells of the immune system, which are critical for maintaining a balanced homeostatic environment in the central nervous system (CNS). Moreover, these cells are known to participate in inflammatory events within the CNS by secreting cytokines such as transforming growth factor-beta (TGF-beta). In this study we have investigated the ability of TGF-beta to influence astrocyte functions. TGF-beta 1 mRNA is constitutively expressed by astrocytes in vitro, and when cultures are stimulated with exogenous TGF-beta 1 an increase in the expression of this mRNA can be shown, suggesting both autocrine and paracrine regulation. In in vitro assays, TGF-beta 1 is chemotactic for astrocytes in a dose-dependent fashion and inhibits astrocyte proliferation. These results indicating signal transduction by TGF-beta 1-prompted studies to explore receptor-ligand interactions on isolated astrocyte populations. In a receptor binding assay, we demonstrate that astrocytes appear to express three distinct TGF-beta receptor subtypes with nearly 10,000 receptors per cell. Thus, TGF-beta may play an important role in regulating astrocyte functions pivotal to the evolution of intracerebral immune responses including recruitment and activation of glial cells at local inflammatory sites within the CNS.

Role of Kupffer cells in developing streptococcal cell wall granulomas. Streptococcal cell wall induction of inflammatory cytokines and mediators.

Hepatic granulomas are induced by intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats. Kupffer cells rapidly clear SCW from the blood, and the authors examined Kupffer cells further for a role in SCW-hepatic inflammation. Isolated Kupffer cells cultured with SCW secreted high levels of tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), transforming growth factor beta (TGF beta), and prostaglandin E2 (PGE2). SCW transiently induced increased steady-state levels of IL-1 beta and TNF alpha mRNA; in contrast, constitutive expression of TGF beta 1 mRNA in Kupffer cells was not affected by SCW. Low concentrations of SCW induced the accumulation of intracellular IL-1 and TGF beta bioactivity, with intracellular IL-1 bioactivity remaining high through at least 72 hours of culture. Kupffer cells isolated 1, 7, and 21 days after SCW injection did not express IL-1 beta or TNF alpha mRNA greater than control levels and exhibited marked hyporesponsiveness to secondary in vitro stimulation with SCW or LPS. SCW transiently induces Kupffer cells to secrete a variety of soluble mediators that contribute to hepatic inflammation by inducing leukocyte recruitment and activation and fibroproliferation. The transient nature of the Kupffer cell response and the hyporesponsiveness to secondary stimulation may be a mechanism by which the hepatic inflammation is negatively regulated.

Kupffer cells express type I TGF-beta receptors, migrate to TGF-beta and participate in streptococcal cell wall induced hepatic granuloma formation.

Intraperitoneal injection of Group A streptococcal cell wall (SCW) fragments into female Lewis rats results in the induction of an acute hepatic inflammation that progresses to granulomatous lesions. Kupffer cells have been shown to rapidly clear circulating SCW which triggers production of TGF-beta. In this study, we examined Kupffer cells for the expression of TGF-beta receptors to determine if these cells might be modulated in an autocrine/paracrine fashion by TGF-beta during SCW-hepatic inflammation. By receptor crosslinking and subsequent SDS-PAGE analysis we demonstrate that Kupffer cells express Type I TGF-beta receptors, but not Types II and III. Scatchard analysis indicated a receptor density of approximately 1100 receptors per cell. Functionally, TGF-beta was found to be chemotactic for Kupffer cells in vitro and this chemotactic response was higher in cells isolated from rats 1-21 days post SCW-injection. Although TGF-beta 1 mRNA is constitutively expressed by Kupffer cells, in vitro stimulation of the cultures with purified TGF-beta augments the expression of TGF-beta 1 mRNA and protein synthesis suggesting autocrine/paracrine regulation. These results indicate that TGF beta secreted by Kupffer cells during SCW-induced hepatic inflammation may amplify its own expression and regulate Kupffer cell functions relevant to the formation of granulomatous lesions within the liver.

Modulation of monocyte type I transforming growth factor-beta receptors by inflammatory stimuli.

The regulatory mechanisms which control the wide array of cellular responses to transforming growth factor beta (TGF beta) are not understood. This report presents evidence that down-regulation of TGF beta receptors on human monocytes may be one mechanism by which the effects of TGF beta are regulated. Treatment of monocytes with interferon gamma (IFN gamma) and lipopolysaccharide for 18 h reduced monocyte receptor number (approximately 400/cell) in a dose-dependent fashion by 89 and 78%, respectively, as determined by 125I-TGF beta binding. Incubation with other cytokines (granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor-1, interleukin-1, tumor necrosis factor alpha) did not alter the amount of TGF beta bound. The decrease in 125I-TGF beta binding could not be attributed to competition for receptor sites by secreted TGF beta. Instead, the decline in binding was due to a loss of type I TGF beta receptors, the subtype primarily expressed by monocytes, with no decrease in receptor affinity. Lipopolysaccharide-induced receptor loss was rapid (1-4 h), in contrast to the prolonged (12 h) decline induced by IFN gamma. Loss of receptors was accompanied by a diminished ability of the cells to respond to TGF beta with an induction of TNF alpha mRNA. Thus, this monocyte system is the first example of a heterologous agent causing the down-regulation of TGF beta receptors with a concomitant decline in a TGF beta-stimulated function.

Type I transforming growth factor-beta receptors on neutrophils mediate chemotaxis to transforming growth factor-beta.

Participation of human polymorphonuclear neutrophils in the inflammatory response is mediated, in part, by soluble factors such as chemotactic peptides and cytokines. Although the cytokine, transforming growth factor beta (TGF-beta), has been shown to recruit monocytes and promote the inflammatory process, its effects on neutrophils are unknown. In this investigation, [125I]TGF-beta 1 affinity binding studies were employed to show that neutrophils express TGF-beta receptors (350 +/- 20 receptors/cell), which exhibit high affinity for the ligand (dissociation constant, 50 pM). Affinity cross-linking studies identified the receptors to be primarily of the type I class. In contrast to the receptors on monocytes, neutrophil TGF-beta receptors were not down-regulated by exposure to specific inflammatory mediators. Additional studies examined whether exposure of neutrophils to TGF-beta could enhance specific functions, as occurs with monocytes. TGF-beta was shown to cause directed migration of neutrophils at femtomolar concentrations, thus it is the most potent neutrophil chemotactic factor yet identified. Neutrophil production of reactive oxygen intermediates was not stimulated by TGF-beta, nor did TGF-beta enhance or depress subsequent PMA- or FMLP-stimulated superoxide production. However, the stable expression of neutrophil TGF-beta receptors, and the capacity of this cytokine to stimulate neutrophil chemotaxis, suggest that the pro-inflammatory effects of TGF-beta are mediated by neutrophils in addition to monocytes.

IL-4 antagonizes induction of Fc gamma RIII (CD16) expression by transforming growth factor-beta on human monocytes.

The multifunctional cytokines, transforming growth factor (TGF)-beta 1 and beta 2, have the capability of inducing human peripheral blood monocytes to express the type III receptor for the Fc portion of IgG (Fc gamma RIII/CD16). In this study we show that the T cell-derived cytokine, IL-4, antagonizes the ability of TGF-beta to induce the expression of CD16. Furthermore, this ability to down-regulate expression of CD16 is completely abrogated after treatment with polyclonal anti-IL-4, suggesting that IL-4 is solely responsible for the observed inhibition. The mechanism for negating the effect of TGF-beta is not due to decreased expression of surface receptors for TGF-beta, but appears to occur at the mRNA level. Nuclear run-off assays indicate that regulation occurs predominantly through a posttranscriptional mechanism(s), although a transcriptional process cannot be ruled out. Normally, CD16 appears on only a small population of circulating monocytes, however, expression is apparent on the majority of mature tissue and inflammatory macrophages likely due to the release of TGF-beta in these sites. Inasmuch as this receptor binds immune complexes and opsonized particles, it is associated with enhanced immunophagocytosis. Suppression of CD16 expression and its ability to suppress a number of other monocyte functions suggests that IL-4 may play an important role in the resolution of inflammatory and tissue repair responses.

Transforming growth factor beta 1 suppresses acute and chronic arthritis in experimental animals.

Systemic administration of the cytokine, TGF beta 1, profoundly antagonized the development of polyarthritis in susceptible rats. TGF beta 1 administration (1 or 5 micrograms/animal), initiated one day before an arthritogenic dose of streptococcal cell wall (SCW) fragments, virtually eliminated the joint swelling and distortion typically observed during both the acute phase (articular index, AI = 2.5 vs. 11; P less than 0.025) and the chronic phase (AI = 0 vs. 12.5) of the disease. Moreover, TGF beta 1 suppressed the evolution of arthritis even when administration was begun after the acute phase of the disease. Histopathological examination of the joint revealed the systemic TGF beta 1 treatment greatly reduced inflammatory cell infiltration, pannus formation, and joint erosion. Consistent with the inhibition of inflammatory cell recruitment into the synovium, TGF beta 1 reversed the leukocytosis associated with the chronic phase of the arthritis. Control animals subjected to the same TGF beta 1 dosing regimen displayed no discernable immunosuppressive or toxic effects even after 4 wk of treatment. These observations not only provide insight into the immunoregulatory effects of TGF beta, but also implicate this cytokine as a potentially important therapeutic agent.

Cytokine modulation of bacterial cell wall-induced arthritis.

Bacterial cell wall-induced arthritis in experimental animals is dependent on mononuclear cell infiltration and accumulation. These mononuclear cells secrete cytokines which promote synovial hyperplasia and erosive destruction of bone and cartilage. Whereas local administration of cytokines may exacerbate these events, systemic administration of TGF-beta or gamma IFN effectively suppresses both the acute and chronic phases of the disease.

The production of alveolar macrophage-derived growth-regulating proteins in response to lung injury.

Tissue injury elicits an inflammatory response, one element of which is the activation of the local macrophage population. Macrophages are recognized as the source of multiple growth-regulating proteins, and are thus thought to play an important role in wound healing. Injury to the lung by exposure to oxidant gases, particulates, chemicals or drugs is often followed by replication of the cells of the alveoli. The growth-regulating proteins released by alveolar macrophages (AM) may be one mechanism which controls the proliferation of these cells. This article describes the AM growth factors, the cell types which they affect, and the injuries known to cause their release. In view of the multiplicity and overlapping functions of the macrophage growth factors, potential mechanisms which might regulate the growth response of the surrounding cells are also considered.

Stimulated rabbit alveolar macrophages secrete a growth factor for type II pneumocytes.

The type II pneumocyte plays a principle role in the maintenance and repair of the pulmonary alveolar epithelium by increasing its rate of proliferation under conditions of epithelial damage. This investigation examined the role of the alveolar macrophage in the control of type II cell division through its ability to produce specific growth factors when activated in vitro. Type II cells were isolated from adult male rabbits and cultured in the presence of media and matrix that support cell proliferation. Proliferation was assessed by cell counting and pulsing with [3H]thymidine, followed by measurements of labeling index and TCA-insoluble radioactivity. Alveolar macrophages were cultured in serum-free media in the presence of a particulate stimulus. Conditioned media was diluted and added to type II cell cultures. Conditioned media from stimulated macrophage cultures was found to double basal type II cell proliferation, whereas media from unstimulated macrophage cultures had no effect. Macrophage production of type II cell growth-promoting activity was dependent on the concentration of the stimulus and the length of the incubation. Investigation into the identity of the growth-regulating protein established that it is heat labile, insensitive to reduction and acidic conditions, and sensitive to trypsin digestion. Its molecular weight appears to be greater than or equal to 25 kD. Addition of several characterized growth factors to type II cell cultures demonstrated that other known growth-promoting products of macrophages do not act as type II cell growth factors. The evidence presented suggests that in vitro activated alveolar macrophages produce a type II cell growth factor that may play a critical role in mediating repair of the alveolar epithelium.

Induction of the stress response by isolation of rabbit type II pneumocytes.

Protein synthesis by isolated rabbit type II pneumocytes was evaluated by incorporation of labeled amino acids, gel electrophoresis, and fluorography. Type II alveolar epithelial cells preferentially synthesize a small number of proteins immediately following isolation and simultaneously exhibit a depression of all other protein synthesis. Examination of the time course of the appearance and decline of these proteins showed them to be actively synthesized during the first 14 h after isolation. Decline in their production was accompanied by a gradual enhancement in the synthesis of other proteins. Actin synthesis was not measurable 1 h after isolation, but increased to 9% of total label incorporated by 14 h. This recovery was independent of attachment to an extracellular matrix. Treatment with actinomycin D immediately following cell isolation abolished the synthesis of these proteins, suggesting a requirement for active mRNA production. A subset of these proteins are induced by heat treatment of cultured type II cells. The half-life, intracellular localization, isoelectric points, apparent molecular weights, and heat inducibility of this group of proteins are similar to stress proteins found in other mammalian systems. These stress proteins are unique in that their synthesis is induced by the cell isolation process. Possible mechanisms by which cell isolation may induce the stress response are discussed.