Fibroblast stimulation in schistosomiasis. IV. Isolated egg granulomas elaborate a fibroblast chemoattractant in vitro

Hepatic fibrosis complicates the chronic granulomatous inflammatory reaction to Schistosoma mansoni eggs, and is the major cause of morbidity and mortality in human schistosomiasis. We previously presented evidence that schistosomal egg granulomas secreted factors that can stimulate fibroblast proliferation and collagen synthesis in vitro. We now report that serum-free supernatants from cultures of hepatic egg granulomas isolated from S. mansoni-infected mice contained activity that stimulated the directional migration of human and guinea pig dermal fibroblasts in modified Boyden chambers. This fibroblast chemotactic activity was also detected in culture supernatants of granuloma adherent cells highly enriched for macrophages (95% latex-ingesting) but not in culture supernatants from resident peritoneal macrophages of uninfected or infected mice. This suggests that granuloma macrophages are a source of the chemotactic activity. The chemoattractant had the properties of large molecular weight (greater than 200,000 daltons; Sephadex G-200 gel filtration), pl approximately 4.5 (preparative flatbed isoelectrofocusing in granular matrix), heat stability (56 degrees C; 45 min), and trypsin sensitivity. Since preincubation of the partially purified granuloma and adherent-cell derived chemoattractants with rabbit anti-human fibronectin antibody abolished their chemotactic activity, it appears that the factor is antigenically similar to fibronectin. We propose that egg granuloma macrophages are activated in vivo to secrete a fibronectin-like molecule with activity that stimulates the directional migration of fibroblasts. This factor may therefore play a role in the local recruitment of fibroblasts and, in concert with other granuloma-derived factors, may play an important role in the pathogenesis of hepatic fibrosis in schistosomiasis.     

Leptin requires canonical migratory signaling pathways for induction of monocyte and macrophage chemotaxis

The growing worldwide obesity epidemic is frequently linked to an increased risk of developing diseases such as diabetes, cardiovascular disease, and cancer. These diseases are associated with the infiltration of macrophages in white adipose tissue (WAT), the artery wall, and tumors, respectively; and these macrophages likely contribute to disease progression and pathogenesis. Abdominal WAT, adipose tissue surrounding the heart and artery wall, as well as carcinoma cells, secrete many factors that could induce macrophage infiltration. Leptin is an adipocyte-secreted hormone, and deficiency of either leptin or its receptor has been shown to cause morbid obesity in animals and in humans. However, what is more commonly noted in human obesity is the presence of central leptin resistance leading to hyperleptinemia. As leptin receptors are present on macrophages, we hypothesized that leptin could act as a monocyte/macrophage chemoattractant. Our current study demonstrates: 1) leptin is a potent chemoattractant for monocytes and macrophages, inducing maximal chemotactic responses at 1 ng/ml; 2) leptin-mediated chemotaxis requires the presence of full-length leptin receptors on migrating cells; 3) leptin causes increased influx of intracellular calcium in macrophages; and 4) activation of janus kinase/signal transducers and activators of transduction (JAK/STAT), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) pathways are all necessary for leptin-induced macrophage migration. Taken together, these data demonstrate that leptin is a potent monocyte/macrophage chemoattractant in vitro and that canonical cell motility machinery is activated upon macrophage exposure to leptin. These data have implications for the impact of hyperleptinemia on obesity-related pathophysiological conditions such as diabetes, cardiovascular disease, and cancer.     

Evaluation of monocyte chemotactic responsiveness in uraemic patients undergoing haemodialysis with different dialytic membranes

Recent data show that monocyte chemotactic peptide-1 (MCP-1), a chemotactic factor specific for monocytes, may play a central role in regulating the activation of these cells. For this reason, the production of MCP-1 in peripheral blood mononuclear cell (PBMC) cultures of eight healthy subjects, six chronic uraemic subjects under conservative treatment and six chronic uraemic patients undergoing haemodialysis (HD), was assessed. In the latter group of individuals, complement-activating membranes such as cuprophan (CU) were used for 1 month followed by biocompatible non-complement-activating membranes, like polymethylmetacrylate (PMMA) for the next 30 days. The chemotactic index (CI) elicited by PBMC supernatants from patients undergoing dialysis was found to be significantly higher than that obtained by supernatants recovered from normal subjects or uraemic patients on conservative therapy. Furthermore, the CI from PBMC supernatants having had contact with CU membranes was higher than that obtained from PBMC activated by PMMA. Finally, the increased chemotactic ability in the supernatants was closely correlated with the augmented MCP-1 gene expression and production, as assessed by in vitro hybridization studies.     

Regulation by PGE2 of the production of oxygen intermediates by LPS-activated macrophages

The regulation by prostaglandin E2 (PGE2) of production of oxygen radicals by bacterial lipopolysaccharide-(LPS) activated macrophages was studied in vitro. A 48-hr incubation of murine thioglycollate-elicited macrophages with LPS (0.1 micrograms/ml) resulted in an enhanced ability of these cells to produce oxygen radicals when challenged with phorbol myristate acetate (PMA). Macrophages incubated for 48 hr without LPS did not produce measurable amounts of oxygen radicals when exposed to this triggering stimulus. Thus, PMA-triggered production of oxygen radicals was the result of macrophage activation by LPS. The PMA-triggered production of oxygen radicals by the LPS-activated macrophages was inhibited when PGE2 (10(-5) to 10(-9) M) was present during the incubation with LPS. Inhibition by PGE2 occurred during the early stages of macrophage activation, since the addition of PGE2 24 hr after LPS no longer inhibited the production of oxygen radicals by the macrophages. This inhibitory effect of PGE2 on the LPS-induced activation of macrophages could be reproduced by cyclic-adenosine-monophosphate (cAMP) agonists, such as isoproterenol and cholera toxin as well as by the cAMP analog dibutyryl-cAMP, suggesting a cAMP-mediated mechanism for the inhibitory effect of PGE2 on macrophage activation by LPS. Previous reports have implicated prostaglandins as mediators of destructive processes associated with chronic inflammation. Our findings suggest that PGE2 may, on the other hand, reduce tissue damage in a chronic inflammatory site by inhibiting the production of oxygen radicals by macrophages activated in the sera.     

Fibroblast stimulation in schistosomiasis. V. Egg granuloma macrophages spontaneously secrete a fibroblast-stimulating factor

Isolated intact egg granulomas from the liver of Schistosoma mansoni-infected mice have been previously shown to elaborate factors in vitro that can stimulate fibroblasts for biological functions that are of potential importance in the pathogenesis of hepatic fibrosis in schistosomiasis. We report here that cell cultures obtained from monodispersed granuloma cell suspensions, and specifically enriched for macrophages (95% to 100%) spontaneously elaborated fibroblast proliferation-stimulating activity in vitro. These cells possessed functional and phenotyptic characteristics of activated macrophages. In contrast, control peritoneal macrophages from uninfected mice lacked such phenotypic characteristics, and did not spontaneously elaborate fibrogenic activity in vitro. The granuloma macrophage activity was present, pre-formed within the isolated cells, and was continuously elaborated during 72 hr of incubation. By gel infiltration chromatography (Sephacryl S-200 sf), fibroblast-stimulating activity was identified in two pooled fractions, one with estimated molecular radius (Mr) of 46 kd to 57 kd and the other with Mr of 10 kd to 16 kd. Preparative isoelectric focusing in granular gel of crude macrophage culture supernatants identified peak activity in fractions with pI approximately 5. Two different serine esterase inhibitors had no effect on the ability of crude granuloma macrophage supernatants to stimulate fibroblast proliferation. Whereas crude and chromatographed fractions of granuloma macrophage supernatant were active for fibroblasts, they had minimal or no interleukin 1 (IL 1) activity when tested in a thymocyte proliferation assay. In contrast, resident peritoneal macrophages from the same infected mice spontaneously secreted substantial IL 1 and fibroblast-stimulating activity in vitro. We conclude that egg granuloma macrophages are activated in vivo to secrete fibrogenic molecules functionally distinct from IL 1, which might contribute to the pathogenesis of hepatic fibrosis in schistosomiasis.     

Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these growth factors might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and fibroblasts as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and fibroblasts to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in fibroblasts in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of fibroblasts and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and fibroblasts from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney fibroblasts. The results suggest that TGF-beta transiently attracts fibroblasts into the wound and may stimulate collagen synthesis directly. In contrast, PDGF is a more potent chemoattractant for wound macrophages and fibroblasts and may stimulate these cells to express endogenous growth factors, including TGF-beta, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.     

Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

Tumor associated macrophages are known to be closely linked with tumor progression and metastasis. On the other hand, clusterin is overexpressed in several tumor types and regarded as a putative tumor-promoting factor due to this overexpression and the subsequent induction of chemoresistance. In our previous study, clusterin was found to induce the expression of matrix metalloproteinase-9 (MMP-9) in macrophages, and MMP-9 is known to be essential for tumor cell migration and invasion via basement membrane breakdown. Because paracrine interactions between tumor cells and surrounding macrophages regulate metastasis, these findings raise the possibility that clusterin promotes the secretion of cytokines in macrophages in addition to MMP-9. Here, we demonstrate that clusterin upregulates the expressions of chemotactic cytokines, that is, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), regulated upon activation, normal T cell expressed and secreted (RANTES), and tumor necrosis factor-α (TNF-α) in Raw264.7 macrophages. In particular, clusterin stimulated TNF-α secretion via the activations of ERK, JNK, and PI3K/Akt pathways in a time and dose-dependent manner. Furthermore, clusterin-induced TNF-α secretion was found to play a critical role in the chemotactic migration of Raw264.7 macrophages. It was also found that clusterin acts directly as a chemoattractant for macrophages. Together, these results suggest that clusterin stimulates the expression and secretion of TNF-α, which plays a critical role in promoting macrophage chemotaxis, via ERK, JNK, and PI3K/Akt pathways. Collectively, these findings describe a novel function for clusterin as an inducer of TNF-α in macrophages and their chemotactic migration, and suggest that clusterin has a tumor-promoting effect.     

Macrophage activation for tumor cytotoxicity: induction of tumoricidal macrophages by supernatants of PPD-stimulated Bacillus Calmette-Guérin-immune spleen cell cultures.

Resident peritoneal macrophages from normal mice were activated for tumor cytotoxicity in vitro by co-cultivation with BCG1-immune spleen cells and PPD and by incubation with supernatants of PPD-stimulated BCG-immune spleen cell cultures (lymphokine supernatants). Lymphokine activation of macrophages occurred in unfractionated PC suspensions as well as in macrophage monolayers depleted of nonadherent PC. Tumor cytotoxicity by lymphokine-activated macrophages was evident by 3 to 4 hr of culture in active supernatants, reached maximal levels by 8 to 12 hr. and was absent by 20 hr. Continued incubation in lymphokines or even re-exposure after washing did not maintain macrophage cytotoxicity. The capacity of normal resident macrophages to be activated by lymphokines in vitro progressively decreased and was absent by 20 hr in culture. This decrease did not necessarily reflect cell death; macrophage viability as estimated by exclusion of trypan blue or by phagocytic responses did not change over the 20-hr culture period. The short lived nature of both macrophage tumoricidal capacity and capacity of precursor cells to be activated by lymphokines may function as negative feedback mechanisms in immune reactions.     

NK-leukocyte chemotactic factor (NK-LCF): a large granular lymphocyte (LGL) granule-associated chemotactic factor

A chemotactic factor was identified in the supernatants of human large granular lymphocytes (LGL) activated by a glutaraldehyde-fixed NK-sensitive tumor, K562. The factor stimulated migration of human LGL, rat alveolar macrophage (RAM), and human monocytes and neutrophils (PMN). The locomotor response was chemotactic and chemokinetic on the basis of unidirectional migration in concentration gradients. The cell producing the factor was detected exclusively in LGL-rich Percoll fraction coincident with the peak of NK lytic activity and HNK-1+ cells. The monoclonal phenotype of the cell was HNK-1+, partially OKT-11+, OKM-1-, OKT-3-, OKT-4-, and OKT-8-. The factor was released by LGL within 20 min of incubation with Sr++, a cation that is able to induce LGL degranulation. A powerful chemoattractant was also detected in the granules of the rat LGL leukemia, RNK. Chemotactic activity coincided with granule enzyme beta-glucuronidase and cytolysin after RNK nitrogen cavitation and Percoll fractionation of subcellular constituents. The RNK granule chemoattractant induced unidirectional migration of human LGL and was also active against rat alveolar macrophages and human PMN. Anti-RNK granule antibody conjugated to Sepharose 4B was able to deplete the chemotactic activity from both K562-induced LGL supernatants and solubilized RNK granules. These observations indicate that a leukocyte chemotactic factor (NK-LCF) is present in NK cell granules and is probably released after tumor-induced granule exocytosis.