Delayed mast cell mediated mitogenic reactivity in diabetic rats

Rats with diabetes of 4 weeks' duration have previously demonstrated increased mitogenesis in normal connective tissue cells following mast cell secretion. The appearance of this augmented mast cell-mediated mitogenic reactivity was studied in the mesentery of insulin-deficient rats, 3, 7, and 28 days after they had been rendered diabetic by a single dose of streptozotocin. On day 7 mast cell secretion induced a subnormal mitogenic response which, however, increased above normal on day 28. This time lag in the augmentation of mitogenic responsiveness may be important since proliferative lesions in diverse mesenchymal tissues typically develop with a similar delay in both experimental and clinical diabetes.     

LOCAL MITOGENIC EFFECT OF TISSUE MAST CELL SECRETION

The effect of drug-induced mast cell secretion on proliferation was studied in fibroblast-like and mesothelial-like cells in organ-cultured rat mesentery. Mast cell degranulation achieved by Compound 48/80 was followed by a marked mitogenic reaction in the surrounding tissue cells. The drug itself lacked mitogenic effect on cultured guinea-pig mesentery, the mast cells of which are unresponsive to the drug, and on a human normal fibroblast-like cell line. In contrast, histamine at about 10^?10 M, a major mast cell component, induced marked mitogenesis in guinea-pig mesentery without causing degranulation of mast cells. It is concluded that secreting rat-tissue mast cells release a mitogenic factor or factors acting locally on nearby tissue cells.     

Acute hyperglycaemia and mast-cell-dependent mitogenesis in rat

Hyperglycaemia is considered to be of possible aetiological significance for proliferative lesions in diabetes. We recently found that mast-cell-mediated proliferation in mesentery and skin is augmented in hyperglycaemic rats with longstanding diabetes. We now report a study to examine whether acute hyperglycaemia affects the proliferative response of fibroblasts and mesothelial cells that follows local mast-cell secretion in healthy rats. Hyperglycaemia was achieved by continuous intravenous infusion of glucose over a period of 2.5 h preceding and 9.5 h following administration of the mast-cell secretagogue compound 48/80. The 48/80 was given intraperitoneally which causes rapid mast-cell secretion and prompt subsequent mesenteric mitogenic stimulation. Hyperglycaemia thus persisted throughout the prereplicative period, during which stimulated cells are recruited into the cell cycle from a quiescent state. Because the ensuing mesenteric mitogenic response, estimated by specific DNA activity and mitosis counting, was unaffected by glucose infusion, acute hyperglycaemia itself appears to be of no aetiological significance for the accelerated mast-cell-dependent mesenteric mitogenesis in the diabetic rat.     

Further studies on the delayed mast-cell-mediated mitogenesis in diabetic rat

We have investigated whether the delayed augmented mast-cell-dependent mitogenesis that we recently demonstrated in the true mesentery of diabetic rats may occur also in a tissue which is essentially different both functionally and structurally. Cutaneous proliferation in insulin-deficient rats following a single intradermal injection of compound 48/80 was assessed by the specific DNA activity and mitotic index, on days 7 and 28 after giving streptozotocin. The proliferation variables, which ran parallel, indicated that mast-cell secretion was not followed by proliferation on day 7, whereas there was markedly augmented proliferation on day 28. Mitosis counting showed that cell production following mast-cell secretion was significantly increased not only in the entire cutis but also in epithelial structures (epidermis and epidermal appendages). These findings are closely similar to the delayed augmentation of mast-cell-dependent mesenteric mitogenesis in diabetic rats. It is concluded that there may be a general pattern of delay in the appearance of augmented mast-cell-mediated mitogenesis in rats with chemically induced diabetes. It is questioned whether or not this delayed mitogenesis may be connected with the postponed proliferation in a variety of tissues and organs (such as arteries, eyes, and kidneys) that occurs in experimental and clinical diabetes.     

Molecular aspects of mast-cell-mediated mitogenesis in fibroblasts and mesothelial cells in situ

Mast-cell-mediated mitogenesis in intact tissues is a paracrine reaction the molecular mechanisms of which still have to be elucidated. One strategy worth exploring is to study the mitogenic reaction under as defined conditions as possible. The present study demonstrates that in the virtually avascular rat mesentery, organ-cultured in a biochemically-defined medium, activation of mast cells induced a mitogenic reaction in fibroblasts and mesothelial cells, the two predominant, morphologically distinct neighboring cell types. Thus the system provides a means of studying the influence of defined molecules in the growth medium on the outcome of a mitogenic response in these two cell types in situ. It was further observed that exogenous platelet-derived growth factor (PDGF) was not essential for this mast-cell-mediated mitogenic reaction to occur in the tissue-bound fibroblasts and mesothelial cells.     

On the role of arachidonic acid metabolites in mast-cell mediated mitogenesis in the rat

We investigated whether the mitogenic response induced by local mast-cell secretion in the rat mesentery was affected by suppression of phospholipase A2, lipoxygenase, or cyclooxygenase in arachidonic acid metabolism. Enzyme inhibitor was given in a single intravenous dose 5 min before intraperitoneal injection of the mast-cell secretagogue 48/80. Mepacrine, a phospholipase A2 inhibitor, suppressed the generation of both leukotrienes (SRS) and prostaglandins (PG), whereas the lipoxygenase inhibitor BW 755C reduced the generation of SRS, and the cyclooxygenase inhibitor indomethacin significantly suppressed the generation of PG. None of the enzyme inhibitors affected the basal mesenteric histamine content or histamine release in the mesentery after exposure to 48/80, and none of them affected mast-cell-mediated mitogenesis in the mesentery as judged by specific DNA activity and mitosis counting. The stimulation of DNA synthesis and mitosis initiated by secreting mast cells is apparently not mediated or modulated by synthesis of leukotrienes, prostaglandins, or other known arachidonic acid metabolites.     

Aging-associated shifts in functional status of mast cells located by adult and aged mesenteric lymphatic vessels

We had previously proposed the presence of permanent stimulatory influences in the tissue microenvironment surrounding the aged mesenteric lymphatic vessels (MLV), which influence aged lymphatic function. In this study, we performed immunohistochemical labeling of proteins known to be present in mast cells (mast cell tryptase, c-kit, prostaglandin D(2) synthase, histidine decarboxylase, histamine, transmembrane protein 16A, and TNF-α) with double verification of mast cells in the same segment of rat mesentery containing MLV by labeling with Alexa Fluor 488-conjugated avidin followed by toluidine blue staining. Additionally, we evaluated the aging-associated changes in the number of mast cells located by MLV and in their functional status by inducing mast cell activation by various activators (substance P; anti-rat DNP Immunoglobulin E; peptidoglycan from Staphyloccus aureus and compound 48/80) in the presence of ruthenium red followed by subsequent staining by toluidine blue. We found that there was a 27% aging-associated increase in the total number of mast cells, with an ～400% increase in the number of activated mast cells in aged mesenteric tissue in resting conditions with diminished ability of mast cells to be newly activated in the presence of inflammatory or chemical stimuli. We conclude that higher degree of preactivation of mast cells in aged mesenteric tissue is important for development of aging-associated impairment of function of mesenteric lymphatic vessels. The limited number of intact aged mast cells located close to the mesenteric lymphatic compartments to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response.     

Low-molecular-weight peptides in bone extracts that stimulate osteoblast mitogenesis

Although a majority of regulatory peptides elaborated by neuroendocrine cells are small, i.e., less than 50-60 residues, no low-molecular-weight, bone-derived mitogenic peptides have been described. We have size-fractionated extracts of neonatal mouse calvaria, a rapidly forming bone, and assayed for osteoblast proliferation. Mitogenic peptides with estimated sizes of 1,600, 1,050, and 770 daltons were detected. Their protein nature was demonstrated by the reduction in mitogenic activity following protease treatment. Fibroblast mitogenesis was not stimulated by any of the peptides. These data indicate that there are mitogenic peptides in bone smaller than any previously described locally-derived bone cell growth factor.     

The SH3 domain, but not the catalytic domain, is required for phospholipase C-γ1 to mediate epidermal growth factor-induced mitogenesis

Phospholipase C-γ1 (PLC-γ1) is a multiple-domain protein and plays an important role in epidermal growth factor (EGF)-induced cell mitogenesis, but the underlying mechanism is unclear. We have previously demonstrated that PLC-γ1 is required for EGF-induced mitogenesis of squamous cell carcinoma (SCC) cells, but the mitogenic function of PLC-γ1 is independent of its lipase activity. Earlier studies suggest that the Src homology 3 (SH3) domain of PLC-γ1 possesses mitogenic activity. In the present study, we sought to determine the role of the SH3 domain of PLC-γ1 in EGF-induced SCC cell mitogenesis. We examined the effect of overexpression of PLC-γ1, a catalytically active PLC-γ1 mutant lacking the SH3 domain or a catalytically inactive PLC-γ1 mutant lacking the X domain on EGF-induced SCC4 (tongue squamous cell carcinoma) cell mitogenesis. We found that overexpression of PLC-γ1 enhanced EGF-induced SCC4 cell mitogenesis. This enhancement was abolished by deletion of the SH3 domain but not by deletion of the X catalytic domain. These data suggest that the SH3 domain, but not the catalytic domain, is required for PLC-γ1 to mediate EGF-induced SCC4 cell mitogenesis.     

Long term increase of mucosal mast cells in the rat induced by administration of Compound 48/80

Summary Administration of Compound 48/80 to rats for 5 days resulted in an increase of the specific type of mucosal mast cell, while connective tissue mast cells elsewhere were almost completely degranulated. The number of mucosal mast cells increased slowly for another 5 days and then returned to the control level, in an exponential manner. The half life of the newly formed mast cells was calculated to be about 40 days. This value may be taken as an estimate of the half life of mucosal mast cells. These cells, therefore, constitute a fraction of mast cells with rapid turnover. Available evidence indicates that the classical connective tissue mast cell has a very long life span, without significant turnover in terms of cell death and cell renewal. We suggest that the increase of mucosal mast cells is an indirect effect of Compound 48/80, related to its effect on other mast cells and mediated by material(s) released from these cells during the secretory process.Supported by grants from the Swedish Medical Research Council, Project no 2235     

Analysis of the activation of lymphoid cells by mitogens in vitro with limiting dilution methods: adherent peritoneal cells suppress B-cell activation and synergize with WEHI-3 in the activation of T cells by Con A

Adherent peritoneal cells (APC) have often been used as a pure and effective macrophage population. Using partition analysis and small numbers of lymphoid cells activated by mitogens (concanavalin A for T cells (in the presence of TCGF) and LPS + DxS for B cells) we found that APC were accessory cells for T cell activation and growth but were not effective for B cells. Although APC were effective in assisting T-cell mitogenesis, they were not especially efficient. However, when APC were mixed with irradiated WEHI-3 cells (a tissue culture line previously shown to exhibit accessory cell activity in vitro for mitogenic activation T and B cells), the APC and WEHI-3 showed apparent synergy. One reason for failure of APC to assist B-cell mitogenesis was traced to the presence of a suppressor cell population which overcame the accessory cell help given by irradiated WEHI-3 cells to LPS-DxS stimulated murine B cells. It is thus possible to find "helper" effects (synergy of APC and WEHI-3 assisting the mitogenesis of T cells), as well as suppressor effects within the range of cells found in adherent accessory cells.     

Morphological and functional characteristics of peritoneal mast cells from young rats

To study why neonatal and young rats are resistant to the effects of some secretagogues, such as compound 48/80 and 2.5-S nerve growth factor, we examined peritoneal mast cells from 14–15-day-old rats (young rats) and compared them to peritoneal mast cells from adults. Peritoneal mast cells from young rats contain approximately one-tenth of the amount of histamine observed in adult peritoneal mast cells. However, both cell populations contained similar low levels of the mucosal mast cell-associated protease rat mast cell protease II. Histochemical analysis of peritoneal mast cells from young rats using safranin O and berberine sulphate suggested that only a portion of the granules of these cells contained heparin. At an ultrastructural level the young rat peritoneal mast cell contains relatively few granules. The majority of mast cells from young rats have a bilobed or indented nucleus which is only rarely observed in adult cells. Functionally, the young rat peritoneal mast cell demonstrates a significantly reduced histamine release in response to the connective tissue mast cellspecific secretagogues compound 48/80 and 2.5-S nerve growth factor. In contrast, the percent histamine release in response to the neurotransmitter substance P, which degranulates both connective tissue mast cells and intestinal mucosal mast cells, was similar in the adult cells and the young rat cells. This study demonstrates substantial differences between the young rat and adult peritoneal mast cells which may explain the ability of very young animals to withstand large doses of certain secretagogues.     

Is cholesterol the receptor for polyene antibiotic-induced B-lymphocyte activation

Stimulation induced by polyene antibiotics has previously been considered to be mediated via binding to cell membrane cholesterol. However, stimulation by polyenes such as nystatin was not affected differently than other polyclonal B-cell activators by the addition of cholesteral-blocking drugs such as digitonin, tomatin, or pimaricin, nor did cholesterol-containing serum diminish its mitogenic properties. Incubation of cells with lecithin-cholesterol vesicles, thereby increasing total cell cholesterol, greatly reduces cell reactivity toward polyenes to the same extent as toward other B- or T-cell activators. We therefore, tentatively suggest that polyene-induced mitogenesis is not mediated via binding to cell membrane cholesterol but via interaction with a distinct mitogen receptor on the cell surface.     

The effect of insulin on delayed augmented mast-cell mediated mitogenesis in diabetic rats

Delayed augmented mitogenic reactivity follows mast-cell secretion in mesentery and skin in streptozotocin-diabetic rats with 4-week insulin-deficiency (Norrby 1982; Norrby et al. 1982; Norrby 1983). In such rats the basal proliferation is essentially unchanged in the mesentery and skin, whereas it is significantly increased in the small gut and significantly decreased in the kidney. On treating rats with 4-week-old diabetes with very-long-acting insulin for over 3 days (16 U/kg X 2 daily) the basal proliferation of the small gut and the kidney apparently became normal, the body weight increased, and the blood glucose concentration dropped substantially and progressively. However, insulin-treatment did not affect the mast-cell-dependent mitogenesis in the mesentery following intraperitoneal injection of the mast-cell secretagogue compound 48/80, as judged by specific DNA activity and mitosis counting. We conclude that some metabolic or cellular feature of diabetes which is not restored by 3-day insulin treatment, and not insulin deficiency itself, is the cause of the delayed increased mitogenic reactivity that follows mast-cell secretion in diabetic rat.     

Factor Xa induces mitogenesis of vascular smooth muscle cells via autocrine production of epiregulin

Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell.     

Foreign Body Response to Subcutaneous Implants in Diabetic Rats

Implantation of synthetic matrices and biomedical devices in diabetic individuals has become a common procedure to repair and/or replace biological tissues. However, an adverse foreign body reaction that invariably occurs adjacent to implant devices impairing their function is poorly characterized in the diabetic environment. We investigated the influence of this condition on the abnormal tissue healing response in implants placed subcutaneously in normoglycemic and streptozotocin-induced diabetes in rats. In polyether-polyurethane sponge discs removed 10 days after implantation, the components of the fibrovascular tissue (angiogenesis, inflammation, fibrogenesis, and apoptosis) were assessed. Intra-implant levels of hemoglobin and vascular endothelial growth factor were not different after diabetes when compared with normoglycemic counterparts. However, there were a lower number of vessels in the fibrovascular tissue from diabetic rats when compared with vessel numbers in implants from non-diabetic animals. Overall, the inflammatory parameters (neutrophil accumulation - myeloperoxidase activity, tumor necrosis factor alpha, and monocyte chemotactic protein-1 levels and mast cell counting) increased in subcutaneous implants after diabetes induction. However, macrophage activation (N-acetyl-β-D-glucosaminidase activity) was lower in implants from diabetic rats when compared with those from normoglycemic animals. All fibrogenic markers (transforming growth factor beta 1 levels, collagen deposition, fibrous capsule thickness, and foreign body giant cells) decreased after diabetes, whereas apoptosis (TUNEL) increased. Our results showing that hyperglycemia down regulates the main features of the foreign body reaction induced by subcutaneous implants in rats may be relevant in understanding biomaterial integration and performance in diabetes.     

Anesthetic effects on pulmonary allergic responses in rats: changes in sensitivity to serotonin.

Subsequent to observations that pulmonary responses to antigen challenge are of different magnitudes in sensitized rats that are anesthetized with different drugs, we conducted studies to test whether the alterations in responses were due to changes in airway responsiveness to cholinergic or serotonergic challenge, opioid-receptor mediated events, or changes in mast cell mediator release. Immunoglobulin E-sensitized rats anesthetized with ketamine/urethan had larger changes in lung resistance and plasma histamine after pulmonary antigen challenge compared with rats anesthetized with fentanyl-droperidol. Blockade of opioid receptors with naloxone did not affect the responses. In unsensitized rats, airway responses to aerosolized methacholine were similar for the two anesthetics, indicating unchanged smooth muscle responsiveness; however, airway responses to intravenous serotonin were enhanced by ketamine and ablated by droperidol. We conclude that ketamine- and droperidol-induced alterations of pulmonary allergic responses are due to changes in sensitivity to serotonin and in mast cell mediator release. We speculate that mast cell mediator release may be modulated by a serotonin receptor-linked mechanism.     

The role of mast cell degranulation products in mast cell hyperplasia. I. Mechanism of action of nerve growth factor

A variety of mast cell degranulating agents have previously been shown to induce mast cell hyperplasia in adult rats. In neonates 2.5 S nerve growth factor (NGF) induces a hyperplasia of both mucosal and connective tissue mast cells (MMC and CTMC). We have examined the role of the potent mast cell degranulating properties of NGF on its ability to induce mast cell hyperplasia. Administration of NGF in combination with the mast cell stabilizing agent disodium cromoglycate was found to abrogate the CTMC hyperplasia induced by NGF alone. Treatment of neonatal rats with the alternate degranulating agent compound 48/80 was found to induce a limited CTMC but not a MMC hyperplasia. A supernatant obtained by degranulating purified adult rat peritoneal mast cells with anti-IgE was found to induce hyperplasia of the CTMC population similar to that observed with NGF administration. However, this degranulation product supernatant only induced a limited MMC hyperplasia as judged by RMCP II content of the tissues. These results suggest that NGF has dual action inducing mast cell hyperplasia; CTMC hyperplasia being dependent on the ability of NGF to degranulate mast cells. MMC hyperplasia induced by NGF is independent of CTMC degranulation. Degranulation products from peritoneal mast cells act to increase both MMC and CTMC populations in the neonate. These data suggest that the CTMC population may be regulated by an autocrine positive feedback mechanism in vivo.     

Mast cell heterogeneity: effects of neuroenteric peptides on histamine release

Recent reports suggesting that the actions of certain neuroenteric peptides may be mediated in part by the secretion of histamine and other mast cell contents could have important implications for gastrointestinal motility and secretion. However, evidence for a mast cell-hormonal interaction is based on studies using peritoneal or cutaneous mast cells. Because intestinal mucosal mast cells (MMC) differ functionally from peritoneal mast cells (PMC), we compared the effects of several neurotransmitters and intestinal hormones on histamine secretion from two mast cell types in the rat. MMC hyperplasia was induced in rats by infection with the nematode Nippostrongylus brasiliensis, and MMC were isolated from the small intestine by collagenase digestion. Substance P, somatostatin, vasoactive intestinal polypeptide (VIP), neurotensin, and bradykinin had a potent secretagogue effect on (10(-7) to 10(-4)M) PMC which was temperature-, energy-, and calcium-dependent. In contrast to PMC, MMC released significant amounts of histamine only when challenged with substance P. Acetylcholine, bombesin, motilin, and pentagastrin had no secretory effect on either PMC or MMC. The differences between PMC and MMC in responsiveness to peptides could not be attributed to the MMC isolation procedure because PMC treated similarly or mixed with MMC suspensions retained their responsiveness to these stimuli. Our results extend the concept of neurocrine control of mast cell function, but indicate that mast cells from different sites have distinct profiles of responsiveness to regulatory peptides.