Mustafa Kh. Dabbous

Mast cells and matrix degradation at sites of tumour invasion in rat mammary adenocarcinoma.

Significant numbers of mast cells have been demonstrated histologically around the periphery of the invasive rat mammary adenocarcinoma 13672NF. The number of mast cells at microfoci along the tumour:host tissue junction was significantly greater than that found in normal mammary tissues, and few mast cells were detected within the tumour itself. Mast cell degranulation, often associated with disruption and lysis of the connective tissue matrix, was a common feature in later stages of tumour proliferation. When soluble products derived from purified rat peritoneal mast cells were added to monolayer cultures of rat stromal fibroblasts or tumour cells they stimulated a significant increase in total collagenase production, and the mast cell products were also capable of activating the latent collagenases thus produced. Histological examination indicated that degradation of local collagenous matrix was a common feature of mast cell degranulation, an observation possibly explained by the release of mast cell enzymes and/or the potential of this cell to modulate the expression of collagenolytic activity by surrounding cells. These observations suggest that, at least in some tumours, mast cells contribute to the connective tissue breakdown commonly associated with tumour invasiveness and metastatic spread.

Chemotactic response of rat mammary adenocarcinoma cell clones to tumor-derived cytokines

A cytokine with an apparent molecular weight of 53,000 daltons was isolated from serum-free medium conditioned by MTLn3 cells or from homogenates of MTLn3 cells, a highly metastatic variant of the rat 13762NF mammary adenocarcinoma. The chemotactic responses of MTLn3 and the low metastatic variant MTLn2 cells to this cytokine were tested in vitro using modified Boyden chambers. Both the chemotactic and chemokinetic movements of MTLn3 cells were stimulated by the MTLn3-derived cytokine. In addition, the MTLn3-derived cytokine stimulated a relatively small, but significant chemotactic migration of MTLn2 tumor cells, while these cells did not respond to medium conditioned by MTLn2 cells. MTLn3 cells themselves did not respond chemotactically to type I collagen or medium conditioned by MTLn2 cells. These results suggest that the chemotactic response may be a function of metastatic potential of the invading tumor cells. The production of tumor cytokines that enhance tumor cell motility may thus represent a phenotypic difference between 13762NF tumor cell subpopulations of high and low metastatic potential.

Cyclooxygenase-2 Inhibitors Decrease Interleukin-1β–Stimulated Prostaglandin E2 and IL-6 Production by Human Gingival Fibroblasts

BACKGROUND Previous work showed that normal and aggressive periodontitis (AgP) gingival fibroblasts produce the boneresorbing cytokine IL-6. PGE2 is important in regulating IL-6 production. Non-steroidal anti-inflammatory drugs inhibit PG synthesis via COX-1 and/or COX-2 isoenzymes and may inhibit periodontal destruction. COX-2 is induced after cellular activation (i.e., by inflammatory cytokines such as IL-1β). Little is known about IL-1β-stimulated AgP fibroblast IL-6 and PGE2 production and their regulation by COX inhibitors. The objective of this study was to determine the effects of COX-2 inhibitors on amounts of PGE2 and IL-6 made by IL-1β-stimulated gingival fibroblasts. METHODS Gingival fibroblasts (2.5 × 104 ) from healthy or severe periodontitis patients were cultured in serum-free medium, with or without IL-1β (10-11 M) for 24 hours, with or without the COX-1/2 inhibitor indomethacin or the selective COX-2 inhibitors NS-398, celecoxib, or rofecoxib. PGE2 and IL-6 in culture supernatants were determined by specific enzyme-linked immunosorbent assay (ELISA)s. RESULTS All of the COX inhibitors caused dose-dependent decreases in IL-1β-stimulated PGE2 , to a maximum of >90% in all cell lines (P ≤0.0001). The selective COX-2 inhibitors, but not indomethacin, caused partial (generally up to approximately 60%), dose-dependent decreases in IL-1β-stimulated IL-6 in all cell lines (P ≤0.003). When exogenous PGE2 was added concurrently with COX-2 inhibitors before addition of IL-1β, IL-6 production returned to levels at or approaching that produced by cells exposed only to IL-1β (P ≤0.04). CONCLUSION The results suggest that COX-2 inhibition may be useful in helping to control fibroblast production of IL-6 in patients with severe periodontitis. J Periodontol 2003;74:1754-1763.

Mast cell modulation of tumour cell proliferation in rat mammary adenocarcinoma 13762NF

Mast cells were shown to accumulate around the periphery of the invasive and metastatic rat mammary adenocarcinoma (MTLn3), and histological evidence of mast cell degranulation was observed during the later stages of this model. To assess the physiological role of mast cells in vivo we have used the mast cell-stabilising compound FPL 55618 applied i.p. daily at 1 mg kg-1 for 23 days. Using groups of 12 rats we have found that this compound inhibited tumour growth at the primary site by as much as 70% in most of the treated animals compared with the control group which received equivalent volumes of saline. When the drug treatment was stopped after 23 days, tumour growth of the test group accelerated over the next 7 days and reached a similar tumour size to that of control animals. Histological studies of the tumour and contiguous host tissue at day 24 of the experiment revealed numerous extra-tumoural mast cells often showing signs of degranulation at several sites around the tumour periphery in the control animals. Such observations were not seen in those animals receiving FPL 55618 where, in contrast to controls, numerous intact mast cells were often seen within the tumour mass. Following cessation of the MC-stabilising treatment progressive mast cell activation was evident within 2-4 days, primarily at the tumour periphery. In vitro studies have shown that drug concentrations equivalent to five times the in vivo dose had no effect on the proliferative rate or viability of the MTLn3 cells. Moreover, the proliferative rate of these cells in culture was significantly increased when exposed to soluble mast cell products. Thus our data indicate that a mast cell-stabilising compound has significant benefits in reducing tumour growth in vivo, an observation which supports the concept that mast cell:tumour cell interactions are important for the growth and invasive properties demonstrated by this model of breast carcinoma.

Interaction of Salivary Fibronectin with Oral Streptococci

Immunoreactive Fibronectin (Fn) has been demonstrated in stimulated human parotid saliva by western blot analysis and also found to be a component of the artificial tooth pellicles derived from hydroxyapatite (HA) beads coated with parotid saliva. Saliva depleted of gelatin-binding components showed a significantly lower degree of reactivity with anti-Fn antibodies than did the control saliva when tested by an enzyme-linked immunosorbent assay (ELISA). Depletion of gelatin-binding components from saliva was also found to affect the degree of saliva-mediated aggregation of four of the seven oral streptococci tested [Streptococcus mutans strains GS-5 and OMZ 176, S. sobrinus, and S. rattus]. Similarly, the adherence of the same four micro-organisms to the artificial tooth pellicles (derived from saliva which had previously been depleted of gelatin-binding component) was significantly inhibited ( 37-53%) when compared with the control saliva-coated HA beads. Pre-treatment of streptococci with 100 μg of soluble Fn also caused a 34—57% inhibition of adherence of the same oral streptococci to saliva-treated HA beads. Quantitation of Fn in human parotid saliva showed that the amounts of immunoreactive Fn varied from 2 to 6 μg/mL of parotid saliva. Furthermore, the Fn from parotid saliva was found to be adsorbed onto the bacterial surfaces, as demonstrated by immunofluorescence and ELISA. The presence of Fn in parotid saliva and its ability to bind to HA beads (artificial pellicles), in conjunction with the ability of soluble Fn to inhibit the adherence of streptococcal strains to the artificial tooth pellicles, suggest that the microbial ecology of the oral cavity may, in part, be influenced by the interactions mediated by salivary fibronectin.

Host-mediated effectors of tumor invasion: role of mast cells in matrix degradation.

The role of collagenolytic enzymes in tumor invasion and metastasis has been emphasized, but the source of enzyme activity has remained unclear. Degradation of stromal connective tissue is a common feature of invasive neoplasia, and host-tumor cell interactions are probably important for localized collagenolysis. We have examined the role of mast cells in malignant cell invasion using cells derived from the rat mammary adenocarcinoma 13762NF. Histologic studies have shown increased numbers of mast cells at the zone of tumor invasion. Mast cell products and conditioned medium from such cells stimulated the production of collagenolytic enzymes by stromal fibroblasts as well as certain subpopulations of tumor cells in vitro. The tumor cell response to mast cell-mediated stimulation of collagenolysis appears to be related to the metastatic potential of the tumor cell. A subpopulation of host fibroblasts derived from the invading tumor zone was also found to be more responsive to mast cell factors than normal fibroblasts, as judged by collagenase production. Thus the mast cell has the potential to induce collagenolytic activity from both host fibroblasts and tumor cells.

Effects of mast cell-macrophage interactions on the production of collagenolytic enzymes by metastatic tumor cells and tumor-derived and stromal fibroblasts.

Histological examination of the metastatic rat mammary adenocarcinoma line MTLn3 showed that macrophages and mast cells were frequently localized at the tumor periphery in the stromal tissues adjacent to the zones of tumor invasion. The interactions of these host cells with tumor cells and tumor-associated fibroblasts could be important in stimulating the production of extracellular matrix-degrading enzymes that facilitate tumor invasion and metastatic spread. Therefore, we examined the effects of isolated, activated macrophages and mast cells on the secretion of collagenolytic activities by normal fibroblasts, metastatic mammary adenocarcinoma cells and tumor-associated fibroblasts. Medium from activated macrophages or degranulated mast cells stimulated significant increases in production of collagenolytic activities by normal and tumor-associated fibroblasts and MTLn3 tumor cells. Medium from activated macrophages that had been pretreated with medium from degranulated mast cells, however, were less stimulatory to fibroblasts and tumor cell production of collagenolytic activities than medium from degranulated mast cells alone. We also examined the effects of two cytokines, interleukin-1α and tumor necrosis factor-a on activated macrophage- and degranulated mast cell-stimulation of fibroblast and tumor cell collagenolytic activities. The two cytokines alone or in combination stimulated increased production of collagenolytic activities by fibroblasts and tumor cells. Addition of the cytokines to degranulated mast cell products resulted in secretion of higher collagenolytic enzyme activities by normal fibroblasts (but not by tumor-derived fibroblasts or tumor cells) than with degranulated mast cell product-treatment of either target cell alone. Cytokines used in combination with macrophage-conditioned medium were less effective in stimulating fibroblast and tumor cell collagenase activities than cytokines alone. Thus normal infiltrating host cells such as macrophages and mast cells can have profound effects on the production of degradative enzymes by tumor cells and tumor-associated stromal fibroblasts.

Matrix metalloproteinases in the progression and regression of Kaposi's sarcoma.

Background:  Matrix metalloproteinases (MMPs) are associated with Kaposi’s sarcoma (KS) tumorigenesis. To date, only a few MMPs have been studied in KS lesions. Their role in KS regression has not been investigated. The aim of this study was to evaluate the expression of multiple MMPs in developing and pharmacologically regressed KS lesions.

Separation of VX-2 rabbit carcinoma-derived cells capable of releasing collagenase.

Primary and secondary cultures of VX-2 carcinoma produced high levels of collagenase activity in both active and latent forms in serum-free media. These cultures appeared morphologically heterogeneous in phase-contrast microscopy and revealed the presence of mainly three distinct forms: epithelial-like cells (E cells), fibroblast-like cells (F cells), and large rounded-flat cells which may represent a subclass of the F cells. Cell separation techniques such as brief dispase treatment, Percoll gradient centrifugation, thimerosal treatment, and rabbit serum were used to obtain predominantly one form or the other. The E cells never formed a monolayer but rather grew as limited size clusters of intimately associated cells with large nuclei and often appeared multinucleated. These cells were difficult to maintain in culture or serially passed more than a few times. The F cells, rare in early cultures but having the highest growth potential, appeared in various morphological forms ranging from spindle- to stellate-shaped cells. The cells in their third passage were capable of producing palpable tumors, similar in light and electron microscopic studies to the original tumor from which they were derived, when injected intramuscularly into recipient rabbits and produced specific collagenase activity in active and latent forms in serum-free media. Ultrastructural studies suggested that the E cells were of epithelial origin whereas the F cells were similar to stromal fibroblasts. Cytogenetic studies demonstrated that almost all of the E cells showed both numerical and structural chromosomal changes in a modal number of 54 chromosomes. On the other hand, the major cell population of the F cells resembled normal rabbit fibroblasts; both contained a normal diploid (2n = 44). However, few cells (4-6%) in the F-cell population were hyperdiploid with a modal chromosome number of 54. These cells may represent inadvertent contaminating E cells and account for the apparent limited turmorigenicity observed in early F-cell cultures. The data suggested that the E cells were of tumor origin whereas the majority of the F-cell population appeared to be of host origin. Furthermore, it is suggested that the E cells stimulate tumor-associated stromal cells to produce elevated levels of collagenolytic activity and contribute to collagen degradation during tumor invasion.

Myeloperoxidase-catalyzed chlorination of histamine by stimulated neutrophils

Abstract To investigate neutrophil interactions with mediators released by mast cells at sites of inflammation, stimulated neutrophils were incubated with histamine. No accumulation of chlorinated histamine derivatives was detected in the medium. Instead, histamine inhibited the formation of chloramine derivatives of other amines. Incubation with radiolabeled histamine resulted in rapid uptake of label into the cells, and most of the label could be extracted and recovered as histamine. About 3% of the label taken up was incorporated into acid-precipitable forms. Uptake depended on myeloperoxidase (MPO)-catalyzed formation of chlorinating agents. Uptake was promoted by adding MPO and blocked by the MPO inhibitor dapsone, catalase, scavengers for hypochlorous acid and chloramines, or in a low-chloride medium, but not by histamine receptor antagonists. Incubation of histamine with MPO, hydrogen peroxide, and chloride resulted in formation of mono- and di-chloramine derivatives of the primary amino group. Above pH 7.0, the chloramines were primarily in uncharged, lipophilic forms as indicated by partitioning into organic solvents. Histamine is a cation at neutral pH, but chlorination eliminated the charge on the amino group and shifted the pKa of the imidazole ring, resulting in formation of neutral histamine-chloramines. Incubation of neutrophils or other blood cells with radiolabeled histamine-chloramines resulted in rapid uptake of label, indicating membrane permeation by the uncharged, lipid-soluble forms. Incubation with labeled histamine-dichloramine also resulted in acid-precipitable incorporation. The results indicate that MPO-catalyzed chlorination of histamine could modulate histamine activity, tissue distribution, and metabolism at sites of inflammation.