Inhibition of interleukin-17-stimulated interleukin-6 and -8 production by cranberry components in human gingival fibroblasts and epithelial cells.

BACKGROUND AND OBJECTIVE: Gingival epithelial cells and fibroblasts participate in periodontal inflammation and destruction, producing interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8. IL-17, a product of T-helper 17 cells, may play a role in periodontitis by stimulating cytokine production by gingival cells. The cranberry (Vaccinium macrocarpon) is rich in polyphenols, particularly proanthocyanidins, which have antioxidant and other beneficial properties. Cranberry components inhibit pro-inflammatory activities of lipopolysaccharide-stimulated human macrophages, gingival fibroblasts, and epithelial cells, but little is known of its effects on IL-17-stimulated cytokine production. The objectives were to determine the effects of IL-17 ± cranberry components on IL-6 and IL-8 production by human gingival epithelial cells and fibroblasts. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), which is rich in proanthocyanidins, was derived from cranberry juice. Human gingival epithelial cells and normal human gingival fibroblasts were incubated with NDM (5-50 µg/mL), IL-17 (0.5-100 ng/mL), or NDM + IL-17 in serum-free medium for 6 d. IL-6 and IL-8 in culture supernatants were measured by ELISA. Membrane damage and viability were assessed by lactate dehydrogenase activity released into cell supernatants and activity of a mitochondrial enzyme, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: In both cell lines, IL-17 (≥ ~5-10 ng/mL) significantly stimulated production of IL-6 (p < 0.005) and IL-8 (p < 0.03). Non-toxic levels of NDM inhibited constitutive IL-6 and IL-8 production by epithelial cells (p ≤ 0.01) and fibroblasts (p ≤ 0.03) as well as IL-17-stimulated cytokine production by epithelial cells [IL-6 (maximum ~80% inhibition; p ≤ 0.0001); IL-8 (maximum ~70% inhibition; p ≤ 0.03)] and fibroblasts [IL-6 (maximum ~90% inhibition; p ≤ 0.0001); IL-8 (maximum ~80% inhibition; p ≤ 0.008)]. CONCLUSION: Cranberry NDM inhibition of constitutive and IL-17-stimulated IL-6 and IL-8 production by gingival fibroblasts and epithelial cells suggests that cranberry components could be useful as a host modulatory therapeutic agent to prevent or treat periodontitis.

Myeloperoxidase-catalyzed chlorination of histamine by stimulated neutrophils.

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 dichloramine 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.

Autocrine transforming growth factor beta stimulation of extracellular matrix production by fibroblasts from fibrotic human gingiva.

Hereditary gingival fibromatosis (HGF) is a fibrotic enlargement of the gingiva. HGF gingiva contains large amounts of collagen and other extracellular matrix (ECM) molecules. In vitro, HGF fibroblasts produce greater amounts of the ECM components fibronectin (FN) and type 1 collagen than normal human gingival (GN) fibroblasts. Transforming growth factor beta (TGF beta) is a cytokine important in regulating tissue repair and regeneration after injury, and stimulating fibroblast proliferation and the production of FN and collagens. The objective of this study was to determine whether HGF fibroblasts produce TGF beta and, with the use of neutralizing antibodies to TGF beta isoforms, if their increased expression of FN and type 1 collagen is under autocrine TGF beta control. The HGF strains produced greater amounts of TGF beta1 and TGF beta2 (P < or = 0.003) as well as FN (P < or = 0.04) and type 1 collagen (P < or = 0.03) (measured by specific ELISA) than the GN strains. Treatment of HGF fibroblasts with anti-TGF beta1, beta2, or beta3, as well as a combination of all 3 antibodies, decreased their FN production by up to 60% (P < or = 0.04), and was able to decrease FN production by HGF fibroblasts to the levels of the GN fibroblasts. When used alone, the neutralizing antibodies decreased type 1 collagen production by the HGF fibroblasts by up to 40% (P = 0.014), and treatment with all 3 antibodies caused decreases of up to 55% (P = 0.0005). The results suggest that autocrine stimulation by the increased amounts of TGF beta isoforms made by HGF fibroblasts contributes to their increased production of FN and type 1 collagen.

Increased proliferation, collagen, and fibronectin production by hereditary gingival fibromatosis fibroblasts.

HEREDITARY GINGIVAL FIBROMATOSIS (HGF) is a fibrotic enlargement of the gingiva. HGF gingiva contains large amounts of interstitial collagen and other extracellular matrix (ECM) molecules. Increased proliferation and elevated production of the ECM molecules type I collagen and fibronectin (FN) could contribute to the clinical increased bulk of HGF gingiva. Fibroblast strains from HGF gingiva and normal human gingival fibroblast strains (GN) were used in this in vitro study. Fibroblast proliferation was determined by ELISA which measured the incorporation of 5-bromo-2'-deoxyuridine into DNA. The results showed that HGF fibroblast strains proliferated more rapidly than GN fibroblasts (68% to 488% increase, depending on the strains) (P < or = 0.01), the only exception being one HGF strain versus one normal strain. All HGF strains produced greater amounts of FN (measured by ELISA) than all of the normal fibroblast strains (23% to 49% increase, depending on the strain) (P < or = 0.04). Similarly, all HGF strains made significantly greater (P < or = 0.3) amounts of type I collagen (also measured by ELISA) than all of the normal strains (55% to 235% increase, depending on the strain). The results show that, in vitro, HGF fibroblasts display several phenotypic characteristics of activated fibroblasts: increased proliferative rates as well as increased production of FN and type I collagen, consistent with in vitro studies of fibroblasts derived from other types of fibrotic tissue. These results suggest that the increased proliferation of HGF fibroblasts and their increased production of extracellular matrix molecules such as collagen and FN may contribute to the clinical gingival enlargement characteristics of HGF.

Effects of nicotine on proliferation and extracellular matrix production of human gingival fibroblasts in vitro.

Normal function of gingival fibroblasts is essential for maintenance of the gingival extracellular matrix (ECM), but under inflammatory conditions in gingival tissue which may occur with tobacco use, they can also act in its destruction. The purpose of this study was to determine the effects of nicotine, a major component of tobacco, on gingival fibroblast proliferation, the production of fibronectin (FN), and the production and breakdown of type I collagen to elucidate its role in periodontal destruction associated with its use. A human gingival fibroblast strain derived from a healthy individual with non-inflamed gingiva was used in this study. Nicotine at concentrations > 0.075% caused cell death, and at 0.075% and 0.05% it caused transient vacuolization of the fibroblasts. At concentrations of 0.001% to 0.075%, nicotine significantly inhibited proliferation (P < or = 0.03), measured by the incorporation of [3H]-thymidine into DNA. The production of FN and type I collagen was significantly inhibited by nicotine at > or = 0.05% (P < or = 0.001), measured using specific ELISAs. On the other hand, nicotine at > or = 0.025% significantly increased collagenase activity (P < or = 0.008), using [3H]-gly and [14C]-pro-labeled type I collagen gels as substrate. The results show that, in vitro, nicotine inhibits the growth of gingival fibroblasts and their production of FN and collagen, while also promoting collagen breakdown. This suggests that nicotine itself may augment the destruction of the gingival ECM occurring during periodontal inflammation associated with smokeless tobacco use.

Role of saliva and salivary components as modulators of bleaching agent toxicity to human gingival fibroblasts in vitro.

Mild oxygenating agents generating H2O2 are used for effective at-home tooth bleaching, but can cause gingival ulcers in some patients. There are concerns about the possible pathological effects of relatively long-term exposure of oral tissues to bleaching agents. Previous work in our laboratory showed that a bleaching agent, which generates approximately 3% H2O2 from carbamide peroxide, was toxic to human gingival fibroblasts in vitro, but that the toxicity was abolished by treatment with the H2O2-destroying enzyme catalase. The purpose of the present study was to determine if whole saliva, the salivary enzyme lactoperoxidase (LP) (which, like catalase, removes H2O2), or salivary mucin protected fibroblasts from bleaching agent toxicity. The cells were exposed to 0.05% agent with or without saliva, LP, mucin or catalase (as a positive control based on our previous study) and assessed for effect on viability/morphology (by microscopic observation), proliferation (by [3H]-thymidine incorporation), and the production of fibronectin (FN) and type I collagen (by ELISA). While the bleaching agent at 0.05% caused cell death, the cells appeared viable and morphologically normal when treated with the bleaching agent and LP (> or = 0.1 microM), saliva LP, and catalase from agent inhibition of proliferation (P < or = 0.04) and FN production (P < or = 0.01). Mucin had statistically insignificant or no protective effect as assessed by the above parameters. Treatment with saliva, LP, mucin, and catalase gave complete or partial protection from agent-inhibition of collagen production (P < or = 0.04).(ABSTRACT TRUNCATED AT 250 WORDS)

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-1 alpha and tumor necrosis factor-alpha 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.

Effects of a bleaching agent on human gingival fibroblasts.

Mild oxygenating agents generating low concentrations of hydrogen peroxide (H2O2) are effective alternatives to heat-activated 30% H2O2 in bleaching discolored, vital teeth. There are concerns about possible pathological effects of long-term exposure to bleaching agents, and irritation and ulceration of the gingiva and other oral soft tissues can occur. The objective of this study was to determine the effect of one of these agents on gingival fibroblasts in vitro. Microscopic examination revealed that concentrations of 0.05% to 0.025% of the agent appeared to kill most of the cells. At concentrations of 0.025% to 0.017% some morphological changes were noted; the cells appeared normal at concentrations of < or = 0.0125%. The agent significantly (P < or = 0.002) decreased proliferation (measured by incorporation of [3H]-thymidine into cellular DNA) at concentrations as low as 0.006%. The agent also had a dose-dependent effect on fibronectin production, measured by ELISA, causing significant (P < or = 0.03) decreases at concentrations as low as 0.017%. The agent significantly decreased the production of types I (P < or = 0.01) and III (P < or = 0.04) collagens (measured by ELISA) at concentrations as low as 0.0125%. Type V collagen was not detected under any conditions. Catalase, which catalizes the breakdown of H2O2, abolished toxic effects of a 0.05% solution. The results show that in vitro, the agent is toxic to human gingival fibroblasts, inhibiting several cellular functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered collagen metabolism in nifedipine-induced gingival overgrowth.

Fibroblasts from nifedipine-induced fibrotic gingiva (NFG) have been characterized with respect to several cellular functions which could contribute to the characteristic clinical overgrowth of the gingiva: collagen synthesis and breakdown, glycosaminoglycan production, fibronectin synthesis, and proliferation. Histologic examination of NFG tissue revealed a hyperplastic epithelium with elongated, branched rete pegs. The connective tissue consisted of densely-packed collagen fibers and numerous enlarged fibroblasts, as well as regions of thinner, disorganized collagen fibers in the vicinity of scattered inflammatory and mast cells. Results of in vitro experiments showed that the fibroblast strains from the fibrotic gingiva (NFG) produced significantly greater amounts of collagen and lower levels of collagenase activity when compared to age- and sex-matched normal human gingival fibroblast strains. The NFG fibroblasts did not produce significantly greater amounts of fibronectin, and their level of glycosaminoglycan production was less than that of the normal fibroblasts. The NFG fibroblasts did not proliferate significantly more rapidly than the normal fibroblast strains. These findings therefore show that there are defects in the regulation of collagen production by NFG fibroblasts in vitro, and suggest that these alterations in collagen metabolism contribute to the over-deposition of collagen in this tissue, rather than hyperproliferation of the fibroblasts or through the production of increased amounts of fibronectin and glycosaminoglycans.

Binding and subcellular distribution of cyclosporine in human fibroblasts.

The uptake, binding, and subcellular sites of accumulation of [3H]-cyclosporine (CS) in two human gingival fibroblast strains, GN 23 and GN 54, have been examined. GN 23 responds to CS treatment with a decrease in collagenolysis, while GN 54 does not. Binding of the drug was determined using [3H]-CS concentrations ranging from 10(-5) to 10(-8) M in the absence or presence of excess unlabeled CS (1 mM). The binding of the drug to both strains was specific and reached a plateau within 10 min, remaining at that level for up to 1 h. Scatchard analysis of the specific binding of [3H]-CS to the responsive GN 23 strain revealed two dissociation constants: KD = 5 x 10(-8) M (1.2 x 10(7) sites/cell) and KD = 1.4 x 10(-6) M (2.2 x 10(8) sites/cell). GN 54, on the other hand, had only one class of low affinity binding site (KD = 0.47 x 10(-6) M [1.2 x 10(8) sites/cell]). Unlabeled CS (0.01-1 mM) inhibited the binding of [3H]-CS in a dose-dependent manner to both strains, as did the calmodulin antagonist W-7, to a lesser extent. However, W-7 inhibited CS binding much more efficiently in GN 54 than in GN 23, suggesting that calmodulin may be the predominant CS receptor in GN 54. In both strains, 70% of the drug accumulated in the crude nuclear fraction after a 1 min incubation, with very little (< or = 4%) being membrane associated, and the remainder was in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Fibroblast heterogeneity in collagenolytic response to cyclosporine.

To investigate the mechanism of cyclosporine (CS)-induced fibrotic gingival enlargement, the effect of CS on the collagenolytic activities of 14 different human gingival fibroblast strains derived from healthy individuals with non-inflammed gingiva was examined in vitro. There was marked heterogeneity among individuals in basal levels of collagenase activity, and there was also variation among the subpopulations derived from one strain. Fibroblasts from different individuals also varied markedly in their collagenolytic response to CS (0.1 to 0.75 micrograms/ml). In most strains, CS decreased collagenase activity, but in some, the drug caused no change or significantly increased activities. In most of the subpopulations CS significantly decreased collagenolytic activity. Two of the fibroblasts strains and the subpopulations described above were examined for the production of immunoreactive collagenase and tissue inhibitor of metalloproteinase (TIMP). The two strains made similar amounts of collagenase, but differed markedly in TIMP levels; CS affected their collagenase production differently but had similar effects on TIMP. Among the subpopulations there was variation in the production of collagenase, although none made detectable levels of TIMP; they also varied in the production of both proteins in response to CS. In two of the subpopulations and in both strains at some concentrations, the effect of CS on the relative levels of collagenase and TIMP could account for the decreased collagenase activity; i.e., the level of collagenase was unchanged or decreased, and TIMP production was unchanged or increased. This study demonstrates the variation among individuals as well as intrastrain heterogeneity of human gingival fibroblasts with regard to collagenase activity and the production of collagenase and TIMP. The heterogeneity of the collagenolytic response of different gingival fibroblast strains and their subpopulations to CS treatment may partly explain the susceptibility of only some individuals to CS-induced gingival enlargement.

Isolation and characterization of a 180-kiloDalton salivary glycoprotein which mediates the attachment of Actinomyces naeslundii to human buccal epithelial cells.

The adherence of Actinomyces naeslundii to human buccal mucosa is mediated by specific interactions between the bacterial cell surface fimbriae and complementary beta-linked galactoside receptors on the epithelial cell surface. The buccal mucosa and the bacteria that colonize its surface are constantly bathed in saliva. Several salivary components are thought to play an important role in modulating adhesive interactions between oral bacteria and the buccal epithelium. We have observed that pretreatment of isolated buccal epithelial cells (BEC) with human parotid saliva increased the attachment of three different strains of A. naeslundii. By employing affinity chromatography, ion-exchange and high-pressure liquid chromatographic techniques we have isolated a 180 kDa A. naeslundii-binding salivary glycoprotein (An-SPG). This salivary glycoprotein was capable of mediating separate but specific binding interactions with A. naeslundii and BEC. Pretreatment of BEC with increasing amounts of An-SGP resulted in a corresponding increase in the attachment of A. naeslundii. The adherence of A. naeslundii to An-SGP-coated BEC is sensitive to the same inhibitors previously shown to block adherence of A. naeslundii to uncoated BEC, namely lactose- and galactosyl-binding lectins. When a solubilized extract of freshly isolated and washed BEC was reacted on a Western blot with antibodies to An-SGP, a prominent 180 kDa immunoreactive band was detected. Furthermore, the immunoreactive component was demonstrated on the BEC surface when assayed by immunofluorescence using An-SGP-specific antibodies, suggesting that An-SGP or a protein structurally and immunologically identical to the isolated glycoprotein is present on BEC.

Interleukin-1 beta- and tumor necrosis factor-alpha-independent monocyte stimulation of fibroblast collagenase activity.

To investigate the mechanism of cyclosporine (Cs)-induced fibrous gingival enlargement, the indirect effects of Cs on fibroblast collagenolysis via the drug's effect on the synthesis of the fibroblast regulatory monokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) have been studied. Peripheral blood monocytes stimulated with lipopolysaccharide (LPS) for 48 h produced conditioned media (MCM-LPS) that contained 665 pg/ml IL-1 beta and 16 pg/ml TNF alpha and significantly (P less than 0.001) enhanced the collagenase activity of a fibroblast strain (GN 23) derived from a healthy individual with clinically normal gingiva. The concurrent addition of Cs (50, 100, or 150 ng/ml) with LPS to the monocytes (MCM-LPS-Cs) significantly diminished their ability to enhance GN 23 collagenase activity in a dose-dependent manner, with MCM-LPS-Cs (150 ng/ml) causing the greatest effect. Cs also significantly inhibited IL-1 beta and TNF alpha production. Although the greatest inhibition of both cytokines was at 50 ng/ml Cs, the corresponding MCM-LPS-Cs caused the least diminution (16%) of the collagenase stimulation caused by MCM-LPS (no Cs). This suggested that factor(s) other than or in addition to IL-1 beta and TNF alpha might be responsible for the stimulation of GN 23 collagenase activity. MCM-LPS depleted of IL-1 beta by affinity chromatography retained its stimulatory effect on GN 23 collagenolysis, and human recombinant IL-1 beta and TNF alpha, when tested alone or together at levels found in the stimulatory MCM-LPS and MCM-LPS-Cs, did not stimulate GN 23 collagenase activity as did the crude conditioned media. This evidence suggested that the conditioned media contained the complex mixture of cytokines necessary to stimulate collagenase activity of this fibroblast strain and that IL-1 beta and TNF alpha were not necessarily involved. Cs may alter the synthesis of other collagenase-stimulating cytokines, accounting for the diminished ability of Cs-treated monocytes to enhance collagenase activity of susceptible fibroblast strains. Decreased collagenase activity, therefore, resulting from Cs suppression of monokine production, may be an important factor in the development of fibrous gingival enlargement seen in some susceptible patients treated with Cs.

Macrophage and lymphocyte potentiation of syngeneic tumor cell and host fibroblast collagenolytic activity in rats.

The collagenolytic responses of normal rat skin fibroblasts (NRS-F) and rat mammary MTLn3 tumor-derived fibroblasts (Ln3-F) were examined following exposure to rat macrophage (M phi-CM)- and lymphocyte (LYM-CM)-conditioned culture medium and/or tumor cell-conditioned medium. Alveolar, intratumoral, and peritoneal macrophages were prepared from mammary adenocarcinoma-bearing rats, as were the peritoneal lymphocytes. Incubation of the two fibroblast populations with LYM-CM produced a 10- and 7-fold stimulation of collagenolytic activity by NRS-F and Ln3-F cells, respectively. Similarly, exposure of NRS-F and Ln3-F fibroblasts to peritoneal M phi-CM produced a 7- and 4-fold increase in the expression of collagenolytic activity, respectively. Conditioned medium from MTLn2 tumor cells also stimulated the collagenolytic expression of both fibroblast populations. Incubation of tumor-associated Ln3-F or NRS-F fibroblasts with MTLn2 tumor cell-conditioned medium enhanced fibroblast collagenolytic activity approximately 20 and 17 times, respectively. When M phi-CM and LYM-CM were further "conditioned" by a subsequent incubation with MTLn2 tumor cells, each stimulated the expression of collagenolytic activity by both fibroblast populations and this was especially pronounced (120-fold increase) in the response of Ln3-F to LYM-CM further conditioned by MTLn2 tumor cells. The conditioned media derived from M phi, LYM, and MTLn2 tumor cells with or without trypsin activation contained low levels of interstitial-type collagenolytic activity which made no significant contribution to the collagenolytic activity of the stimulated fibroblasts. Some collagenase inhibitory activity, however, was detected in the M phi-CM, suggesting that the actual stimulation of collagenolysis by host fibroblasts is underestimated. We conclude that macrophages, lymphocytes, and tumor cells all have the potential to produce stimulatory factor(s) which enhance the collagenolytic activity of normal fibroblast populations. This study provides further evidence of the multifactorial control of collagenase production and supports the concept that host cell-tumor cell interactions can enhance the expression of collagenolytic enzymes.

A comparative fine structure study on myofibroblasts from a cultured human and an in-situ rat tumor source.

Cultured cells from human embryonal testis (HET 1) and basal-cell (BCE-5) carcinoma and cells from the peripheral region of growing tumors of rat adenocarcinoma (13762NF) were harvested and processed for examination with the electron microscope. Cells from the culture sources were collected from Percoll density fractions of 2-10%, 24% and 35%. The results demonstrate that cells from all sources were morphologically reminiscent of myofibroblasts (Gabbiani et al., 1971). They were elongate, fibroblast-like, appearing cells containing mitochondria, endoplasmic reticulum, Golgi complexes, filaments and microtubules. They also contained filament bundles associated with electron densities typical of myofibroblasts described elsewhere. Furthermore, cells from the 35% Percoll density gradient fraction were characterized by the presence of filament-containing vacuoles whose constituent filaments had a 60-65 A periodicity. It is concluded that cells from all three sources are morphologically similar, and are classifiable as myofibroblasts, and that cells from the 35% Percoll density fraction are also involved in collagen anabolism and/or catabolism.

Heterogeneity of fibroblast response in host-tumor cell-cell interactions in metastatic tumors.

The spread and invasion of tumor cells into host tissues are associated with the release of elevated levels of collagenolytic activity of both host and tumor cell origins. However, the mechanisms of regulation of the enzyme activity is still unresolved. Histological examination of human and animal tumors revealed morphological changes in stromal fibroblasts and mast cells at the tumor periphery. Numerous mast cells appeared at microfoci along the tumor: host tissue junction and mast cell degranulation were associated with collagenolysis. In vitro studies, using rat mammary adenocarcinoma and human lung adenocarcinoma cells, showed that both tumor cells and host fibroblasts participate in matrix degradation. Tumor-associated stromal fibroblasts released higher levels of enzyme activity than normal fibroblasts and were more responsive to stimulation by tumor-conditioned media and soluble mast cell products. Host fibroblasts appear to be heterogeneous populations of responsive and nonresponsive subpopulations based on their response to tumor- or mast-cell-mediated stimulation of collagenase release. Fibroblast subpopulations were obtained by density fractionation of serum-deprived, synchronized confluent fibroblasts on discontinuous Percoll gradient. Density-fractionated fibroblast subpopulations differed in their response to stimulation by mast cell products and tumor-cell-conditioned media. The stimulatory activity of tumor-cell-conditioned media also varied as a function of the metastatic potential of the tumor cells. The data suggest that cellular interactions between tumor cells and select subpopulations of host fibroblasts at the tumor periphery play a key role in host tissue degradation. However, heterogeneity of stromal fibroblasts may determine the site and extent of the tissue damage at foci of tumor invasion.

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.

Interaction of a 60-kilodalton D-mannose-containing salivary glycoprotein with type 1 fimbriae of Escherichia coli.

A 60-kilodalton glycoprotein previously isolated and purified from human saliva (J. B. Babu, E. H. Beachey, D. L. Hasty, and W. A. Simpson, Infect. Immun. 51: 405-413, 1986) was found to interact with type 1 fimbriae and prevent adhesion of type 1 fimbriated Escherichia coli to animal cells in a D-mannose-sensitive manner. Purified salivary glycoprotein agglutinated type 1 fimbriated E. coli and, at subagglutinating concentrations, blocked the ability of type 1 fimbriated E. coli to attach to human buccal epithelial cells or agglutinate guinea pig erythrocytes. Both interactions were inhibited by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside. Complexing of the glycoprotein to type 1 fimbriae was demonstrated by molecular sieve chromatography and modified Western blots. When mixed with type 1 fimbriae, the radiolabeled salivary glycoprotein coeluted with type 1 fimbriae from a column of Sepharose 4B. When blotted from a sodium dodecyl sulfate gel to nitrocellulose sheets, the glycoprotein interacted directly with type 1 fimbriae applied to the blots. Both of the latter interactions also were blocked by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside. Chemical modification of the glycoprotein with sodium metaperiodate abolished its ability to interact with isolated type 1 fimbriae or type 1 fimbriated E. coli. These results suggest that the carbohydrate moiety of the 60-kilodalton glycoprotein serves as a receptor for type 1 fimbriae in the oral cavity, and we postulate that the interaction may cause agglutination and early removal of E. coli, thereby preventing colonization by these organisms of oropharyngeal mucosae and dental tissues.