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Am. J. Respir. Cell Mol. Biol., Volume 22, Number 5, May 2000 557-565

Matrix-Associated Transforming Growth Factor-beta 1 Primes Mouse Bone Marrow-Derived Mast Cells for Increased High-Affinity Fc Receptor for Immunoglobulin E-Dependent Eicosanoid Biosynthesis

René O. Sanchez Mejia, Bing K. Lam, and Jonathan P. Arm

Departments of Medicine, Harvard Medical School and Brigham and Women's Hospital; Partner's Asthma Center, Brigham and Women's Hospital; and the Harvard-MIT Division of Health Sciences and Technology, Boston, Massachusetts


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mast cells at different tissue locations are heterogeneous with respect to histochemical staining characteristics, granule protease and proteoglycan content, and eicosanoid biosynthesis. We used Matrigel, an extract from the Engelbreth-Holm-Swarm mouse sarcoma that is enriched in basement-membrane proteins, to investigate the effect of tissue matrix proteins on the differentiation of mouse mast cells, with particular attention to eicosanoid biosynthesis. Culture of mouse bone-marrow cells in interleukin-3 on Matrigel for 3 to 4 wk provided a population of mast cells with more intense metachromasia and increased safranin counterstaining compared with mast cells derived in the absence of Matrigel (bone marrow-derived mast cells [BMMC]). High-affinity Fc receptor for immunoglobulin E-dependent biosynthesis of prostaglandin D2 and leukotriene (LT) C4 was 6- and 11-fold higher, respectively, from mast cells derived in the presence of Matrigel compared with conventional BMMC derived in its absence. BMMC derived in the presence of Matrigel also generated substantial quantities of 6-trans-LTB4 diastereoisomers and LTB4, which were minimally generated by conventional BMMC. When conventional BMMC derived in the absence of Matrigel were then cultured on Matrigel for 5 d, eicosanoid biosynthesis was upregulated without any change in granule staining characteristics. This upregulation in eicosanoid biosynthesis was inhibited by neutralizing anti- transforming growth factor (TGF)-beta 1-specific antibodies, was reproduced by 1 ng/ml TGF-beta 1, and was attributed to increased expression of cytosolic phospholipase A2.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mast cells are critical effector cells of allergic inflammation, releasing mediators that are preformed (e.g., histamine, serine proteases, and proteoglycans) or newly synthesized (e.g., eicosanoids and cytokines) in response to diverse stimuli including stem-cell factor (SCF) and immunoglobulin (Ig) E and antigen-dependent activation (1). Mast cells are heterogeneous with respect to their histochemical staining characteristics, granule content, and pattern of eicosanoid generation, their phenotype depending on the local tissue microenvironment. Thus, serosal and connective tissue mast cells counterstain with safranin after alcian blue staining whereas mucosal mast cells do not. Mouse mast-cell protease phenotype is influenced by cytokines (2) and the local tissue microenvironment (6) and demonstrates remarkable plasticity during the course of Trichinella spiralis intestinal infestation (7, 8). With respect to eicosanoid generation, rat and mouse serosal mast cells generate prostaglandin (PG) D2 in preference to leukotriene (LT) C4 (9), whereas mast cells isolated from the intestine of T. spiralis- infested rats generate LTC4 in preference to PGD2 (10). Eicosanoid generation by other populations of rodent mast cells has not been studied due to difficulties in obtaining these cells in sufficient numbers or purity.

Bone marrow-derived mast cells (BMMC) from BALB/c mice, developed in WEHI-3 cell-conditioned medium as a source of interleukin (IL)-3 for 3 to 4 wk (11, 12), represent a relatively immature population of committed mast-cell progenitors that reconstitute both connective tissue and mucosal mast cells in mast cell-deficient mice of the WBB6F1/J-W/Wv strain (13). BMMC fail to stain with safranin after alcian blue staining, synthesize chondroitin sulfate E proteoglycan, are poor in histamine content, and preferentially produce LTC4 over PGD2 when activated by perturbation of the high-affinity Fc receptor for IgE (Fcvarepsilon RI) (14) or the receptor tyrosine kinase c-kit (15). In prior studies we have shown that culture of BMMC for 2 to 7 d in SCF led to priming for increased PGD2 synthesis, an effect that was augmented by IL-3, IL-9, or IL-10 (16). In contrast, culture of bone-marrow cells for 4 wk in SCF and IL-10 derived a population of BMMC with poorly developed eicosanoid biosynthetic pathways. Subsequent addition of IL-3 to the culture for 1 to 2 wk elicited a population of mast cells with markedly upregulated IgE-dependent LTC4 generation (17).

In the present study we have evaluated the effect of tissue matrix and basement membrane proteins on mouse mast-cell eicosanoid generation using Matrigel, an extract from the Engelbreth-Holm-Swarm mouse sarcoma that is rich in basement-membrane proteins. These proteins include laminin, type IV collagen, heparan sulfate proteoglycans, entactin, nidogen (18), and, to a lesser extent, fibronectin (19). In addition to these structural proteins, Matrigel is embedded with a variety of growth factors and cytokines that include transforming growth factor (TGF)-beta 1, fibroblast growth factor, tissue plasminogen activator, platelet-derived growth factor, and other poorly characterized factors (20). We demonstrate that BMMC developed in the presence of Matrigel have increased granularity, counterstain with safranin, and have increased IgE-dependent eicosanoid biosynthesis compared with conventional BMMC derived in the absence of Matrigel. Increased IgE-dependent eicosanoid biosynthesis was also seen in conventional BMMC cultured on Matrigel for 5 d, an effect that was reproduced by TGF-beta 1 and inhibited by a neutralizing antibody to TGF-beta 1.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Materials

Matrigel, growth factor-reduced Matrigel, laminin, fibronectin, and type IV collagen were supplied by Collaborative Biomedical Products, Becton Dickinson Labware, Bedford, MA. Chicken IgY anti-TGF-beta 1 and isotype-matched control chicken IgY were supplied by Genzyme, Cambridge, MA. Rabbit antiserum to human cytosolic phospholipase (cPL) A2, which cross-reacts with mouse cPLA2 (16), was a gift from Dr. J. D. Clark, Genetics Institute, Cambridge, MA; rabbit antisera to 5-lipoxygenase (5-LO) and to 5-LO activating protein (FLAP) were gifts from Dr. J. F. Evans, Merck and Co., West Point, PA; and rabbit antiserum to cyclooxygenase (COX) 1 was a gift from Dr. W. L. Smith, Michigan State University, East Lansing, MI.

Culture of BMMC in the Presence of Matrigel

Bone-marrow cells from male BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were cultured at a cell density of 5 × 105 cells/ml for 3 to 4 wk in 50% enriched medium (RPMI 1640 containing 100 U/ml of penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamicin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 10% fetal calf serum) and 50% WEHI-3 cell (American Type Culture Collection, Rockville, MD)-conditioned medium as a source of IL-3 in 25-cm2 flasks (Corning Laboratories, Corning, NY) in the presence of Matrigel. Flasks were coated with Matrigel at 4°C using precooled flasks and pipette tips. Approximately 2.5 ml of Matrigel was added to each 25-cm2 flask. After 1 and 2 wk of culture, cells were treated for 1 h at 37°C with Dispase, a bacillus-derived neutral metallo protease (Collaborative Biosystems, Cambridge, MA), which digested the Matrigel and allowed the isolation of cells adherent to or embedded in the matrix as well as nonadherent cells. Adherent and nonadherent cells were passed together at 5 × 105 cells/ml up to 3 wk. After 3 wk of culture, nonadherent and adherent cells were cultured separately. At 4 wk of culture, both populations of cells gave rise to nonadherent and adherent cells that were studied separately.

Culture of Conventional BMMC

Bone-marrow cells from male BALB/c mice were cultured at a cell density of 5 × 105 cells/ml for 3 to 4 wk in 50% enriched medium and 50% WEHI-3 cell-conditioned medium (12) in 25-cm2 flasks. To determine the effects of connective tissue matrix or its individual components on histochemical staining and IgE-dependent eicosanoid generation, conventional BMMC were cultured on plates coated with Matrigel or growth factor-reduced Matrigel for 5 to 20 d; were cultured on plates coated with 2.5 to 25 µg/cm2 of laminin, fibronectin, or type IV collagen for 5 d; or were cultured in the presence of 0.1 to 10 ng/ml purified human TGF-beta 1 for 5 d. In selected experiments, 1 to 1,000 ng/ml chicken IgY anti-TGF-beta 1 or isotype-matched control chicken IgY was added to the cultures.

Histochemical Staining of Cells

At each passage, 104 cells were applied to glass slides by cytocentrifugation. The slides were air-dried for 10 min and stained with alcian blue and safranin or were fixed in Mota's fixative and stained with toluidine blue (21). The alcian blue/safranin-stained cells were categorized on the basis of the proportion of granules that were positive for alcian blue or safranin. Cells were scored into one of the following groups: (1) containing only alcian blue- positive granules, (2) containing more alcian blue-positive than safranin-positive granules, (3) containing approximately equal numbers of alcian blue-positive and safranin-positive granules, (4) containing more safranin-positive than alcian blue-positive granules, and (5) containing only safranin-positive granules (21).

Fcvarepsilon RI-Dependent Activation

Cells were sensitized with 8 µg/ml monoclonal IgE anti-trinitrophenyl (TNP) at a cell density of 107 cells/ml in BMMC culture medium at 37°C for 30 min, washed, resuspended at 5 × 106 cells/ml in Tyrode's buffer containing 1.8 mM Ca2+, 0.2 mM Mg2+, 0.1% gelatin, and 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (pH 7.2), and activated with 50 ng/ml TNP-conjugated bovine serum albumin (TNP-BSA) at 37°C for 10 min to elicit an immediate phase of eicosanoid generation and secretory granule exocytosis (15).

Measurement of Eicosanoids

The release of PGD2 into the supernatant was measured by radioimmunoassay (Amersham Corp., Arlington Heights, IL). Supernatants were assayed for LT generation by reverse phase-high-performance liquid chromatography (RP-HPLC) (22). Samples were applied to a 5-µm 4.6 × 250-mm C18 Ultrasphere RP-HPLC column (Beckman, Fullerton, CA) equilibrated with a solvent of methanol/acetonitrile/water/acetic acid (10:15:80:0.2, vol/vol), pH 5.6. The column was eluted at a flow rate of 1 ml/min with a programmed concave gradient (System Gold curve 6) to 55% of the equilibrating solvent and 45% methanol over 2.5 min. Beginning at 5 min, the proportion of methanol was increased linearly to 75% over the next 15 min and was maintained at this level for 10 min more. Online ultraviolet absorbance was monitored at 270 and 280 nm. LTs were quantified by calculating the ratio of the area corresponding to the LT peak and the area of the PGB2 internal standard peak with correction for the molar extinction coefficient of each lipid. LT data are expressed in pmol/106 cells. LTA4 generation was assessed by summing its conversion products LTC4, LTB4, and 6-trans LTB4 diastereoisomers (6-t-LTB4).

Measurement of Secretory Granule Exocytosis

The cell pellets were suspended in enriched medium and disrupted by freeze-thawing. beta -hexosaminidase, a marker of mast-cell secretory granule exocytosis, was quantitated in the supernatants and pellets by spectrophotometric analysis of the hydrolysis of p-nitrophenyl-beta -D-2-acetamido-2-deoxyglucopyranoside. The percent release of beta -hexosaminidase was calculated by the formula (S/[S + P]) × 100, where S and P are the beta -hexosaminidase contents of equal portions of each supernatant and cell pellet, respectively (15).

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Immunoblotting

Expression of cPLA2, 5-LO, FLAP, and COX-1 in lysates of BMMC was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting as previously described (16, 17).

Statistics

All experiments were conducted at least four times. Statistical significance was analyzed by performing Student's paired or unpaired t tests, as appropriate. Statistical significance was considered for P < 0.05 (23).

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Derivation of Mast Cells in the Presence of Matrigel

When BALB/c bone-marrow cells were cultured in the presence of WEHI-3 cell-conditioned medium (as a source of IL-3) and Matrigel, both nonadherent cells (present in the tissue culture medium) and adherent cells (embedded in the Matrigel and/or adherent to the tissue culture flask) were obtained. These two populations were passed together until the third week of culture, at which point they were cultured separately. At 1 wk after culturing the two populations separately, each population gave rise to both nonadherent and adherent cells. The nonadherent cells were identical in histochemical staining and eicosanoid generation regardless of adherent or nonadherent origin. Likewise, the adherent cells were identical in histochemical staining regardless of origin (data not shown). Thus, data were pooled to allow for two populations (namely, nonadherent and adherent) regardless of origin.

Histochemical Characterization of BMMC Derived in the Presence of Matrigel

Although nonadherent mast cells derived in the presence of Matrigel did not acquire metachromasia more rapidly than conventional BMMC (Table 1), and mast cells adherent to the Matrigel were contaminated by a population of nonmetachromatic cells, both populations of mast cells contained denser granules on toluidine blue staining compared with conventional BMMC, suggesting a greater degree of morphologic maturity (Figure 1). Further, the nonadherent and adherent populations of BMMC derived in the presence of Matrigel revealed more intense counterstaining with safranin when compared with conventional BMMC (Table 1 and Figure 1). Thus 78 and 75%, respectively, of the nonadherent and adherent BMMC derived in the presence of Matrigel that stained with alcian blue contained some safranin-positive granules at 3 wk of culture, compared with 35% of conventional BMMC (n = 4, P < 0.005) (Table 1). The same pattern was seen at 4 wk.

                              
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TABLE 1
Histochemical staining of BMMC derived in the presence and absence of Matrigel


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  		Figure 1.   Histochemical staining of mast cells derived in Matrigel. Mast cells were derived from culture of bone marrow in the presence (M-BMMC) or absence (BMMC) of Matrigel. BMMC (A and D), nonadherent M-BMMC (B and E), and adherent M-BMMC (C and F ) were stained with toluidine blue (A, B, and C) or with alcian blue and safranin (D, E, and F ).

Fcvarepsilon RI-Dependent Eicosanoid Generation by BMMC Derived in the Presence of Matrigel

The generation of nonadherent cells from an adherent population, and the reciprocal generation of adherent cells from a nonadherent population, suggest that these cells cycled between an adherent and nonadherent state. Because the adherent population of mast cells derived in the presence of Matrigel did not exhibit greater than 50% metachromasia due to contamination with nonmetachromatic macrophage-like cells, because adherent cells needed proteolytic pretreatment for matrix detachment before IgE-dependent activation for eicosanoid generation, and because the conventional BMMC are nonadherent cells, only the nonadherent cells were studied for eicosanoid generation.

After 4 to 6 wk of culture, BMMC derived in the presence of Matrigel generated 6-fold more PGD2 and ~ 30-fold more total LTs in response to activation through Fcvarepsilon RI than did conventional BMMC (Figures 2A-2D). LTC4 was the principal LT product of conventional BMMC and was increased 11-fold in BMMC derived in the presence of Matrigel. BMMC derived in the presence of Matrigel also exhibited significant generation of 6-t-LTB4 and LTB4, which were barely detectable in conventional BMMC (Figure 2). Secretory granule exocytosis, assessed by release of beta -hexosaminidase activity, was not significantly different between BMMC derived in the presence of Matrigel and conventional BMMC (Figure 2E).


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  		Figure 2.   Eicosanoid generation by BMMC derived in Matrigel. Representative RP-HPLC chromatograms of Fcvarepsilon RI-dependent LT generation from conventional BMMC (A) and mast cells derived in the presence of Matrigel (M-BMMC) (B). Fcvarepsilon RI-dependent LT generation (C), PG generation (D), and beta -hexosaminidase release (E) are compared between BMMC (solid bars) and M-BMMC (striped bars) (n = 4; **P < 0.005 compared with control BMMC).

Culture of Conventional BMMC on Matrigel

To further investigate the components of Matrigel responsible for upregulation of arachidonic acid metabolism, a simpler culture system was sought. BMMC were derived in the absence of Matrigel for 4 wk, when they were > 95% toluidine blue-positive, and were then cultured in the presence or absence of Matrigel. Although PG and LT generation were upregulated less dramatically after 5 d of culture on Matrigel compared with BMMC derived in the presence of Matrigel, this was mostly due to variability in the generation of eicosanoids from conventional BMMC. Total LT and PGD2 generation were comparable between BMMC derived in the presence of Matrigel (Figure 2) and conventional BMMC cultured on Matrigel for 5 d (Figure 3). This, therefore, provided an experimental approach to the analysis of which of the basement membrane component(s) that are present in Matrigel contributed to priming for increased eicosanoid generation. Initial experiments focused on whether matrix proteins and/or growth factors associated with those matrix proteins were responsible. Hence, conventional BMMC were cultured for 5 d in the presence or absence of Matrigel or a growth factor-reduced Matrigel and then activated through Fcvarepsilon RI. BMMC treated with Matrigel or growth factor-reduced Matrigel generated approximately equal quantities of PGD2 and total LT products that were increased ~ 2- and ~ 2.5-fold, respectively, compared with conventional BMMC maintained in the absence of Matrigel (Figures 3A and 3B). BMMC treated with both Matrigel and growth factor-reduced Matrigel generated approximately 1.6-fold more LTC4 per million cells compared with untreated cells, and generated significant amounts of 6-t-LTB4 and LTB4, whereas these products were barely detectable in untreated cells (Figure 3A). In contrast, secretory granule exocytosis, measured by the release of beta -hexosaminidase, was approximately equal for all three sets of BMMC (Figure 3C).


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  		Figure 3.   Eicosanoid generation by conventional BMMC cultured for 5 d in the presence or absence of Matrigel. BMMC were derived in the absence of Matrigel in WEHI-3 cell-conditioned medium for 4 wk and then cultured in the absence (Control; open bars) or presence (striped bars) of Matrigel, or growth factor- reduced Matrigel (solid bars) for 5 d before sensitization with IgE anti-TNP and activation with TNP-BSA. The generation of LTA4, LTC4, 6-t-LTB4, and LTB4 (A); the generation of PGD2 (B); and secretory granule exocytosis (C) were assessed as described in MATERIALS AND METHODS (n = 4; *P < 0.05, **P < 0.005 compared with control BMMC).

TGF-beta 1 Upregulates Eicosanoid Generation in BMMC

To examine the contribution of individual matrix components to the upregulation of eicosanoid generation, BMMC were cultured in flasks coated with laminin, type IV collagen, or fibronectin at 2.5, 10, or 25 µg/cm2 for 5 d. Although Matrigel elicited an upregulation in Fcvarepsilon RI-dependent LT and PG generation, as previously seen, none of these extracellular matrix components induced an upregulation in Fcvarepsilon RI-dependent eicosanoid generation when compared with untreated cells (data not shown).

Growth factor-reduced Matrigel still contains physiologic concentrations of TGF-beta 1 (1.7 ng/ml; manufacturer's specification sheet). We therefore investigated whether TGF-beta 1 was contributing to the upregulation of IgE-dependent eicosanoid generation elicited by Matrigel. BMMC derived without Matrigel were treated with or without Matrigel for 5 d, or with 0.01 to 10 ng/ml TGF-beta 1. A dose-dependent upregulation in Fcvarepsilon RI-dependent LT generation was observed in response to priming with TGF-beta 1. Maximal LT generation was observed in BMMC treated with 1.0 ng/ml TGF-beta 1, those cells generating approximately the same amount of LTs as did Matrigel-treated cells (Figures 4A-4D). A dose-dependent increase in PGD2 generation was also observed although this was not statistically significant (Figure 4E). The modest increase in secretory granule exocytosis observed in response to TGF-beta 1 was not statistically significant (Figure 4F).


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  		Figure 4.   Priming by TGF-beta 1 of eicosanoid generation by BMMC. BMMC were derived in the presence of WEHI-3 cell- conditioned medium and treated with 0.01 to 10.0 ng/ml of TGF-beta 1 or with Matrigel for 5 d. They were sensitized with IgE anti-TNP and activated with TNP-BSA. The generation of LTA4 (A), LTC4 (B), 6-t-LTB4 (C), LTB4 (D), PGD2 (E), and secretory granule exocytosis (F ) were assessed as described in MATERIALS AND METHODS (n = 4; *P < 0.05, **P < 0.005 compared with control BMMC).

Anti-TGF-beta 1 Neutralizing Antibody Inhibits the Upregulation of Eicosanoid Generation Elicited by Matrigel

To confirm that matrix-associated TGF-beta 1 was responsible for the increase in eicosanoid generation observed in Matrigel-treated BMMC, we evaluated the effect of a neutralizing antibody to TGF-beta 1. BMMC derived in the absence of Matrigel were treated with 1.0 ng/ml TGF-beta 1 and various concentrations of a chicken anti-TGF-beta 1-specific neutralizing IgY antibody or an isotype-matched control antibody. The anti-TGF-beta 1 antibody, but not the control IgY, acted in a dose-dependent manner to inhibit the upregulation in Fcvarepsilon RI-dependent LT generation elicited by 1 ng/ml TGF-beta 1; maximal inhibition was observed with 100 to 1,000 ng/ml anti-TGF-beta 1 (data not shown). Incorporation of 1,000 ng/ml anti-TGF-beta 1 in the Matrigel matrix inhibited the priming of BMMC by Matrigel for increased LT and PG generation to approximately the same levels as cells cultured in the absence of Matrigel, whereas the isotype-matched antibody was without effect (Figures 5A-5E). The neutralizing antibody had no effect on secretory granule exocytosis (Figure 5F).


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  		Figure 5.   The effect of a neutralizing antibody to TGF-beta 1 on Matrigel-induced priming of BMMC for increased eicosanoid generation. BMMC were derived in WEHI-3 cell-conditioned medium and then treated with Matrigel in the presence or absence of 1,000 ng/ml anti-TGF-beta 1 neutralizing antibody or an isotype-matched control antibody for 5 d. The generation of LTA4 (A), LTC4 (B), 6-t-LTB4 (C), LTB4 (D), PGD2 (E), and secretory granule exocytosis (F ) were assessed as described in MATERIALS AND METHODS (n = 5; *P < 0.05, **P < 0.005 compared with control Ab).

Upregulation of cPLA2 by Matrix-Associated TGF-beta 1

Culture of BMMC in the presence of Matrigel therefore elicited an increase in IgE-dependent generation of LTC4, LTB4, 6-t-LTB4 (the nonenzymatic product of LTA4), and PGD2 that was abrogated by a neutralizing antibody to TGF-beta 1 (Figure 5). These data suggested that the site of upregulation of eicosanoid biosynthesis was in the supply of arachidonic acid to downstream enzymes. Several species of PLA2 that liberate free arachidonic acid from cell-membrane phospholipids have been described in BMMC, including the low molecular-weight group IIA and group V enzymes (24, 25) and 85-kD cPLA2 (16). Gene disruption experiments indicate that cPLA2 is essential for eicosanoid generation in BMMC (26). We therefore evaluated the effect of Matrigel in the presence or absence of neutralizing antibodies on expression of cPLA2, 5-LO, FLAP, and COX-1 in BMMC. Culture of conventional BMMC on Matrigel for 5 d led to an increase in expression of cPLA2 but not of 5-LO, FLAP, or COX-1 (Figure 6). The increase in expression of cPLA2 in response to Matrigel was reduced to basal levels in a dose-dependent manner by the anti-TGF-beta 1 antibody, which was without effect on the expression of FLAP (Figure 7).


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  		Figure 6.   The effect of Matrigel on expression of cPLA2, 5-LO, FLAP, and COX-1. BMMC were derived in WEHI-3 cell-conditioned medium and were cultured for 5 d in the absence (-) or presence (+) of Matrigel. BMMC were lysed and analyzed for expression of cPLA2, 5-LO, FLAP, and COX-1 by SDS-PAGE immunoblotting. A total of 2 × 105 cell equivalents were loaded per lane.


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  		Figure 7.   The effect of Matrigel and of a neutralizing antibody to TGF-beta 1 on expression of cPLA2. BMMC were derived in WEHI-3 cell-conditioned medium and were cultured for 5 d in the absence (lane 1) or presence (lanes 2-5) of Matrigel with (lanes 3-5) or without (lanes 1 and 2) a neutralizing chicken IgY anti-TGF-beta 1. BMMC were then lysed and analyzed for expression of cPLA2 and FLAP by SDS-PAGE immunoblotting. Lanes 3, 4, and 5 represent cells cultured in Matrigel in the presence of 10, 100, and 1,000 ng/ml, respectively, of chicken IgY anti-TGF-beta 1. A total of 2 × 105 cell equivalents were loaded in each lane. Equal loading of lanes is indicated by immunostaining for FLAP.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mast cells are critical effector cells of inflammation whose phenotype is determined by the local tissue microenvironment (6). Although the protease phenotype of murine mast cells can be assessed on a cell-by-cell basis using immunohistochemical techniques (6), the pattern of eicosanoid generation is not amenable to such exquisite phenotyping. Thus, LT and PG generation has been assessed in bulk preparations of partially purified tissue mast cells and in immature progenitor BMMC exposed to certain cytokines. Our previous studies have demonstrated that SCF favors the development of the COX-1/PGD2 synthase pathway for PGD2 generation (16) whereas IL-3 is critical for the development of the 5-LO/LTC4 synthase pathway of LTC4 generation (17).

The present study extends these studies of the microenvironmental regulation of eicosanoid generation in mouse mast cells to evaluate the effect of connective tissue matrix and basement membrane components on eicosanoid generation by BMMC. When bone-marrow cells were cultured for 3 to 4 wk in WEHI-3 cell-conditioned medium, as a source of IL-3, in the presence of Matrigel, a population of mast cells was derived with enhanced maturity and a shift toward a connective-tissue mast-cell phenotype (Figure 1 and Table 1). BMMC derived in SCF, with (17) or without IL-10 (2), also exhibited an increase in safranin staining, which in the latter case was accompanied by an increase in histamine content and heparin proteoglycan biosynthesis (2). The individual component(s) in Matrigel responsible for the increased density of granules and counterstaining with safranin were not determined due to the complexity of the Matrigel culture system.

Eicosanoid biosynthesis was markedly upregulated in BMMC derived in the presence of Matrigel compared with conventional BMMC (Figure 2). Indeed, the Matrigel protocol provided enough substrate to overwhelm the terminal enzymes of LT biosynthesis, leading to substantial generation of 6-t-LTB4, nonenzymatic degradation products of LTA4 that are not typically generated in significant quantity by conventional BMMC (27) (Figures 2A-2C). To further elucidate the component(s) of the basement membrane responsible for the upregulation in Fcvarepsilon RI- dependent eicosanoid products, we developed a less complicated culture system in which conventional BMMC were cultured with Matrigel for 5 d. This elicited upregulation of both PGD2 and LT generation to levels similar to those seen from BMMC developed over 4 wk in the presence of Matrigel (Figures 3 and 5). Culture of BMMC for 5 d with individual matrix proteins present within Matrigel demonstrated that laminin, type IV collagen, and fibronectin were not responsible for upregulated eicosanoid biosynthesis. Nevertheless, culture of BMMC on either Matrigel or growth factor-reduced Matrigel for 5 d primed for an upregulation of Fcvarepsilon RI-dependent eicosanoid generation (Figures 3A and 3B). Although the concentrations of epidermal growth factor, insulin-like growth factor 1, and platelet-derived growth factor are reduced in growth factor-reduced Matrigel, TGF-beta 1 is present at physiologic concentrations. Indeed, the capacity of Matrigel to prime BMMC for increased eicosanoid biosynthesis was reproduced by 1 ng/ml TGF-beta 1 (Figure 4), a concentration similar to that present in Matrigel or growth factor-reduced Matrigel (~ 2.3 and ~ 1.7 ng/ml, respectively). Further, the priming effect of Matrigel was inhibited by a neutralizing antibody to TGF-beta 1 (Figure 5). Thus, TGF-beta 1 is the major factor responsible for the priming effect of Matrigel on Fcvarepsilon RI-dependent eicosanoid biosynthesis by BMMC.

Neither Matrigel nor TGF-beta 1 affected secretory granule exocytosis of BMMC (Figures 2-5). This is consistent with the observation that TGF-beta 1 did not affect the release of histamine from mouse peritoneal mast cells in response to the calcium ionophore A23187 (28, 29) and indicated that increased eicosanoid generation was not due to a general upregulation in signal transduction through Fcvarepsilon RI. The increase in both prostanoid and LT generation and the increase in generation of proximal LT products (Figures 2C and 2D) suggested that matrix-associated TGF-beta 1 primed BMMC for increased eicosanoid generation through upregulation of PLA2. BMMC from mice deficient in group IIA PLA2 showed no impairment of eicosanoid generation (24), whereas eicosanoid generation was completely ablated in mice with disruption of the gene for cPLA2 (26). The upregulation of cPLA2 in response to culture in Matrigel and the inhibition of this upregulation by an antibody to TGF-beta 1 (Figures 6 and 7) are consistent with these observations. An increase in total LT biosynthesis might also be accounted for by increased expression of 5-LO/FLAP (17). Matrigel elicited no consistent increase in expression of these proteins (Figure 6) nor of COX-1, the first committed enzyme of immediate PGD2 generation (15, 16). We have previously reported that the upregulation of eicosanoid generation by BMMC in response to priming over 2 to 7 d with SCF was accompanied by increased expression of cPLA2 (16) that was inhibited by IL-4 (30) and that was not accompanied by changes in expression of 5-LO. The effect of SCF was most marked on PGD2 biosynthesis and also elicited an increase in expression of COX-1 (16). In contrast, the IL-3-dependent increase in LTC4 generation by BMMC was associated with increased expression of 5-LO and FLAP (17). Thus, SCF, IL-3, and TGF-beta 1 each elicit upregulation of eicosanoid generation by BMMC through different effects on the expression of eicosanoid biosynthetic enzymes.

TGF-beta 1 is a versatile, 25-kD cytokine derived from cells of all hematopoietic lineages as well as cells of mesenchymal origin such as fibroblasts and endothelial cells (31, 32). Latent TGF-beta 1 is anchored to a fibrillin-like protein termed latent TGF-beta 1 binding protein (LTBP) (31), presumably through interactions with type IV collagen and fibronectin (33). Mast-cell chymase has been shown to mediate the cleavage of LTBP, releasing latent TGF-beta 1 from extracellular matrix (34). Treatment of BMMC derived in IL-3, SCF, and IL-9 with TGF-beta 1 led to increased expression and release of the chymase mouse mast-cell protease 1 (35). Although latent TGF-beta 1 is not activated by mast-cell chymase (34) it may be activated by type I, II, and III receptors for TGF-beta 1 (31, 32), expressed by mast cells (36). Mast cells may also store and release TGF-beta 1 upon activation (37).

TGF-beta 1 is pleotrophic in its effects on immune cells with both proinflammatory and anti-inflammatory actions whose effects are dependent on the context in which it acts (31, 32, 38, 39). TGF-beta 1 is chemotactic for mast cells (36). It has been reported to inhibit the SCF-dependent induction of gelatinase B expression in a dog mastocytoma cell line (40). TGF-beta 1 inhibits proliferation of IL-3- and IL-4- dependent cultured mouse peritoneal mast cells and BALB/c BMMC grown in IL-3 (28). Although TGF-beta 1 inhibited the SCF rescue of BALB/c BMMC from apoptosis after IL-3 deprivation, it did not affect the viability and function of BALB/c BMMC grown in IL-3 (28, 29). Our experiments confirmed that at concentrations up to 1 ng/ ml of TGF-beta 1, viability of IL-3-dependent BMMC was not affected although proliferation was somewhat diminished (data not shown). These observations reveal a complex dynamic interaction between mast cells and TGF-beta 1 in which mast cells may store, release, and activate TGF-beta 1, which has pleotropic effects on mast-cell phenotype and function.

Murine mast cells originate from hematopoietic progenitors in the bone marrow, circulate as progenitors, and then reversibly change their phenotype in response to local microenvironments in peripheral tissues (6, 7). Mast cells are exposed to basement membranes when they exit the circulation and when they aggregate beneath the epithelial surfaces of the skin, the respiratory system, the gastrointestinal and genitourinary tracts, and the connective tissue adjacent to blood or lymphatic vessels, providing an early defense to a variety of pathogens. Murine BMMC, activated by phorbol myristate acetate or SCF, and certain transformed mouse mast-cell lines, adhere to fibronectin (41, 42), vitronectin (43), and laminin (44, 45). Thus, mast cells and their progenitors may interact with matrix-associated or basement membrane-associated TGF-beta 1 during migration and maturation in peripheral tissues, inducing phenotypic changes and priming for eicosanoid biosynthesis. It is notable that several connective tissue disorders such as rheumatoid arthritis (46), scleroderma (47), myelofibrosis (48, 49) and pulmonary fibrosis (50) demonstrate mast-cell hyperplasia in connection with increased levels of TGF-beta 1 transcript and protein. Further, expression of TGF-beta 1 is increased in the airways of asthmatic subjects (51, 52), suggesting that it may contribute to the priming for increased LT generation observed in the asthmatic airways (53). In conclusion, our data suggest that matrix-associated TGF-beta 1 may not only recruit mast cells to sites of tissue inflammation but also substantially augment the capacity of mast cells to elicit or perpetuate an inflammatory response through provision of proinflammatory eicosanoids.

    Footnotes

Address correspondence to: Jonathan P. Arm, Div. of Rheumatology, Immunology & Allergy, Brigham and Women's Hospital, Smith Research Bldg., Rm. 638B, One Jimmy Fund Way, Boston, MA 02115. E-mail: jarm{at}rics.bwh.harvard.edu

(Received in original form August 13, 1999 and in revised form December 3, 1999).

Abbreviations: 5-lipoxygenase, 5-LO; 6-trans LTB4 diastereoisomers, 6-t-LTB4; bone marrow-derived mast cell, BMMC; bovine serum albumin, BSA; cyclooxygenase, COX; cytosolic phospholipase, cPL; high affinity Fc receptor for IgE, Fcvarepsilon RI; 5-LO activating protein, FLAP; immunoglobulin, Ig; interleukin, IL; leukotriene, LT; prostaglandin, PG; reverse phase- high-performance liquid chromatography, RP-HPLC; stem-cell factor, SCF; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; transforming growth factor, TGF; trinitrophenyl, TNP.

Acknowledgments: This work was supported in part by National Institutes of Health Grant HL36110; by American Cancer Society Grant RPG97-001-BE; by an American Academy of Allergy, Asthma, and Immunology Summer Fellowship Award to an author (R.O.S.M.); and by a Burroughs Wellcome Developing Investigator Award to an author (J.P.A.).
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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