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Departments of
*
Pathology and
Internal Medicine, Division of Pulmonary and Critical Care, University of Michigan Medical School, Ann Arbor, MI 48109; and
National Cancer Institute, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The interaction of mast cells with surrounding cell populations during inflammatory events may be critical for the progression of these responses. For example, in chronic fibrotic lesions, a correlation exists between the number of mast cells and the severity of fibrosis (16, 17). Since mast cells depend upon stem cell factor (SCF)3 to avoid apoptosis (18), the production of SCF by surrounding cell populations is critical. Adding to the complexity of mast cell requirements for SCF is the fact that SCF can be found in either a soluble or transmembrane form and may differentially contribute to mast cell activation (19). In addition, SCF has also been shown to mediate mast cell adherence events that allow mast cells to bind to other structural cell populations (20, 21), which is important to their survival and activation (22, 23, 24, 25). Furthermore, data have shown that incubation of mast cells with fibroblasts can induce proliferation and differentiation of mast cells (26). Finally, recent data have indicated that mast cell interaction with fibroblasts can induce collagen gene activation (27, 28) and, therefore, contribute to fibrosis during disease progression.
Few studies have examined either the mechanisms that are operative during the cellular interactions or the cytokines that are produced during fibroblast-mast cell interactions. In particular, chemokines that are produced during chronic inflammation may be critical to the progression of the pathologic responses. Interestingly, the eosinophil has been identified as a potential effecter cell in both chronic allergic responses and fibrotic diseases. The identification of C-C family chemokines that are produced during the cell-to-cell interaction may be pertinent to the overall inflammatory response. Eotaxin, which appears to specifically recruit eosinophils, may play a primary role in these chronic diseases (29, 30). Since both mast cells and fibroblasts are able to produce chemokines, the cell-to-cell interaction may promote considerable production and therefore cause increased leukocyte accumulation at the site of the response. In the present studies, we have used fibroblast-mast cell cocultures to examine the role and production of histamine and eotaxin from the two cell populations. Interestingly, eotaxin was specifically up-regulated during the cocultures by transmembrane SCF, possibly indicating a specific activational role during disease, which relates to eosinophil accumulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mast cell lines were derived from the bone marrow of pathogen-free CBA/J mice (The Jackson Laboratory, Bar Harbor, ME), which was perfused from femurs (31). The cells were incubated with Dulbecco’s MEM supplemented with 1 mM L-glutamine, 10 mM HEPES, antibiotics, and 10% FCS with IL-3 (10 ng/ml) and SCF (10 ng/ml). By the end of 2–3 wk, a nonadherent population of large granular cells was observed. These isolated cells appeared homogeneous in cytospin preparations stained by Diff Quik (Baxter Diagnostics, McGaw Park, IL) with a typical mast cell granular appearance. The homogeneity of these cell lines was determined by flow cytometric analysis of surface markers, by histamine release assays (IgE + Ag), and by electron microscopy. In particular, these cells were c-kit positive (SCF receptor) but were negative for CD3, CD4, CD8, CD23, B220, and F480 by flow cytometry. These cell lines were routinely expanded, as described above, for 4 to 6 wk.
Pulmonary-derived fibroblasts
Whole mouse lungs were excised from a normal CBA/J mouse, cut in
1-mm sections, and washed twice with PBS (pH 7.2). The lung pieces were
incubated in 75-mm2 flasks in DMEM supplemented with 1
mM L-glutamine, 10 mM HEPES, antibiotics, and 10% FCS.
After fibroblasts began growing out from the primary lung explants, the
tissue pieces were removed. The fibroblasts were allowed to grow until
near confluency and were then trypsinized (0.25%) off the flask and
transferred to a 2 x 150-mm2 culture flask. This was
repeated once more before plating the fibroblast cells into 35-mm
plates. Fibroblasts were allowed to grow in the culture dishes until
they were near confluency. The expansion process required 
28 days,
and <1% of passenger cells (i.e., macrophages) were observed by
nonspecific esterase stain.
Fibroblast-mast cell cocultures
Cultured mast cell populations (2 x 105) were added to six-well plates with or without confluent primary fibroblast cultures. After 24 h, the culture supernatants were harvested and frozen at -20°C. In some cultures, the cell populations were separated by a 0.45-µm membrane in a Trans-Well culture dish (Costar, Kennebunkport, ME) or coincubated with anti-SCF serum (1:100 dilution). In some experiments, fibroblasts were preincubated with TNF for 6 to 8 h before mast cell coculture.
Production of anti-SCF and anti-eotaxin Abs
Rabbit anti-murine SCF and anti-eotaxin Abs were
prepared by multiple-site immunization of New Zealand White rabbits
with recombinant murine SCF or eotaxin (Genzyme, Cambridge, MA) in CFA.
Polyclonal Abs were titered by direct ELISA and specifically verified
by the failure to cross-react to mIL-3, mIL-1
, mTNF, mMIP-1,
IL-6, mJE, mMIP-1ß, hMCP-1, hIL-8, hRANTES, hMIP-1, hTNF, and
hMIP-1ß. The IgG portion of the serum was purified over a protein A
column and used in a sandwich ELISA.
ELISAs
Mouse-specific cytokines were quantitated by ELISA using a
modification of a double-ligand method as previously described (32).
Briefly, flat-bottom 96-well microtiter plates (Immuno-Plate I 96-F;
Nunc, Roskilde, Denmark) were coated with 50 µl/well of rabbit
anti-SCF or anti-eotaxin Ab (in 0.6 M NaCl, 0.26 M
H3B04, and 0.08 N NaOH, pH 9.6) for
16 h at 4°C and then washed with PBS, pH 7.5, and 0.05%
Tween-20 (wash buffer). Nonspecific binding sites were blocked with 2%
BSA in PBS and incubated for 90 min at 37°C. Standards were
1/2-log dilutions of recombinant cytokines from 10 pg/ml to 100
ng/ml. This ELISA method consistently detected cytokine concentrations
above 50 pg/ml. The specific Ab ELISAs did not cross-react with each
other or with other cytokines or chemokines, including mIL-3, mIL-1,
mTNF, mMIP-1, IL-6, mJE, mMIP-1ß, hMCP-1, hIL-8, hRANTES,
hMIP-1, hTNF, and hMIP-1ß.
Histamine release assays
Histamine levels in supernatants were determined by ELISA (Amac, Westbrook, ME). As positive controls, mast cells were exposed to compound 48/80 (Sigma) and a mast cell degranulator, and cells were sonicated to determine total intracellular histamine levels.
Measurement of SCF from pulmonary fibroblasts
Confluent 35-mm plate cultures were coincubated with various
cytokines (IL-1, TNF, IL-4, IFN-
, and IL-10) at various
concentrations (0.1, 1.0, and 10 ng/ml). After 24 h, supernatants
were harvested, and the adherent fibroblast cells were lifted off the
plates using Ca2+- and Mg2+-free HBSS at
4°C for 15 min. Once the fibroblasts were lifted from the culture
dishes, the cells were lysed by sonication and the cytoplasmic and
membrane portions of the cells were separated by ultracentrifugation
(100,000 x g). After separation, the membrane
fractions were lysed with PBS containing Triton X-100 (0.1%). The
culture supernatant, cytoplasmic, and lysed membrane fractions were all
assayed by specific SCF ELISA. No problem was encountered in the ELISA
with the nonionic detergent Triton X-100.
mRNA analysis and antisense therapy
Total cellular RNA from the cultures was isolated by homogenizing in a solution containing 25 mM Tris, pH 8.0, 4.2 M guanidine isothiocyanate, 0.5% Sarkosyl, and 0.1 M 2-ME. After homogenization, the suspension was added to a solution containing an equal volume of 100 mM Tris, pH 8.0, 10 mM EDTA, and 1.0% SDS. The mixture was then extracted two times each with phenol-chloroform and chloroform-isoamyl alcohol. The RNA was alcohol precipitated, and the pellet was dissolved in diethyl pyrocarbonate water. Total RNA was determined by spectrometric analysis at a 260-nm wavelength. Five µg of total RNA was reversed transcribed into cDNA utilizing a BRL reverse transcription kit (BRL, Gaithersburg, MD) and oligo(dT)12–18 primers. The cDNA was then amplified using specific primers for ß-actin, soluble SCF, and a transmembrane form of SCF. The primers used were 5'-GTG GGG CGC CCC AGG CAC CA-3' (sense) and 5'-GCT CGC CCG TGG TGG TGA AGC-3' (antisense) for ß-actin (350 bp), 5'-CAC TCA GCT TGA CTA CTC TT-3' (sense) and 5'-GTC ATT CCT AAG GGA GCT GG-3' (antisense) for soluble SCF (358 bp), and 5'-CAC TCA GCT TGA CTA CTC TT-3' (sense) and 5'-TTG CGG CTT TCC CTT TCT CG-3' (antisense) for transmembrane SCF (302 bp). The amplification buffer contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), and 2.5 mM MgCl. Specific oligonucleotide primer was added (200 ng/sample) to the buffer, along with 1 µl of the reverse-transcribed cDNA samples. The cDNA was amplified after determining the optimal number of cycles. The mixture was first incubated for 5 min at 94°C and then was cycled 30 times at 95°C for 30 s, followed by 58°C for 45 s, and elongated at 72°C for 75 s. This format allowed optimal amplification with little or no nonspecific amplification of contaminating DNA. After amplification, the sample (20 µl) was separated on a 2% agarose gel containing 0.3 µg/ml (0.003%) of ethidium bromide. The bands were visualized and photographed using UV transillumination.
Phosphorothioate-linked antisense and sense SCF oligonucleotides were used at 20 µM in tissue culture by preincubating the fibroblasts overnight before and during mast cell coculture. This level was shown to significantly inhibit the level of SCF protein produced by the lung fibroblasts in preliminary experiments with the antisense but not with the sense oligonucleotide. Sequences were 5'-CTGGATCGCAGCGCTGCCTTTCCTTATGA-3' for the sense and 5'-TCATAAGGAAAGGCAGCGCTGCGATCCAG-3' for the antisense oligonucleotide.
Statistics
Statistical significance was determined by ANOVA, and significance was determined with p values <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stromal cells and mast cells are in intimate contact within
tissues where mast cells reside. During chronic disease pathology, such
as in fibrosis and allergic diseases, the numbers of both mast cells
and stromal cells increase. Previous studies have demonstrated a
significant role for fibroblasts in mast cell differentiation,
activation, and proliferation (22, 23, 24, 25, 33). Figure 1
demonstrates that during
fibroblast-mast cell interaction, a substantial activational event
occurs as monitored by histamine release from 24-h cocultures. The
degranulation appears to require cell-to-cell contact, as separation of
the cell populations with a 0.45 µm Trans-Well culture dish
significantly decreased histamine release. Interestingly, there was
still a significant histamine release in Trans-Well culture dishes when
fibroblasts were present in the lower chamber. Finally, if fibroblasts
were incubated with TNF 6 h before the addition of the mast cells
and then washed, the histamine release response was significantly
augmented. Preactivation of the fibroblasts with other cytokines,
including IL-4, IL-1, and IFN-, had no effect on histamine release
(data not shown).
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) when the cell populations were
combined. Preactivation of the fibroblast populations with increasing
levels of TNF induced significant increases in the release of eotaxin
during cell-to-cell interactions (Fig. 2); however, TNF did not induce
eotaxin production in either fibroblasts or mast cells alone (data not
shown). Preactivation of the fibroblasts with other cytokines, such as
IL-1 and IFN-, had no effect on the eotaxin production (data not
shown). Altogether, these studies verify earlier studies demonstrating
that fibroblast-mast cell interaction leads to an
activation/degranulation event.
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Previous studies have indicated that fibroblast populations can
augment mast cell activation and differentiation. Separation of the two
cell populations by a Trans-Well membrane significantly attenuated the
eotaxin production (Fig. 3), indicating
that cell-to-cell interactions were required. These responses may be
exaggerated by the production of a major mast cell growth and survival
factor, SCF. Preincubation of the fibroblast with anti-SCF
demonstrated that histamine release in 24-h-accumulation cultures could
be attenuated (Fig. 1
). Likewise, regulation of eotaxin production was
observed by the anti-SCF treatment during the cell-to-cell
interaction (Fig. 3). In addition, we have also constructed sense and
antisense oligonucleotides specific for SCF and inhibited the
production of eotaxin by preincubation (overnight) of fibroblasts with
the antisense, but not sense, SCF-specific nucleotides (Fig. 4). The use of the antisense SCF
oligonucleotide demonstrated a significant reduction in isolated
membrane SCF production by the fibroblasts compared with the
sense-treated cells (0.48 ± 0.24 vs 2.0 ± 0.12 ng/ml,
respectively). To determine whether SCF on its own was enough to drive
eotaxin production, we stimulated mast cells with soluble SCF or by
SCF-coated plates to simulate the transmembrane form. The data in Table I indicate that eotaxin was produced only
on SCF-coated plates and not by soluble SCF after 24 h of
incubation. Interestingly, other degranulating stimuli, such as 48/80
and IgE + Ag, demonstrated no eotaxin production from the mast
cells, while they did induce histamine release (15 and 12% of total,
respectively) as previously described (34), further demonstrating the
importance of SCF-mediated activation in eotaxin production.
Interestingly, when soluble SCF was added into the mast cells plated
onto the plate-bound cultures, an inhibition of eotaxin production was
observed, suggesting a blockage of the cross-linking of
c-kit. These data suggest that SCF can drive eotaxin
production primarily in its solid phase form and that the different
forms of SCF may have specific regulatory functions in chemokine
production.
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To better define the role of SCF during fibroblast-mast cell
interaction, we have examined pulmonary fibroblasts stimulated with
various inflammatory stimuli, including IL-1, IL-4, TNF, IFN-, and
IL-10. After 24 h poststimulation, cell-free culture supernatants
were harvested. Sonicated fibroblasts were separated by
ultracentrifugation to separate membrane from soluble components. The
membrane portion was dispersed with PBS containing 0.1% Triton X-100.
Little or no SCF protein was measured in the supernatant fraction of
the stimulated fibroblasts (<100 pg/ml). However, nearly all of the
detectable SCF was in the membrane fractions of the stimulated
fibroblasts. Interestingly, substantial levels of SCF were detected in
control fibroblasts (2.0–2.5 ng/ml), while only TNF-stimulated
fibroblasts produced increases in membrane SCF (Fig. 5). All of the SCF protein from the
sonicated fibroblasts was detected in the membrane fraction, and none
was found in the cytoplasmic portion. These data help to explain why
cell-to-cell interactions were required above for the SCF-mediated mast
cell activation, histamine release, and eotaxin production. To verify
this aspect, the lung fibroblasts were assayed for SCF-specific mRNA
using specific primers that differentially recognize soluble or
transmembrane forms of SCF. Both forms of SCF were detectable by
reverse transcription-PCR techniques (data not shown). Altogether, it
appears that the mast cell activation described above is driven, at
least in part, by the transmembrane form of SCF that is found
constitutively and can be up-regulated by TNF in normal lung
fibroblasts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fact that fibroblast cell populations can produce a distinct form of SCF may indicate a divergence in the function of soluble vs transmembrane SCF. In the present studies, plate-bound, but not soluble, SCF drove eotaxin production. The production of membrane SCF from stromal cell populations may contribute to the normal maintenance and/or to the normal healing process within tissue. The overzealous production of transmembrane SCF may lead to augmented and persistent mast cell activation, eotaxin production, eosinophil recruitment, and tissue pathology. SCF activates mast cells via c-kit, a protein tyrosine kinase-coupled receptor. It appears from the present studies that eotaxin production is dependent upon cross-linking of these receptors in a solid phase. These events would be indicative of the overproduction of eotaxin, leukocyte recruitment and activation, and, finally, tissue pathology. Although there is a paucity of data to support these concepts, the idea of SCF in chronic disease progression has been demonstrated in other investigations. SCF has been linked to eosinophil accumulation not only in allergic models of inflammation (36) but also in infectious parasitic models in which the neutralization of SCF decreased the accumulation of eosinophils, the effecter cells in many parasitic infections (37). Thus, although SCF may play a detrimental effect in chronic inflammatory diseases such as asthma, it may be beneficial in infectious parasitic diseases.
The relationship between mast cell activation and eosinophilia repeats itself throughout various types of chronic inflammatory events. In allergic inflammation, the persistent activation of mast cells appears to be a primary event that may directly lead to the accumulation of eosinophils (38). This would be mediated by the level and the type of chemokines that are produced during the mast cell activation. In the most severe cases of allergic asthma, a considerable level of stromal cell proliferation and "airway thickening" is observed within the large airways, where mast cells are predominantly found. The interrelationship between mast cells, transmigrating eosinophils, and the increased numbers of activated stromal cells may contribute to the chronicity and severity of the asthmatic responses. In chronic fibrotic diseases, it has now been reported that a significant influx of eosinophils may participate in the maintenance of the fibrotic response (39, 40). Interestingly, there may be a strong relationship between the severity of the fibrotic event and the number of mast cells and eosinophils that are present. This would be exemplified in these studies by the SCF-dependent expression of eotaxin, a C-C chemokine with potent eosinophil chemoattractant and activating functions (41, 42, 43). The persistent activation of mast cells and eotaxin production within these disease states may relate to the constant influx of leukocytes (eosinophils) that appear to mediate the tissue pathology even in the cases in which an Ag stimulus may not be apparent. Although previous studies have not observed eotaxin production from mast cells (44, 45), the transmembrane SCF-induced mechanism was not examined. In addition, there may be an effect due to the maturity of the mast cell population, as studies have shown that a heterogeneous population of mast cells likely exists within any culture of mast cells grown from bone marrow cells (46).
The results from these studies have outlined a novel relationship between mast cell and fibroblast populations. The production of histamine and chemokines during this interaction indicates a possible mechanism for disease progression. Transmembrane SCF may have a role in exacerbation of mast cell activation (histamine release) and eotaxin production, which could subsequently lead to increased eosinophil accumulation and tissue pathology. Future studies will examine the expression of SCF in fibroblast populations from inflammatory and fibrotic lesions, as up-regulated production of SCF may exacerbate and maintain the altered disease status.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nicholas W. Lukacs, Ph.D., Department of of Pathology, University of Michigan Medical School, 1301 Catherine, Ann Arbor, MI 48109-0602.
3 Abbreviations used in this paper: SCF, stem cell factor.
Received for publication August 27, 1997. Accepted for publication February 17, 1998.
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