The high affinity receptor for IgG (FcγRI, CD64) is expressed on human mast cells, where it is up-regulated by IFN-γ and, thus, may allow mast cells to be recruited through IgG-dependent mechanisms in IFN-γ-rich tissue inflammation. However, the mediators produced by human mast cells after aggregation of FcγRI are incompletely described, and it is unknown whether these mediators are distinct from those produced after activation of human mast cells via FcεRI. Thus, we investigated the release of histamine and arachidonic acid metabolites and examined the chemokine and cytokine mRNA profiles of IFN-γ-treated cultured human mast cells after FcγRI or FcεRI aggregation. Aggregation of FcγRI resulted in histamine release and PGD2 and LTC4 generation. These responses were qualitatively indistinguishable from responses stimulated via FcεRI. Aggregation of FcεRI or FcγRI led to an induction or accumulation of 22 cytokine and chemokine mRNAs. Among them, seven cytokines (TNF-α, IL-1β, IL-5, IL-6, IL-13, IL-1R antagonist, and GM-CSF) were significantly up-regulated via aggregation of FcγRI compared with FcεRI. TNF-α mRNA data were confirmed by quantitative RT-PCR and ELISA. Furthermore, we confirmed histamine and TNF-α data using IFN-γ-treated purified human lung mast cells. Thus, aggregation of FcγRI on mast cells led to up-regulation and/or release of three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Some cytokines, such as TNF-α, were released and generated to a greater degree after FcγRI aggregation, suggesting that selected biologic responses of mast cells may be preferentially generated through FcγRI in an IFN-γ-rich environment.
The high affinity receptors for IgE (FcεRI) (1, 2, 3, 4) and IgG (FcγRI) (1) are both now known to be expressed on human mast cells. Activation of human mast cells through FcεRI is believed to be responsible for allergen-dependent allergic responses in a Th2 environment (5). FcγRI, which is also known to be expressed on monocytes (6, 7), may in turn allow human mast cells to be recruited into IFN-γ-rich tissue inflammation, such as observed in a Th1 environment, and where the biologic contribution of the mast cell may be distinct from its role in allergic inflammation. Supporting this possibility is an increasing body of evidence in animal models that mast cells may be involved in the pathogenesis of vasculitis generated by immune complexes (8, 9, 10) and that mast cells participate in host defense mechanisms against bacteria (11, 12, 13, 14).
The induction of mast cell activation through FcγRI in addition to FcεRI in an IFN-γ-rich microenvironment, thus, appears to present an additional mechanism by which mast cells may be recruited to produce a diversity of inflammatory mediators, including arachidonic acid metabolites, chemokines, and cytokines. To explore this possibility, human mast cells were examined for the release of histamine; PGD2 and leukotriene (LT)4 C4; and the expression of cytokines and chemokines after aggregation of FcγRI or FcεRI. As will be shown, aggregation of FcγRI is followed by histamine release, and LTC4, PGD2, and chemokine generation; and that these responses were qualitatively indistinguishable from those stimulated by FcεRI. Aggregation of FcγRI did lead to a significant enhancement of the expression of TNF-α, IL-1β, IL-5, IL-6, IL-13, IL-1R antagonist (IL-1Ra), and GM-CSF over that which followed FcεRI aggregation in an IFN-γ-rich environment.
Materials and Methods
CD34+ cell culture
Human peripheral blood CD34+ progenitor cells were obtained and processed, after informed consent, as described (15), and placed in serum-free medium (StemPro-34 SFM; Life Technologies, Grand Island, NY) supplemented with 2 mM l-glutamine, 50 μg/ml streptomycin, 100 IU/ml penicillin, 100 ng/ml recombinant human (h) stem cell factor (SCF), 100 ng/ml rhIL-6, and 30 ng/ml rhIL-3 (first week only) (Peprotech, Rocky Hill, NJ) (15, 16, 17). Half of the culture medium was replaced every 7 days. Mast cell percentages were assessed by metachromatic staining of cytopreparations with acidic toluidine blue (pH 1.0). Greater than 95% of the cells were identified as mast cells 8–10 wk after the initiation of the culture (15, 16, 17) To remove contaminating monocytes/macrophages, cultured cells were incubated in a culture dish (35 × 10 mm) for 2 h and nonadherent cells were harvested. The final purity of mast cells was >99%.
Purification of human lung mast cells
Macroscopically normal human lung resected during surgery was obtained from the National Disease Research Interchange (Philadelphia, PA). Lung mast cells were dispersed from chopped lung specimens by an enzymatic procedure and were purified by magnetic bead affinity selection using the anti-Kit mAb, YB5.B8 (BD PharMingen, San Diego, CA) as described (18). Mast cell percentages and numbers were assessed by counting using a Neubauer hemocytometer after metachromatic staining with the Kimura stain (19). The final purity of lung mast cells was >98%.
Abs and flow cytometric analysis
The following mAbs were purchased: mouse anti-human FcγRI (clone 10.1, subclass IgG1) (Caltag, Burlingame, CA); F(ab′)2 fragments (F(ab′)2) of mouse anti-human FcγRI (clone 22, subclass IgG1; Medarex, Annandale, NJ); and mouse anti-human CD117 (subclass IgG1; Immunotech, Miami, FL). FACS analysis was performed as described (1, 16, 17). In some experiments, mast cells were preincubated with 1 μg/ml of human myeloma IgE (Calbiochem, San Diego, CA) for 16 h. Mast cells were then resuspended in a mixture of PE cyanine 5-conjugated c-kit (CD117), biotin-conjugated goat anti-human IgE ε-chain (BioSource International, Camarillo, CA) and FITC-conjugated mouse anti-human FcγRI mAb for 30 min at 4°C. Cells were next washed and incubated with streptavidin-allophycocyanin (BD Pharmmingen) for 20 min at 4°C. Cell analysis was performed using a FACSCalibur (BD Becton Dickinson, San Jose, CA) and CellQuest software (BD Becton Dickinson).
For FcγRI and FcεRI-dependent activation, mast cells were preincubated with rhIFN-γ-1b (15 ng/ml; Genentech, South San Francisco, CA) for 48 h. The increased expression of FcγRI on IFN-γ-treated cells was confirmed by FACS analysis, as was the continued expression of FcεRI, which was unchanged by IFN-γ. The cells were next washed and resuspended with culture medium in 96-well culture plates. Cells were incubated with F(ab′)2 of mouse anti-human FcγRI mAb (clone 22, 0–3 μg/ml for mediator assays and 1.0 μg/ml for RNase protection assays) or mouse F(ab′)2 of IgG (1 μg/ml) (Jackson ImmunoResearch, West Grove, PA) for 30 min at 37°C. The cells were washed and resuspended with culture medium. For aggregation of FcγRI, mast cells were exposed to goat F(ab′)2 of anti-mouse F(ab′)2 of IgG (0–30 μg/ml for the histamine release assay and 10 μg/ml for PGD2 and LTC4 and RNase protection assays; Jackson ImmunoResearch) for 30 min for histamine assay, for 45 min for PGD2 and LTC4 analysis, or for 0, 2, 4, and 8 h for RNase protection studies. For aggregation of FcεRI, cells were incubated with anti-4-hydroxy-3-nitrophenylacetyl (NP)-IgE (0–3 μg/ml for mediator assays and 1.0 μg/ml for RNase protection assays; Serotec, Raleigh, NC) for 16 h and then washed. Cells were activated with NP-BSA (0–100 ng/ml for histamine assay and 10 ng/ml for PGD2 and LTC4 and RNase protection assays; Sigma, St. Louis, MO) for an additional 30 min for histamine assay, for 45 min for PGD2 and LTC4 analysis, or for 0, 2, 4, and 8 h for RNase protection studies. The reaction was stopped by centrifugation at 4°C. Culture supernatants and cell pellets were collected for mediator assay and for total RNA isolation and kept at −80°C.
In kinetic studies of histamine release, mast cells were incubated with 0.3 μg/ml of anti-NP-IgE for 16 h or with 0.3 μg/ml F(ab′)2 of anti-FcγRI mAb for 30 min at 37°C. Cells were then washed and either 10 ng/ml of NP-BSA or 10 μg/ml of goat F(ab′)2 of anti-mouse F(ab′)2 of IgG added at 37°C. At 0 s, 30 s, 1 min, 3 min, 5 min, 10 min, 15 min, and 30 min after adding either NP-BSA or goat F(ab′)2 of anti-mouse F(ab′)2 of IgG, ice-cold physiological HBSS was added to stop the reaction, the mixture centrifuged, and supernatants and cell pellets kept at −80°C.
In the experiments to examine the effect of IFN-γ on FcεRI-mediated TNF-α and IL-8 production, mast cells were preincubated with or without 15 ng/ml of IFN-γ for 48 h and with 1.0 μg/ml of anti-NP-IgE for 16 h. Cells were then incubated with or without 10 ng/ml of NP-BSA for 6 h, supernatants harvested, and concentrations of TNF-α and IL-8 measured.
Histamine in the supernatants and cell pellets was measured using an enzyme immunoassay kit (Immunotech). The net percentage of histamine release was calculated from the ratio of each sample with spontaneous release (<5%) subtracted against total histamine.
PGD2 and LTC4 assays
PGD2 and LTC4 in the supernatants were measured using an enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI).
Isolation of RNA and RNase protection assay
Total cellular RNA was isolated from mast cells with RNeasy Mini Kits (Qiagen, Valencia, CA), according to the manufacturer’s specifications. The purity of RNA was assessed on the basis of the A260/A280 ratio, and the integrity of RNA was verified by agarose gel electrophoresis. The yield of RNA per 106 mast cells was 5.1 (3.1–7.5) μg (median with range; n = 13). The hCK-1, 2, 3, 4, and 5 multiprobe template sets (BD PharMingen) were used for multiple cytokine/chemokine gene RNase protection assays. Total RNA (1 μg/sample) was applied and RNase protection was performed following the manufacturer’s recommendations (BD PharMingen). In addition to human control RNA (BD PharMingen), total RNA from human lymphocytes activated with 1 μM of calcium ionophore for 24 h was used as a positive control. Yeast tRNA (1 μg) was used as a negative control. The quantification of each cytokine was determined by measuring the relative density of expression of each cytokine with respect to the ribosomal protein L32 following background subtraction with the aid of ImageQuant 5.0 (Molecular Dynamics, Sunnyvale, CA).
To avoid the possibility that intact goat F(ab′)2 of anti-mouse F(ab′)2 of IgG in the cell supernatants might directly bridge one mouse anti-cytokine mAb to another in the ELISA, mouse F(ab′)2 of IgG (10 μg/ml) were added to cell supernatants just before performing the ELISA. We confirmed that mouse F(ab′)2 of IgG reduced the background of ELISA data in a concentration-dependent manner and that 10 μg/ml of mouse F(ab′)2 of IgG completely blocked the intact goat F(ab′)2 of anti-mouse F(ab′)2 of IgG in the cell supernatants. Furthermore, 10 μg/ml of mouse F(ab′)2 of IgG did not affect the standard curves. The data were confirmed by an additional method described by Edberg et al. (20). Wells of tissue culture plates were coated with absorbed protein (10 μg/ml of goat F(ab′)2 of anti-mouse F(ab′)2 of IgG) in carbonate-bicarbonate buffer (Sigma) for 30 min at 37°C and overnight at 4°C. The wells were washed with 0.05% Tween 20 in PBS. Cells were then incubated with 0–3 μg/ml of F(ab′)2 of anti-FcγRI mAb for 30 min at 37°C and washed to remove excess anti-FcγRI mAb. The cells were next transferred to the coated wells and incubated for the indicated duration before TNF-α or IL-8 assay. There was no significant difference in cytokine measurements between these two methods. Levels of TNF-α and IL-8 in diluted culture supernatants were quantitated by ELISA kits for TNF-α (sensitivity <0.18 pg/ml; R&D Systems, Minneapolis, MN) and IL-8 (sensitivity <10 pg/ml; R&D Systems).
Statistical significance of differences was performed using the two-tailed unpaired Student’s t test. Differences were considered significant when the probability (p) was <0.05. Data are expressed as mean ± SEM.
Histamine release by FcεRI and FcγRI aggregation
The expression of FcεRI and FcγRI on IFN-γ-treated human mast cells was first confirmed by FACS analysis (Fig. 1, a and b). FcεRI+ cells expressed Kit (CD117) and FcγRI+ cells also expressed FcεRI and Kit (data not shown). To assess immunologic histamine release after FcγRI aggregation, IFN-γ-treated human cultured mast cells were first incubated for 30 min with 0–3 μg/ml of F(ab′)2 of mouse anti-human FcγRI mAb before challenge with 0 to 30 μg/ml of goat F(ab′)2 of anti-mouse F(ab′)2 of IgG. The aggregation of FcγRI caused histamine release in a concentration-dependent manner, which reached 47.2 ± 1.8% with 3 μg/ml of mouse anti-human FcγRI mAb and 30 μg/ml of anti-mouse F(ab′)2 of IgG, the maximum concentrations used (Fig. 1,b; n = 4 donors). For comparison, mast cells were passively sensitized for 16 h with 0–3 μg/ml anti-NP-IgE before challenge with 0–100 ng/ml NP-BSA. Anti-NP-IgE and NP-BSA induced a concentration-related release reaching 40.2 ± 2.8% with 1 μg/ml anti-NP-IgE and 100 ng/ml NP-BSA (Fig. 1 a; n = 4 donors).
For comparison of the time courses of histamine release after either FcεRI or FcγRI aggregation, we used a concentration of stimuli that generated similar histamine release; 0.3 μg/ml of anti-NP-IgE and 10 ng/ml of NP-BSA, which caused 36.0 ± 4.0% net histamine (Fig. 1,a) and 0.3 μg/ml of anti-FcγRI mAb and 10 μg/ml of anti-mouse F(ab′)2 of IgG, which induced 39.8 ± 0.8% net histamine (Fig. 1,b). In time course studies, histamine release, thus, induced by either FcεRI or FcγRI aggregation reached a plateau 15 min after challenge with a t1/2 of 3.1 min and 2.6 min, respectively (Fig. 1 c). Thus, human mast cell degranulation induced by FcεRI and FcγRI aggregation under these conditions caused an approximately similar amount of maximum histamine release and comparable kinetics of histamine release.
Analysis of PGD2 and LTC4 release by human mast cells
Human mast cells produce PGD2 and LTC4 that contribute to inflammatory reactions (5, 21). To examine whether the aggregation of FcγRI on the human mast cell surface is capable of inducing eicosanoid synthesis, we activated IFN-γ-treated mast cells through FcεRI and FcγRI and followed PGD2 and LTC4 release. As expected, FcεRI aggregation on mast cells induced PGD2 (84.9 ± 16.9 ng/106 mast cells at 0.1 μg/ml of anti-NP-IgE) and LTC4 release (49.8 ± 17.1 ng/106 mast cells at 0.3 μg/ml of anti-NP-IgE) (Fig. 2, a and c). Mast cells activated with F(ab′)2 of mouse anti-human FcγRI mAb cross-linked with goat F(ab′)2 of anti-mouse F(ab′)2 of IgG (Fig. 2, b and d) produced comparable concentration-related PGD2 and LTC4 release, reaching 74.2 ± 13.2 ng/106 mast cells and 62.6 ± 17.6 ng/106 mast cells with 0.1 and 0.3 μg/ml of mouse anti-human FcγRI mAb, respectively. These studies, thus, detected no significant differences in the amount of eicosanoid release after FcεRI and FcγRI aggregation under the conditions of this assay.
Chemokine and cytokine mRNA profiles in mast cells after either FcεRI or FcγRI aggregation
In vitro studies with human mast cells and mast cell lines have demonstrated that these cells produce cytokines and chemokines (22, 23, 24, 25, 26, 27). Therefore, we next compared 38 cytokine and chemokine mRNA profiles using an RNase protection assay in IFN-γ-treated mast cells activated by either FcγRI or FcεRI aggregation (Fig. 3,a). In these experiments, mRNA was not detectable for IL-2, IL-4, IL-7, IL-9, IL-12p35, IL-12p40, IL-14, IFN-β, IFN-γ, lymphotoxin-β, TGF-β2, TGF-β3, TNF-β, and lymphotactin. mRNAs for IL-10 and IL-15 were expressed minimally (at a ratio to L32 of <0.1), and no conclusions could be made relative to these cytokines. Up-regulation of mRNAs for IL-1α, IL-3, IL-8, G-CSF, LIF, M-CSF, oncostatin M (OSM), SCF, TGF-β1, IFN-γ-induced protein (IP)-10, I-309, monocyte-inflammatory protein (MIP)-1α, MIP-1β, monocyte chemotactic protein-1, and RANTES were detected in human mast cells after either FcεRI or FcγRI aggregation. In every case, message peaked between 2 to 4 h after stimulation and peak levels did not differ whether mast cells were activated through FcγRI or FcεRI. mRNA levels were greater after FcγRI aggregation compared with levels after FcεRI aggregation for IL-1β, IL-1Ra, IL-5, IL-6, IL-13, TNF-α, and GM-CSF (Fig. 3, c –i). mRNA again peaked between 2 and 4 h for IL-5, IL-8, IL-13, TNF-α, and GM-CSF. mRNA levels for IL-1β, IL-1Ra, and IL-6 continued to increase over 8 h.
Thus, aggregation of FcεRI or FcγRI led to an induction or up-regulation of mRNA for 22 of 38 cytokines and chemokines. Among them, seven cytokines expressed, including TNF-α, IL-1β, IL-5, IL-6, IL-13, IL-1Ra, and GM-CSF, were significantly up-regulated via aggregation of FcγRI compared with FcεRI under the conditions used.
Release of TNF-α and IL-8 from mast cells
To explore whether the results of the RNase protection assay could predict proteins released by activated mast cells; because of reports (5, 14, 22) documenting TNF-α production and release from mast cells; and with the implication that TNF-α has a central role in mast cell-dependent inflammation, we measured released TNF-α. As may be seen in Fig. 4,a, aggregation of FcεRI on IFN-γ-treated mast cell surfaces led to TNF-α release (21.3 ± 11.7 pg/106 mast cells at 1.0 μg/ml of anti-NP-IgE; n = 4 donors), as has been reported for human lung mast cells (28). Concentration response studies showed the optimal concentration of NP-BSA for release was 10 ng/ml (Fig. 4,b), the same as required for optimal histamine release. In agreement with the up-regulation of TNF-α mRNA by FcγRI aggregation, TNF-α was also released from mast cells activated via FcγRI activation (98.4 ± 19.3 pg/106 mast cells at 0.3 μg/ml of anti-FcγRI mAb; n = 4 donors) (Fig. 4,c). The optimal concentration of goat F(ab′)2 of anti-mouse F(ab′)2 of IgG for optimal TNF-α release was 10 μg/ml (Fig. 4,d), the same as required for optimal histamine release. Thus, the doses of stimuli used in the RNase protection assay based on optimal histamine release were also optimal for TNF-α production. We also performed kinetic studies of TNF-α release, which showed that TNF-α protein was first noted 2 h after challenge and continued through 8 h (Fig. 4 e). Comparison of the amount of TNF-α released between these two stimuli also showed that aggregation of FcγRI led to significantly more release at 2, 4, and 8 h (p < 0.01 or p < 0.001; n = 8 donors) compared with release after aggregation of FcεRI.
We next measured a released C-X-C chemokine, IL-8, which is well known as a mast cell chemokine (22, 23). In agreement with the results by RNase protection assay, the results by kinetic studies with IL-8 production showed no significant difference in IL-8 production between these two stimuli (Fig. 4 f).
It has been reported that an IFN-γ-rich Th1 environment itself may alter FcεRI-dependent cytokine and chemokine responses (29). To verify this observation, we examined IL-8 and TNF-α protein release from FcεRI-activated human mast cells in the presence or absence of IFN-γ. Similar to the previous report (29), IFN-γ down-regulated IL-8 secretion (2742 ± 576 without IFN-γ vs 1362 ± 464 pg/106 mast cells with IFN-γ; n = 5; p < 0.01). TNF-α secretion was unchanged (3.3 ± 0.5 without IFN-γ vs 8.4 ± 4 pg/106 mast cells with IFN-γ; n = 5, not significant). Thus, a Th1-like environment may down-regulate some FcεRI-dependent responses so that in such an environment not only may human mast cells now produce mediators after FcγRI aggregation, but also FcεRI-dependent responses appear to be selectively down-regulated as reported (29).
Histamine and TNF-a release from purified human lung mast cells after FcεRI or FcγRI aggregation
Because data obtained using cultured human mast cells may not reflect normal mature tissue mast cell responses, we purified human lung mast cells using a magnetic affinity selection method and examined histamine release from IFN-γ-treated or nontreated lung mast cells after aggregation of FcγRI and compared this data with results obtained after aggregation of FcεRI. Thus, aggregation of FcγRI on IFN-γ-treated lung-derived mast cells led to 30% net histamine release compared with 4.9% from lung-derived human mast cells that had not been treated with IFN-γ (Fig. 5,a). TNF-α release was similarly verified using IFN-γ-treated lung-derived mast cells after aggregation of either FcγRI or FcεRI. As can be seen in Fig. 5 b, TNF-α release increased 4-fold after aggregation of FcγRI compared with release observed in cells activated after aggregation of FcεRI.
Upon binding of Ag to IgE on the surface of mast cells, FcεRI becomes cross-linked and activates intracellular second messengers (5). This is followed by the release or generation of three classes of mediators by mast cells: biogenic amines (histamine); lipid mediators, such as PGD2 and LTC4; and cytokines, such as TNF-α and IL-5 (5, 22). In this article, we demonstrate that FcγRI aggregation is similarly followed by mast cell degranulation as measured by histamine release (Fig. 1), the de novo synthesis and release of phospholipid-derived mediators (Fig. 2) and the up-regulation of mRNAs for a number of cytokines and chemokines (Fig. 3). We further demonstrate that mRNAs for seven cytokines, including TNF-α, IL-1β, IL-6, IL-1Ra, IL-5, IL-13, and GM-CSF, were significantly up-regulated via aggregation of FcγRI compared with mRNA levels after aggregation of FcεRI (Fig. 3). This demonstration that aggregation of a high affinity IgG receptor on mast cells is followed by the release not only of histamine, but also of arachidonate metabolites, chemokines, and cytokines adds weight to the possibility that mast cells not only participate in innate immunity (14), but also in IgG-mediated immune responses in an IFN-γ-rich microenvironment.
Activation of human mast cells through FcγRI leads to PGD2 and LTC4 production by human mast cells (Fig. 2,b). The maximum release of these lipid mediators by human mast cells after aggregation of either FcγRI or FcεRI was similar. FcγRI aggregation induced a concentration-dependent PGD2 and LTC4 release, reaching 0.21 nmol/106 mast cells and 0.10 nmol/106 mast cells, respectively (Fig. 2,b). Similarly, aggregation of FcεRI caused a concentration-related PGD2 and LTC4 release reaching 0.24 nmol/106 mast cells and 0.08 nmol/106 mast cells, respectively (Fig. 2 a). Upon IgE-mediated activation of human lung mast cells, only a small portion (<5%) of cellular arachidonic acid was reportedly released and converted mainly to PGD2 and LTC4 in approximately equal amounts (0.15 nmol/106 cells) (21, 30, 31). Human skin mast cells produce PGD2 in amounts similar to lung mast cells, but reportedly generate very little LTC4 (32). In this regard, human peripheral blood CD34+-derived mast cells treated with IFN-γ, thus, appear to resemble lung mast cells in lipid mediator profiles.
Aggregation of FcεRI or FcγRI on IFN-γ-treated human mast cells led to an induction or accumulation of mRNAs for some chemokines and cytokines (Fig. 3). These include C-X-C chemokine transcripts, such as IP-10 and IL-8; C-C chemokine transcripts, such as RANTES, MIP-1β, MIP-1α, and monocyte chemotactic protein-1 and I-309; mRNAs for mediators and regulators of innate immunity, such as IL-1α, IL-1β, IL-1Ra, IL-6, and TNF-α; mediators and mRNAs for regulators of specific immunity, such as TGF-β1, IL-5, and IL-13; and mRNAs for mediators and regulators of immature leukocyte growth and differentiation, such as GM-CSF, LIF, M-CSF, OSM, IL-3, G-CSF, and SCF. Among these 22 cytokines and chemokines, seven specific cytokine mRNAs, including TNF-α, IL-1β, IL-5, IL-6, IL-13, IL-1Ra, and GM-CSF, were expressed to a greater degree in mast cells activated via FcγRI aggregation compared with FcεRI aggregation. TNF-α mRNA up-regulation was confirmed by real-time quantitative RT-PCR assays (data not shown). IL-10 and IL-15 mRNAs were weakly expressed and they were not affected by FcγRI or FcεRI cross-linking. IL-2, IL-4, IL-7, IL-9, IL-12p35, IL-12p40, IL-14, IFN-β, IFN-γ, lymphotactin, lymphotoxin-β, TNF-β, TGF-β2, and TGF-β3, mRNAs were not detectable in this assay. In addition, we detected the up-regulation of several cytokine transcripts by both FcγRI and FcεRI aggregation that, to our knowledge, have not been previously described in human mast cells, including phorbol ester-treated human mast cell line 1 (22, 23, 24, 25, 26, 27); i.e., mRNAs for IP-10, LIF, M-CSF, OSM, and G-CSF. Up-regulation of message for all chemokines and cytokines was determined after aggregation of receptors under conditions that lead to maximal histamine release and TNF-α generation (Fig. 4). We cannot rule out the possibility under other culture conditions and using other reagents that maximal up-regulation of message might be seen at less than optimal histamine release and TNF-α production.
To explore in part whether the results of RNase protection assay could predict proteins released from mast cells after FcγRI and FcεRI aggregation, we chose the mast cell cytokine and chemokine, TNF-α and IL-8, for measurement by ELISA (Fig. 4). The results of this assay demonstrated that at least for TNF-α and IL-8, the expressions of mRNAs for these cytokines and for released TNF-α and IL-8 were in agreement (Figs. 3 and 4). Because we have not measured all cytokine and chemokine proteins, we cannot exclude the possibility that some cytokines whose mRNAs are detected may not produce the final protein product.
The observations presented in this article are directed to the demonstration that mast cells exposed to IFN-γ in a Th1-like environment acquire the ability to release histamine and generate other mediators through FcγRI-dependent mechanisms. However, there is another consequence of IFN-γ on human mast cells, which is the down-regulation of FcεRI-mediated responses by IFN-γ (29). Although we have reported that IFN-γ does not affect IgE-mediated histamine release from human mast cells (1), we have confirmed that IFN-γ decreased FcεRI-mediated IL-8 production as has been reported by others (29). We found no change in FcεRI-mediated TNF-α production by IFN-γ, which was not examined in the previous study (29). These data reinforce the conclusion that IFN-γ, by up-regulating FcγRI on human mast cells, allows human mast cells to be recruited by IgG-dependent mechanisms in addition to IgE-dependent mechanisms in a Th1-like environment and that, at least in the case of some cytokines, such as IL-8, IFN-γ may down-regulate FcεRI-mediated responses. In a Th2 environment, only FcεRI-IgE-mediated activation would occur. These observations demonstrate that mast cell responses may occur in both a Th1 and Th2 microenvironment, although responsiveness is modulated through receptor expression and specific mediator production.
FcγRI is expressed on the cell surface in association with the γ-chain (6, 7). The γ-chain cytoplasmic domain contains immunoreceptor tyrosine-based activation motifs (ITAMs) and the γ-chain cytoplasmic domain is both necessary and sufficient for FcγRIα-induced functions (33, 34). Biochemical studies have shown that both cross-linking of FcεRIα-γ-chain complex and the FcγRIα-γ-chain complex result in activation of Src family kinases and the tyrosine kinase p72Syk (4, 35, 36, 37, 38). Biological responses triggered by FcR with ITAMs seem to depend on the cell type more than on the receptor (4). Different receptors with ITAMs triggered the same responses when expressed in the same cell (4). Consequently, biological responses appeared primarily determined by the tissue specificity of FcR. This may explain why some responses via FcγRI were qualitatively indistinguishable from responses stimulated via FcεRI in IFN-γ-treated mast cells. However, we also demonstrate that mRNAs for some cytokines, such as TNF-α, were significantly up-regulated via aggregation of FcγRI compared with mRNA levels after aggregation of FcεRI. With regard to this observation, recent evidence suggests that the cytoplasmic domain of FcγRIα may recruit distinct signaling elements to the receptor complex (20). It has been shown by comparison of responses in wild-type human FcγRIα with a cytoplasmic domain deletion mutant of FcγRIα expressed at comparable levels in stable transfectants of the murine macrophage cell line that the FcγRIα-γ-chain complex-induced responses, such as IL-6 production and phagocytosis, are altered (20). The basis for these differences is still unknown, but this might explain significant enhancement of some cytokine mRNAs expression via FcγRI aggregation compared with FcεRI aggregation.
Thus, we have shown that human mast cells may be activated through FcγRI and induce three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Aggregation of FcγRI led to a significant enhancement of the expression of TNF-α, IL-1β, IL-5, IL-6, IL-13, IL-1Ra, and GM-CSF over that which followed FcεRI aggregation. Our findings of histamine release and TNF-α production in human cultured mast cells after exposed to IFN-γ were reproducible using human lung-derived mature mast cells (Fig. 5). Thus, IFN-γ production associated with specific disease states including bacterial or viral infections (39, 40) and autoimmune disease (41) provides a novel means by which the mast cells may be recruited into inflammation associated with both immunologic and infectious diseases.
This work was supported by the National Institute of Allergy and Infectious Diseases Intramural Program.
Abbreviations used in this paper: LT, leukotriene; IL-1Ra, IL-R antagonist; h, human; SCF, stem cell factor; NP, nitrophenylacetyl; OSM, oncostatin M; IP-10, IFN-γ- induced protein-10; MIP, monocyte-inflammatory protein; ITAM, immunoreceptor tyrosine-based activation motif.
Abbreviations used in this paper: LT, leukotriene; IL-1Ra, IL-R antagonist; h, human; SCF, stem cell factor; NP, nitrophenylacetyl; OSM, oncostatin M; IP-10, IFN-γ-induced protein-10; MIP, monocyte-inflammatory protein; ITAM, immunoreceptor tyrosine-based activation motif.