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Stem cell factor enhances IgE-mediated histamine and TNF-α release from dispersed canine cutaneous mast cells
Veterinary Immunology and Immunopathology 75 (2000) 97±108
Stem cell factor enhances IgE-mediated histamine and TNF-a release from dispersed canine cutaneous mast cells P. BrazõÂsa, M. Queralta, F. de Moraa, Ll. Ferrerb, A. Puigdemonta,* a
Departament de Farmacologia, Universitat AutoÁnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain b Departament de Patologia i Produccio Animals, Universitat AutoÁnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain Received 19 October 1999; received in revised form 30 March 2000; accepted 30 March 2000
Abstract Stem cell factor (SCF), the c-kit receptor ligand, plays a critical role in mast cell (MC) development and differentiation. In addition, SCF has recently been found to both modulate and induce MC activation. To investigate the effect of SCF on canine cutaneous MC function, we have characterized the ability of SCF to modulate the release by mature canine MC of preformed (histamine) and newly generated (TNF-a) mediators. Mature MC were isolated from skin and cultured in the absence or presence of exogenous SCF (6 ng/ml) for up to 5 days and then challenged with anti-IgE (1 mg/ml) alone for 30 min or with a combination of SCF (50 ng/ml) and anti-IgE. SCF alone failed to trigger either histamine or TNF-a release at any time. However, we observed that SCF used as a co-stimulus signi®cantly potentiated histamine and TNF-a release in canine MC activated through FceRI regardless of whether or not SCF was added to the medium during culturing. Thus, the mean histamine release (%) and TNF-a production (pg/ml) were found to be signi®cantly higher if cells were maintained in culture in SCFsupplemented medium compared with cells cultured in the absence of exogenous SCF. We also observed that MC responsiveness to immunological stimulation increased with culture time, the percentage of histamine released being higher in cells cultured for at least 3 days when compared to freshly isolated MC. Taken together these ®ndings suggest that canine skin MC releasability can be enhanced independently either through prolonged incubation with SCF and/or through anti-IgE and SCF co-stimulation. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Canine mast cell; Stem cell factor; Histamine; Tumor necrosis factor
*
Corresponding author. Tel.: 34-93-5811896; fax: 34-93-5812006. E-mail address: [email protected] (A. Puigdemont) 0165-2427/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 0 ) 0 0 1 8 8 - 4
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Abbreviations: DAB, diaminobenzidine; DMEM, Dulbecco's modified Eagle's minimum essential medium; FceRI, high-affinity IgE receptor; FCS, fetal calf serum; PBS, phosphate buffered solution; IgE, immunoglobulin E; MEM, minimum essential medium; MC, mast cells; PCA, perchloric acid; OPT, Ophthaldialdehyde; SCF, stem cell factor; SDS, sodium dodecyl sulfate; TBS, tris-buffered saline solution; TNF-a, tumor necrosis factor-a
1. Introduction The function of cutaneous mast cells (MC) has been widely studied and their central role as immunoregulatory cells in allergic reactions is well known. MC activation through the high af®nity immunoglobulin E (IgE) receptor (FceRI) triggers the immediate release of several preformed mediators and induces the transcription of in¯ammatory cytokines and leukocyte-speci®c chemokines, which are in part responsible for the late phase allergic reaction (Gordon and Galli, 1990). In addition to IgE, other factors have been shown to induce MC activation. A primary growth factor involved in MC function has been identi®ed as the c-kit ligand or stem cell factor (SCF) (Tsai et al., 1991). SCF plays a key role in MC survival and promotes the differentiation and maturation of MC precursors (Tsai et al., 1991; Valent, 1994). Studies carried out in dogs have demonstrated that long-term incubation of canine bone marrow cells in medium supplemented with SCF can increase the survival and proliferation of progenitor cells (Shull et al., 1992). Moreover, Thomas et al. (1996) reported that canine mastocytoma cells incubated over the course of 12 weeks with SCF showed increased granularity and higher expression of TNF-a. In addition to its development-related properties, SCF has the ability to stimulate and upregulate the secretory activity of mature MC stimulated through the FceRI. Studies performed on human cutaneous MC have shown that SCF can trigger the release of preformed (histamine) and de novo synthesized mediators (Columbo et al., 1992). The degranulating activity of SCF on MC was also observed by Wershil et al. (1992) when injected into the skin of mice in vivo. Moreover, in vitro studies have indicated that MC co-stimulation with SCF and anti-IgE can strongly potentiate FceRIdependent activation in rat peritoneal MC (Lin et al., 1996) and human intestinal MC (Bischoff et al., 1997), among other cell populations (Frenz et al., 1997). Since little data are available on mature canine MC, the aim of the present study was ®rstly, to evaluate the effects of recombinant canine SCF as a co-stimulus on FceRIactivated canine skin MC mediator release and secondly, to analyze whether the addition of SCF as a growth factor on a prolonged culture in¯uences the MC integrity and response to immunological stimulation. To evaluate MC secretory response, we have chosen to assess the release of preformed mediators such as histamine, and the production of newly formed cytokines such as TNF-a upon stimulation through the FceRI receptor. For this purpose, we have isolated MC from dog skin and have cultured them for up to 5 days with or without addition of exogenous canine recombinant SCF to the medium. Thus, we hypothesized that culturing and maintaining viable mature skin MC in a SCFsupplemented medium would provide a suitable in vitro model to evaluate the effect of microenvironmental changes in the functional, biochemical and morphological properties of MC.
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2. Materials and methods 2.1. Materials The following chemicals and enzymes were purchased from Sigma. (Madrid, Spain): O-phthaldialdehyde (OPT), histamine diphosphate salt, collagenase (type I), hyaluronidase (type I-S), protease (pronase E, type XIV), bovine albumin (fraction V), soybean trypsin inhibitor (STI), cycloheximide, poly-L-lysine solution and diaminobenzidine (DAB). Penicillin±Streptomycin, Dulbecco's modi®ed Eagle's minimum essential medium (DMEM), fetal calf serum (FCS), glutamine, histidine, trypsin-EDTA and sodium dodecyl sulfate (SDS) were obtained from Gibco (Life Technologies, Barcelona, Spain). Avidin/Biotin blocking kit and Vectastain ABC kit were purchased from Vector Laboratories (Burlingame, Canada). Polyclonal anti-canine IgE was purchased from Bethyl Laboratories (Montgomery, TX, USA). Recombinant murine TNF-a and polyclonal antibody anti-TNF-a were obtained from Genzyme (Cambridge, MA, USA). Recombinant canine SCF was kindly provided by Dr. Morstyn (Amgen, Thousand Oaks, CA, USA). 2.1.1. Isolation and culture of skin MC Skin biopsies were obtained from the abdominal area of dogs euthanized in the Animal Care Facility of Barcelona. MC were isolated by means of a procedure developed in our laboratory and described in detail elsewhere (de Mora et al., 1993). Brie¯y, skin samples were chopped into 0.5±1 mm3 fragments, after removal of the subcutaneous fat tissue. The skin fragments were washed twice by centrifugation (400 g, 10 min) and then were enzymatically digested for 180 min in 15 ml of MEM per gram of skin containing 22.3 mg of collagenase, 18 mg of hyaluronidase and 12 mg of protease supplemented with bovine albumin and antibiotics. After digestion, the cutaneous cells were ®ltered, washed once with MEM, and resuspended in DMEM supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamycin and 5% FCS. Cells were then incubated with or without exogenous SCF (6 ng/ml) for up to 5 days in histamine release experiments, and for up to 4 days for TNF-a determination. 2.1.2. Sensitization and activation of cutaneous MC For histamine release experiments, adherent cells were harvested with trypsin-EDTA and counted by staining with Kimura's metachromatic stain (Kimura et al., 1979). The viability was assessed by trypan blue dye being always higher than 90%. MC were then incubated in MEM (378C, 2 h) containing 5% IgE-rich serum from Beagle dogs spontaneously sensitized to Ascaris suum to achieve maximal occupancy of the IgE receptors. After washing, MC were resuspended in PBS containing 1 mM Ca2 and 1 mM Mg2 and distributed in aliquots (2104 MC/500 ml). Cells were stimulated either immediately after the digestion process or after 3 or 5 days of culturing. For MC stimulation, cells were preincubated with or without SCF (50 ng/ml) for 15 min followed by addition of anti-canine IgE (1 mg/ml) for 30 min. Some cells were also stimulated with SCF alone (10, 50, 100 ng/ml) for 30 min. The 15 min preincubation period with SCF was chosen based on other studies performed on skin MC (Columbo et al., 1992). We selected 50 ng/ml of SCF as an optimal concentration to use in co-stimulation with anti-
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IgE based on a preliminary experiment in which several concentrations of SCF (10, 20, 50, 100 ng/ml) were tested alone or in combination with anti-IgE on MC cultured overnight. MC stimulation was halted at 08C and supernatants were centrifuged at 2630 g, 20 min at 48C and mixed with 0.5 ml of 0.8 N perchloric acid (PCA) to precipitate proteins. The samples were then centrifuged at 10900 g for 4 min before the histamine assay was performed. To assess TNF-a production, harvested cells were resuspended in DMEM containing STI (200 mg/ml) in the absence of FCS and were activated in 96 well plate aliquots (2104 MC/100 ml) with anti-IgE (1 mg/ml) alone or after a 15 min preincubation-period with SCF (50 ng/ml). Cell supernatants were collected at different times after stimulation (1, 2, 4, 6 and 8 h) to assess TNF-a activity by a cytotoxicity bioassay. All experiments were performed in duplicate. 2.1.3. Histamine release assay The classic spectro¯uorimetric method of Shore et al. (1959) with modi®cations (Sullivan et al., 1975) was used. Brie¯y, the samples were transferred to tubes containing 0.6 g of NaCl, to which 1 ml PCA (0.4 N), 0.25 ml NaOH (5 N) and 2 ml butanol were added. Samples were mixed and centrifuged at 600 g for 10 min at 208C and the upper organic phase was transferred to tubes containing 1.5 ml of NaOH (0.1 N) and stirred; 1.3 ml of HCl (0.1 N) and 1.9 ml of n-heptane were added to the organic phase and mixed. The phases were separated and 1 ml of the lower aqueous phase was made alkaline (pH 12.5) with 200 ml of NaOH (0.1 N). The histamine derivative was formed by incubating the samples at room temperature with 50 ml of OPT (10 mg/ml methanol) for 4 min. The histamine-OPT reaction was halted at pH 3.5 by addition of 50 ml of H3PO4 (2.5 N) and ¯uorescence was assessed at 345 nm excitation and 435 nm emission wavelengths. 2.1.4. TNF-a cytotoxicity bioassay TNF-a activity was assessed in cell supernatants using a cytotoxicity bioassay modi®ed from a method previously described (Gordon and Galli, 1991) employing the TNF-a-sensitive L929 (mouse ®broblast) cell line. Brie¯y, 50 ml/well of 1106 L929 cells/ml were seeded into ¯at-bottom plates and cultured overnight in RPMI medium supplemented with 5% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin and 1% Hepes 1 M at 378C. The medium was then replaced with 50 ml/well of fresh medium containing 5 mg/ml of cycloheximide and 100 mg/ml of STI. Mouse recombinant TNF-a was used to construct a standard curve ranging from 2 to 2000 pg/ml. Thereafter, 50 ml/well amounts of samples and standards were added, and plates were incubated for 18 h at 378C. Then 10 ml/well of MTT (5 mg/ml) was added, and after a 4 h incubation, 50 ml of a solution of 50% N, N-dimethylformamide and 20% SDS, pH 7.4, was added to dissolve the MTT and the mixture was incubated overnight. The plates were read in an ELISA reader at 570 nm, and results were extrapolated from the standard curve constructed using recombinant murine TNF-a. In order to verify the sensitivity of the bioassay to TNF-a, we attempted to neutralize its bioactivity with a rabbit polyclonal antibody against human TNF-a, which has been shown to cross react with canine TNF-a. The addition of 1 ml of rabbit anti-human TNF-a to 99 ml of the cell supernatant sample led to 100% neutralization of the canine TNF-a
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activity. In contrast, the addition of 1 ml of normal rabbit serum had no effect on the supernatant TNF-a activity in the L929 assay. 2.1.5. Immunocytochemistry Immunostaining was performed to identify the cellular source of TNF-a. Dermal cells were activated with anti-IgE for 2 h in the presence of brefeldin A (10 mg/ml) to allow for accumulation of protein in the cytoplasm. The cell suspension was cytocentrifuged into FCS and poly-L-lysine coated slides, air dried and ®xed in cold acetone. Endogenous peroxidase activity was blocked by incubating slides in 0.1% hydrogen peroxide in methanol. After washing with TBS, endogenous biotin activity was quenched with the avidin/biotin blocking kit for 30 min. Non-speci®c binding was blocked by incubation with normal goat serum. Cells were then incubated overnight at 48C with 100 ml of rabbit polyclonal anti-hTNF-a antibody at 1:500 dilution. After repeated washings with TBS, slides were then incubated with biotinylated goat anti-rabbit IgG for 1 h, washed, and incubated with avidin±biotinylated horseradish peroxidase complex for 1 h at room temperature. Slides were then washed with TBS and DAB was used as a chromogen. Slides were counterstained either with hematoxylin or with alcian blue. Appropriate controls were used to check for non-speci®c binding of the primary antibody. 2.1.6. Data analysis Results are expressed as the arithmetical meanstandard error (meanSEM) of six experiments. Differences between means were tested for signi®cance by a Student's t-test at a level of signi®cance of 0.05. Histamine release was expressed as the net result of subtracting spontaneously-released histamine from the histamine secreted after stimulation with anti-IgE. 3. Results 3.1. Effect of SCF on IgE-dependent histamine release after short-term preincubation As shown in Fig. 1, when cells were activated with SCF alone (10, 50, 100 ng/ml) no histamine release was observed. MC stimulated with anti-IgE released a mean 24.002.4% of their total cellular histamine content. However, in MC preincubated with SCF at concentrations ranging from 10 to 100 ng/ml for 15 min and then costimulated with anti-IgE (1 mg/ml), histamine release increased in a concentrationdependent manner. When cells were preincubated with 50 ng/ml of SCF followed by 1 mg/ml of anti-IgE, the percentage of histamine released reached a plateau. Therefore, 50 ng/ml of SCF was selected as the optimal co-stimulating concentration to be used in the following experiments. 3.2. Effect of SCF on histamine release in freshly isolated and cultured skin MC Fig. 2 shows the net percentage of histamine released from MC cultured for 5 days in medium without exogenous SCF. Freshly isolated MC (0 days) triggered upon anti-IgE alone released 14.231.9% of their total cellular histamine content, and the mean
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Fig. 1. Effect of SCF on anti-IgE-induced histamine release from dispersed canine skin MC. The cells were preincubated with SCF (10, 20, 50, 100 ng/ml) for 15 min and then challenged with anti-IgE. Each bar represents the meanSEM of six independent experiments.
Fig. 2. Histamine release from dispersed skin MC cultured in absence of exogenous SCF, and stimulated upon 50 ng/ml of SCF (black bars), 1 mg/ml of anti-IgE (white bars) or co-stimulated with 50 ng/ml of SCF (15 min) and 1 mg/ml of anti-IgE (grey bars). Each bar represents the meanSEM of six independent experiments (*p<0.05 by comparison with cells not co-stimulated with SCF).
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Fig. 3. Histamine release from dispersed skin MC cultured in presence of exogenous SCF (6 ng/ml), and stimulated upon 50 ng/ml of SCF (black bars), 1 mg/ml of anti-IgE (white bars) or co-stimulated with 50 ng/ml of SCF (15 min) and 1 mg/ml of anti-IgE (grey bars). Each bar represents the meanSEM of six independent experiments (*p<0.05 by comparison with cells not co-stimulated with SCF).
percentage of histamine release signi®cantly increased throughout the days of culture, to reach 26.433.5 and 35.452.3% at 3 and 5 days of culture, respectively. MC preincubation with SCF (50 ng/ml) for 15 min before stimulation with anti-IgE (1 mg/ml) enhanced the histamine release in even freshly isolated (from 14.23 to 21.884.2%) and cultured MC (from 26.43 to 40.485.5% and from 35.45 to 46.74.7% at 3 and 5 days of culture). Stem cell factor by itself had no stimulatory effect. As shown in Fig. 3, an increase in histamine release from MC cultured 5 days in the presence of exogenous SCF (6 ng/ml) was observed throughout the culture time in cells triggered through FceRI (14.231.9% in freshly isolated MC, 38.105.1% after 3 days culture and 46.811.9% after 5 days culture). Again, preincubation with SCF (50 ng/ml) before stimulation with anti-IgE signi®cantly enhanced mediator secretion and led to 21.884.2% of histamine release in freshly isolated MC, and 56.965.2 and 58.155.2% in MC cultured for 3 and 5 days, respectively. Mean percentage of histamine release at each time point was found to be signi®cantly higher if cells were cultured in SCF-supplemented medium compared with cells cultured in absence of exogenous SCF (Fig. 2 versus Fig. 3). 3.3. Effect of SCF on TNF-a production of cultured skin mast cells MC activated with SCF alone (50 and 100 ng/ml) failed to produce TNF-a at any time. Fig. 4 shows TNF-a production (pg/ml) of MC cultured for 4 days without exogenous
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Fig. 4. Time course TNF-a production on dispersed skin MC cultured for 4 days in absence of exogenous SCF and stimulated upon 50 ng/ml of SCF (black bars), 1 mg/ml of anti-IgE (white bars) or co-stimulated with 50 ng/ ml of SCF (15 min) and 1 mg/ml of anti-IgE (grey bars). Each bar represents the meanSEM of six independent experiments (*p<0.05 by comparison with cells not co-stimulated with SCF).
supplementation of SCF. When MC were activated through the FceRI receptor, a timedependent increase in the TNF-a production was observed and maximal TNF-a production (1.780.8 pg/ml) was achieved 2 h after stimulation with anti-IgE. When the same cells were preincubated with SCF (50 ng/ml) for 15 min before activation, IgEdependent TNF-a production was signi®cantly enhanced, and the maximal TNF-a production (7.601.5 pg/ml) was achieved 6 h post-stimulation. Fig. 5 shows that cells cultured for 4 days in presence of exogenous SCF, produced a mean of 8.510.7 pg/ml of TNF-a after 4 h of stimulation with anti-IgE, and at the same time point, preincubation with SCF for 15 min signi®cantly enhanced the TNF-a production to 13.541.6 pg/ml. The production of TNF-a in these cells was two times higher in comparison with cytokine production in MC cultured in a non-supplemented medium (Fig. 4 versus Fig. 5). 3.4. Immunocytochemistry Although slight background staining was observed, the anti-TNF-a antibody, revealed a speci®c reaction when compared to a matched isotype control antibody. Double stained cells, i.e. cells sequentially stained with MC-speci®c alcian blue and immunostaining for TNF-a, indicated that 99% of MC expressed TNF-a in addition to other dispersed canine dermal cells, showing that MC may have been, at least partly, the source of released TNFa identi®ed in the supernatant through the bioassay. No matter what the cell origin was, however, the release of this cytokine was certainly achieved through a MC-mediated mechanism.
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Fig. 5. Time course TNF-a production on dispersed skin MC cultured for 4 days in presence of exogenous SCF (6 ng/ml) and stimulated upon 50 ng/ml of SCF (black bars), 1 mg/ml of anti-IgE (white bars) or co-stimulated with 50 ng/ml of SCF (15 min) and 1 mg/ml of anti-IgE (grey bars). Each bar represents the meanSEM of six independent experiments (*p<0.05 by comparison with cells not co-stimulated with SCF).
4. Discussion We have shown that SCF has the ability to enhance FceRI-mediated degranulation and cytokine production in mature canine cutaneous MC. The enhancing effect of SCF was analyzed at two levels: ®rstly, for an enduring growth/survival effect exerted through the culture of MC with SCF; and secondly, for a co-stimulatory effect observed through simultaneous activation of MC with anti-IgE and SCF. In addition, regardless of the presence or absence of exogenous SCF during the culture, there was a positive correlation between MC histamine releasability and days of culture, an effect possibly mediated by endogenous SCF production and release. In mature skin MC cultured for up to 5 days in a non SCF-supplemented medium, MC histamine secretion gradually increased with culture time. The presence of interstitial cells such as ®broblasts in the MC culture, probably a source of soluble or membranebound SCF, may account for the differences in histamine releasability between cultured and freshly isolated MC. Moreover, the time-dependent increased responsiveness of MC, also investigated by Taylor et al. (1995) in a 48 h study, may re¯ect the fact that cells function properties recovered during culturing, after the aggressive enzymatic isolation process, recovery possibly being at least partly mediated by SCF. In any event, the higher responsiveness together with the high viability of the canine dermal MC primary cultures sustained for up to 5 days, indicates that the method used is an appropriate in vitro model for investigating the effect of modulatory compounds on mature MC activity. Based on
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this observation, we conclude that we did indeed investigate the effect of SCF on our prolonged MC culture system. To investigate the potential role of SCF as a MC survival, growth or regenerating factor, the isolated dermal cells were cultured and studied from days 0 to 5 in medium supplemented with 6 ng/ml of recombinant canine SCF and then studied. The results reported in Fig. 3 show that MC cultured in SCF-supplemented medium, released a higher percentage of histamine at all points in time when compared to cells cultured without exogenous SCF, regardless of whether additional SCF was present or not during the anti-IgE stimulation. Supplementing the culture with SCF probably either improved isolated dermal MC survival and/or further contributed to the restoration of granule mediators. A similar SCF-dependent enhancing effect was observed on MC-mediated TNF-a production and release. TNF-a was shown to be present in skin MC by immunohistochemistry. However, we were not able to detect pre-formed TNF-a in the granules of skin MC (no TNF-a present in dermal MC pellet from non-stimulated cells), suggesting that most of the cytokine released into the medium was newly synthesized upon stimulation. Granule restoration was therefore probably not responsible, in this case, for the increased TNF-a release observed during MC incubation with SCF. In addition, whereas the maximum effect of SCF on histamine release was an 1.5-fold increase, TNF-a release was enhanced up to six times over baseline. The powerful effect of SCF, as a supplementary factor added to the culture medium, on TNF-a production regardless of its presence during stimulation, could re¯ect an overall MC recovery affecting both early and particularly late phase signaling mechanisms. SCF by itself was not capable of triggering MC mediator release in our study. Although Valent et al. demonstrated in 1992 that SCF is an IgE-independent activator of mature tissue MC, no detectable mediator release and cytokine production was observed on either fresh or cultured canine MC challenged with SCF alone. However, when SCF was used as a co-stimulus with anti-IgE, both pre-formed and newly synthesized mediator release was strongly potentiated, and differences were statistically signi®cant at SCF concentrations higher to 20 ng/ml. Again, in our system the effect of SCF used as a costimulator was more potent on TNF-a than on histamine release, suggesting that SCF had a powerful inducing effect (highest 2±6 h post-stimulation) at the gene transcription or protein synthesis levels, in addition to its potential effect on the early signaling cytoplasmic effects. In contrast to our results were those of Lin et al. (1996) who reported a lack of effect of SCF on TNF-a synthesis. Ishuzuka et al. (1998), however, recently showed that the FceRI-mediated TNF-a production is regulated by SCF at the level of cytokine transcription, consistent with our ®ndings. In the same study, the authors investigated the mechanism by which SCF is able to augment FceRI-stimulation of MC, concluding that SCF and anti-IgE induce the activation of similar members of the mitogen-activated protein kinase family. Interestingly, we observed that after culturing the cells in medium supplemented with SCF, the co-stimulatory effect of SCF on MC was not as high as when no SCF was added during culture. This phenomenon was particularly visible when TNF-a production was assessed. We speculate that SCF receptor desensitization on the MC surface may be occurring over the period of culturing, and may be more marked in MC being maintained in medium containing a higher SCF concentration. Consistent with this hypothesis is a
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report by Columbo et al. (1992) showing that 24 h incubation of human skin MC in an SCF-supplemented medium attenuated the potentiating effect of SCF as a MC costimulus. That observation, taken together with ours, implies the existence of interesting new similarities between human and canine MC. Finally, the marked effect of SCF on both MC survival and MC functionality confers to this growth factor a potential role in some pathologic skin conditions (e.g. atopic dermatitis) which are characterized by tissue MC accumulation and increased MC mediator release. Through these mechanisms, an increase in SCF concentration in the skin could contribute to both, MC activation and maintenance of the allergic skin responses through the enhanced release of histamine and TNF-a, one of the most important cytokines in the late phase allergic reactions.
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Stem cell factor enhances IgE-mediated histamine and TNF-a release from dispersed canine cutaneous mast cells P. BrazõÂsa, M. Queralta, F. de Moraa, Ll. Ferrerb, A. Puigdemonta,* a
Departament de Farmacologia, Universitat AutoÁnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain b Departament de Patologia i Produccio Animals, Universitat AutoÁnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain Received 19 October 1999; received in revised form 30 March 2000; accepted 30 March 2000
Abstract Stem cell factor (SCF), the c-kit receptor ligand, plays a critical role in mast cell (MC) development and differentiation. In addition, SCF has recently been found to both modulate and induce MC activation. To investigate the effect of SCF on canine cutaneous MC function, we have characterized the ability of SCF to modulate the release by mature canine MC of preformed (histamine) and newly generated (TNF-a) mediators. Mature MC were isolated from skin and cultured in the absence or presence of exogenous SCF (6 ng/ml) for up to 5 days and then challenged with anti-IgE (1 mg/ml) alone for 30 min or with a combination of SCF (50 ng/ml) and anti-IgE. SCF alone failed to trigger either histamine or TNF-a release at any time. However, we observed that SCF used as a co-stimulus signi®cantly potentiated histamine and TNF-a release in canine MC activated through FceRI regardless of whether or not SCF was added to the medium during culturing. Thus, the mean histamine release (%) and TNF-a production (pg/ml) were found to be signi®cantly higher if cells were maintained in culture in SCFsupplemented medium compared with cells cultured in the absence of exogenous SCF. We also observed that MC responsiveness to immunological stimulation increased with culture time, the percentage of histamine released being higher in cells cultured for at least 3 days when compared to freshly isolated MC. Taken together these ®ndings suggest that canine skin MC releasability can be enhanced independently either through prolonged incubation with SCF and/or through anti-IgE and SCF co-stimulation. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Canine mast cell; Stem cell factor; Histamine; Tumor necrosis factor
*
Corresponding author. Tel.: 34-93-5811896; fax: 34-93-5812006. E-mail address: [email protected] (A. Puigdemont) 0165-2427/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 0 ) 0 0 1 8 8 - 4
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Abbreviations: DAB, diaminobenzidine; DMEM, Dulbecco's modified Eagle's minimum essential medium; FceRI, high-affinity IgE receptor; FCS, fetal calf serum; PBS, phosphate buffered solution; IgE, immunoglobulin E; MEM, minimum essential medium; MC, mast cells; PCA, perchloric acid; OPT, Ophthaldialdehyde; SCF, stem cell factor; SDS, sodium dodecyl sulfate; TBS, tris-buffered saline solution; TNF-a, tumor necrosis factor-a
1. Introduction The function of cutaneous mast cells (MC) has been widely studied and their central role as immunoregulatory cells in allergic reactions is well known. MC activation through the high af®nity immunoglobulin E (IgE) receptor (FceRI) triggers the immediate release of several preformed mediators and induces the transcription of in¯ammatory cytokines and leukocyte-speci®c chemokines, which are in part responsible for the late phase allergic reaction (Gordon and Galli, 1990). In addition to IgE, other factors have been shown to induce MC activation. A primary growth factor involved in MC function has been identi®ed as the c-kit ligand or stem cell factor (SCF) (Tsai et al., 1991). SCF plays a key role in MC survival and promotes the differentiation and maturation of MC precursors (Tsai et al., 1991; Valent, 1994). Studies carried out in dogs have demonstrated that long-term incubation of canine bone marrow cells in medium supplemented with SCF can increase the survival and proliferation of progenitor cells (Shull et al., 1992). Moreover, Thomas et al. (1996) reported that canine mastocytoma cells incubated over the course of 12 weeks with SCF showed increased granularity and higher expression of TNF-a. In addition to its development-related properties, SCF has the ability to stimulate and upregulate the secretory activity of mature MC stimulated through the FceRI. Studies performed on human cutaneous MC have shown that SCF can trigger the release of preformed (histamine) and de novo synthesized mediators (Columbo et al., 1992). The degranulating activity of SCF on MC was also observed by Wershil et al. (1992) when injected into the skin of mice in vivo. Moreover, in vitro studies have indicated that MC co-stimulation with SCF and anti-IgE can strongly potentiate FceRIdependent activation in rat peritoneal MC (Lin et al., 1996) and human intestinal MC (Bischoff et al., 1997), among other cell populations (Frenz et al., 1997). Since little data are available on mature canine MC, the aim of the present study was ®rstly, to evaluate the effects of recombinant canine SCF as a co-stimulus on FceRIactivated canine skin MC mediator release and secondly, to analyze whether the addition of SCF as a growth factor on a prolonged culture in¯uences the MC integrity and response to immunological stimulation. To evaluate MC secretory response, we have chosen to assess the release of preformed mediators such as histamine, and the production of newly formed cytokines such as TNF-a upon stimulation through the FceRI receptor. For this purpose, we have isolated MC from dog skin and have cultured them for up to 5 days with or without addition of exogenous canine recombinant SCF to the medium. Thus, we hypothesized that culturing and maintaining viable mature skin MC in a SCFsupplemented medium would provide a suitable in vitro model to evaluate the effect of microenvironmental changes in the functional, biochemical and morphological properties of MC.
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2. Materials and methods 2.1. Materials The following chemicals and enzymes were purchased from Sigma. (Madrid, Spain): O-phthaldialdehyde (OPT), histamine diphosphate salt, collagenase (type I), hyaluronidase (type I-S), protease (pronase E, type XIV), bovine albumin (fraction V), soybean trypsin inhibitor (STI), cycloheximide, poly-L-lysine solution and diaminobenzidine (DAB). Penicillin±Streptomycin, Dulbecco's modi®ed Eagle's minimum essential medium (DMEM), fetal calf serum (FCS), glutamine, histidine, trypsin-EDTA and sodium dodecyl sulfate (SDS) were obtained from Gibco (Life Technologies, Barcelona, Spain). Avidin/Biotin blocking kit and Vectastain ABC kit were purchased from Vector Laboratories (Burlingame, Canada). Polyclonal anti-canine IgE was purchased from Bethyl Laboratories (Montgomery, TX, USA). Recombinant murine TNF-a and polyclonal antibody anti-TNF-a were obtained from Genzyme (Cambridge, MA, USA). Recombinant canine SCF was kindly provided by Dr. Morstyn (Amgen, Thousand Oaks, CA, USA). 2.1.1. Isolation and culture of skin MC Skin biopsies were obtained from the abdominal area of dogs euthanized in the Animal Care Facility of Barcelona. MC were isolated by means of a procedure developed in our laboratory and described in detail elsewhere (de Mora et al., 1993). Brie¯y, skin samples were chopped into 0.5±1 mm3 fragments, after removal of the subcutaneous fat tissue. The skin fragments were washed twice by centrifugation (400 g, 10 min) and then were enzymatically digested for 180 min in 15 ml of MEM per gram of skin containing 22.3 mg of collagenase, 18 mg of hyaluronidase and 12 mg of protease supplemented with bovine albumin and antibiotics. After digestion, the cutaneous cells were ®ltered, washed once with MEM, and resuspended in DMEM supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamycin and 5% FCS. Cells were then incubated with or without exogenous SCF (6 ng/ml) for up to 5 days in histamine release experiments, and for up to 4 days for TNF-a determination. 2.1.2. Sensitization and activation of cutaneous MC For histamine release experiments, adherent cells were harvested with trypsin-EDTA and counted by staining with Kimura's metachromatic stain (Kimura et al., 1979). The viability was assessed by trypan blue dye being always higher than 90%. MC were then incubated in MEM (378C, 2 h) containing 5% IgE-rich serum from Beagle dogs spontaneously sensitized to Ascaris suum to achieve maximal occupancy of the IgE receptors. After washing, MC were resuspended in PBS containing 1 mM Ca2 and 1 mM Mg2 and distributed in aliquots (2104 MC/500 ml). Cells were stimulated either immediately after the digestion process or after 3 or 5 days of culturing. For MC stimulation, cells were preincubated with or without SCF (50 ng/ml) for 15 min followed by addition of anti-canine IgE (1 mg/ml) for 30 min. Some cells were also stimulated with SCF alone (10, 50, 100 ng/ml) for 30 min. The 15 min preincubation period with SCF was chosen based on other studies performed on skin MC (Columbo et al., 1992). We selected 50 ng/ml of SCF as an optimal concentration to use in co-stimulation with anti-
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IgE based on a preliminary experiment in which several concentrations of SCF (10, 20, 50, 100 ng/ml) were tested alone or in combination with anti-IgE on MC cultured overnight. MC stimulation was halted at 08C and supernatants were centrifuged at 2630 g, 20 min at 48C and mixed with 0.5 ml of 0.8 N perchloric acid (PCA) to precipitate proteins. The samples were then centrifuged at 10900 g for 4 min before the histamine assay was performed. To assess TNF-a production, harvested cells were resuspended in DMEM containing STI (200 mg/ml) in the absence of FCS and were activated in 96 well plate aliquots (2104 MC/100 ml) with anti-IgE (1 mg/ml) alone or after a 15 min preincubation-period with SCF (50 ng/ml). Cell supernatants were collected at different times after stimulation (1, 2, 4, 6 and 8 h) to assess TNF-a activity by a cytotoxicity bioassay. All experiments were performed in duplicate. 2.1.3. Histamine release assay The classic spectro¯uorimetric method of Shore et al. (1959) with modi®cations (Sullivan et al., 1975) was used. Brie¯y, the samples were transferred to tubes containing 0.6 g of NaCl, to which 1 ml PCA (0.4 N), 0.25 ml NaOH (5 N) and 2 ml butanol were added. Samples were mixed and centrifuged at 600 g for 10 min at 208C and the upper organic phase was transferred to tubes containing 1.5 ml of NaOH (0.1 N) and stirred; 1.3 ml of HCl (0.1 N) and 1.9 ml of n-heptane were added to the organic phase and mixed. The phases were separated and 1 ml of the lower aqueous phase was made alkaline (pH 12.5) with 200 ml of NaOH (0.1 N). The histamine derivative was formed by incubating the samples at room temperature with 50 ml of OPT (10 mg/ml methanol) for 4 min. The histamine-OPT reaction was halted at pH 3.5 by addition of 50 ml of H3PO4 (2.5 N) and ¯uorescence was assessed at 345 nm excitation and 435 nm emission wavelengths. 2.1.4. TNF-a cytotoxicity bioassay TNF-a activity was assessed in cell supernatants using a cytotoxicity bioassay modi®ed from a method previously described (Gordon and Galli, 1991) employing the TNF-a-sensitive L929 (mouse ®broblast) cell line. Brie¯y, 50 ml/well of 1106 L929 cells/ml were seeded into ¯at-bottom plates and cultured overnight in RPMI medium supplemented with 5% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin and 1% Hepes 1 M at 378C. The medium was then replaced with 50 ml/well of fresh medium containing 5 mg/ml of cycloheximide and 100 mg/ml of STI. Mouse recombinant TNF-a was used to construct a standard curve ranging from 2 to 2000 pg/ml. Thereafter, 50 ml/well amounts of samples and standards were added, and plates were incubated for 18 h at 378C. Then 10 ml/well of MTT (5 mg/ml) was added, and after a 4 h incubation, 50 ml of a solution of 50% N, N-dimethylformamide and 20% SDS, pH 7.4, was added to dissolve the MTT and the mixture was incubated overnight. The plates were read in an ELISA reader at 570 nm, and results were extrapolated from the standard curve constructed using recombinant murine TNF-a. In order to verify the sensitivity of the bioassay to TNF-a, we attempted to neutralize its bioactivity with a rabbit polyclonal antibody against human TNF-a, which has been shown to cross react with canine TNF-a. The addition of 1 ml of rabbit anti-human TNF-a to 99 ml of the cell supernatant sample led to 100% neutralization of the canine TNF-a
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activity. In contrast, the addition of 1 ml of normal rabbit serum had no effect on the supernatant TNF-a activity in the L929 assay. 2.1.5. Immunocytochemistry Immunostaining was performed to identify the cellular source of TNF-a. Dermal cells were activated with anti-IgE for 2 h in the presence of brefeldin A (10 mg/ml) to allow for accumulation of protein in the cytoplasm. The cell suspension was cytocentrifuged into FCS and poly-L-lysine coated slides, air dried and ®xed in cold acetone. Endogenous peroxidase activity was blocked by incubating slides in 0.1% hydrogen peroxide in methanol. After washing with TBS, endogenous biotin activity was quenched with the avidin/biotin blocking kit for 30 min. Non-speci®c binding was blocked by incubation with normal goat serum. Cells were then incubated overnight at 48C with 100 ml of rabbit polyclonal anti-hTNF-a antibody at 1:500 dilution. After repeated washings with TBS, slides were then incubated with biotinylated goat anti-rabbit IgG for 1 h, washed, and incubated with avidin±biotinylated horseradish peroxidase complex for 1 h at room temperature. Slides were then washed with TBS and DAB was used as a chromogen. Slides were counterstained either with hematoxylin or with alcian blue. Appropriate controls were used to check for non-speci®c binding of the primary antibody. 2.1.6. Data analysis Results are expressed as the arithmetical meanstandard error (meanSEM) of six experiments. Differences between means were tested for signi®cance by a Student's t-test at a level of signi®cance of 0.05. Histamine release was expressed as the net result of subtracting spontaneously-released histamine from the histamine secreted after stimulation with anti-IgE. 3. Results 3.1. Effect of SCF on IgE-dependent histamine release after short-term preincubation As shown in Fig. 1, when cells were activated with SCF alone (10, 50, 100 ng/ml) no histamine release was observed. MC stimulated with anti-IgE released a mean 24.002.4% of their total cellular histamine content. However, in MC preincubated with SCF at concentrations ranging from 10 to 100 ng/ml for 15 min and then costimulated with anti-IgE (1 mg/ml), histamine release increased in a concentrationdependent manner. When cells were preincubated with 50 ng/ml of SCF followed by 1 mg/ml of anti-IgE, the percentage of histamine released reached a plateau. Therefore, 50 ng/ml of SCF was selected as the optimal co-stimulating concentration to be used in the following experiments. 3.2. Effect of SCF on histamine release in freshly isolated and cultured skin MC Fig. 2 shows the net percentage of histamine released from MC cultured for 5 days in medium without exogenous SCF. Freshly isolated MC (0 days) triggered upon anti-IgE alone released 14.231.9% of their total cellular histamine content, and the mean
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Fig. 1. Effect of SCF on anti-IgE-induced histamine release from dispersed canine skin MC. The cells were preincubated with SCF (10, 20, 50, 100 ng/ml) for 15 min and then challenged with anti-IgE. Each bar represents the meanSEM of six independent experiments.
Fig. 2. Histamine release from dispersed skin MC cultured in absence of exogenous SCF, and stimulated upon 50 ng/ml of SCF (black bars), 1 mg/ml of anti-IgE (white bars) or co-stimulated with 50 ng/ml of SCF (15 min) and 1 mg/ml of anti-IgE (grey bars). Each bar represents the meanSEM of six independent experiments (*p<0.05 by comparison with cells not co-stimulated with SCF).
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Fig. 3. Histamine release from dispersed skin MC cultured in presence of exogenous SCF (6 ng/ml), and stimulated upon 50 ng/ml of SCF (black bars), 1 mg/ml of anti-IgE (white bars) or co-stimulated with 50 ng/ml of SCF (15 min) and 1 mg/ml of anti-IgE (grey bars). Each bar represents the meanSEM of six independent experiments (*p<0.05 by comparison with cells not co-stimulated with SCF).
percentage of histamine release signi®cantly increased throughout the days of culture, to reach 26.433.5 and 35.452.3% at 3 and 5 days of culture, respectively. MC preincubation with SCF (50 ng/ml) for 15 min before stimulation with anti-IgE (1 mg/ml) enhanced the histamine release in even freshly isolated (from 14.23 to 21.884.2%) and cultured MC (from 26.43 to 40.485.5% and from 35.45 to 46.74.7% at 3 and 5 days of culture). Stem cell factor by itself had no stimulatory effect. As shown in Fig. 3, an increase in histamine release from MC cultured 5 days in the presence of exogenous SCF (6 ng/ml) was observed throughout the culture time in cells triggered through FceRI (14.231.9% in freshly isolated MC, 38.105.1% after 3 days culture and 46.811.9% after 5 days culture). Again, preincubation with SCF (50 ng/ml) before stimulation with anti-IgE signi®cantly enhanced mediator secretion and led to 21.884.2% of histamine release in freshly isolated MC, and 56.965.2 and 58.155.2% in MC cultured for 3 and 5 days, respectively. Mean percentage of histamine release at each time point was found to be signi®cantly higher if cells were cultured in SCF-supplemented medium compared with cells cultured in absence of exogenous SCF (Fig. 2 versus Fig. 3). 3.3. Effect of SCF on TNF-a production of cultured skin mast cells MC activated with SCF alone (50 and 100 ng/ml) failed to produce TNF-a at any time. Fig. 4 shows TNF-a production (pg/ml) of MC cultured for 4 days without exogenous
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Fig. 4. Time course TNF-a production on dispersed skin MC cultured for 4 days in absence of exogenous SCF and stimulated upon 50 ng/ml of SCF (black bars), 1 mg/ml of anti-IgE (white bars) or co-stimulated with 50 ng/ ml of SCF (15 min) and 1 mg/ml of anti-IgE (grey bars). Each bar represents the meanSEM of six independent experiments (*p<0.05 by comparison with cells not co-stimulated with SCF).
supplementation of SCF. When MC were activated through the FceRI receptor, a timedependent increase in the TNF-a production was observed and maximal TNF-a production (1.780.8 pg/ml) was achieved 2 h after stimulation with anti-IgE. When the same cells were preincubated with SCF (50 ng/ml) for 15 min before activation, IgEdependent TNF-a production was signi®cantly enhanced, and the maximal TNF-a production (7.601.5 pg/ml) was achieved 6 h post-stimulation. Fig. 5 shows that cells cultured for 4 days in presence of exogenous SCF, produced a mean of 8.510.7 pg/ml of TNF-a after 4 h of stimulation with anti-IgE, and at the same time point, preincubation with SCF for 15 min signi®cantly enhanced the TNF-a production to 13.541.6 pg/ml. The production of TNF-a in these cells was two times higher in comparison with cytokine production in MC cultured in a non-supplemented medium (Fig. 4 versus Fig. 5). 3.4. Immunocytochemistry Although slight background staining was observed, the anti-TNF-a antibody, revealed a speci®c reaction when compared to a matched isotype control antibody. Double stained cells, i.e. cells sequentially stained with MC-speci®c alcian blue and immunostaining for TNF-a, indicated that 99% of MC expressed TNF-a in addition to other dispersed canine dermal cells, showing that MC may have been, at least partly, the source of released TNFa identi®ed in the supernatant through the bioassay. No matter what the cell origin was, however, the release of this cytokine was certainly achieved through a MC-mediated mechanism.
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Fig. 5. Time course TNF-a production on dispersed skin MC cultured for 4 days in presence of exogenous SCF (6 ng/ml) and stimulated upon 50 ng/ml of SCF (black bars), 1 mg/ml of anti-IgE (white bars) or co-stimulated with 50 ng/ml of SCF (15 min) and 1 mg/ml of anti-IgE (grey bars). Each bar represents the meanSEM of six independent experiments (*p<0.05 by comparison with cells not co-stimulated with SCF).
4. Discussion We have shown that SCF has the ability to enhance FceRI-mediated degranulation and cytokine production in mature canine cutaneous MC. The enhancing effect of SCF was analyzed at two levels: ®rstly, for an enduring growth/survival effect exerted through the culture of MC with SCF; and secondly, for a co-stimulatory effect observed through simultaneous activation of MC with anti-IgE and SCF. In addition, regardless of the presence or absence of exogenous SCF during the culture, there was a positive correlation between MC histamine releasability and days of culture, an effect possibly mediated by endogenous SCF production and release. In mature skin MC cultured for up to 5 days in a non SCF-supplemented medium, MC histamine secretion gradually increased with culture time. The presence of interstitial cells such as ®broblasts in the MC culture, probably a source of soluble or membranebound SCF, may account for the differences in histamine releasability between cultured and freshly isolated MC. Moreover, the time-dependent increased responsiveness of MC, also investigated by Taylor et al. (1995) in a 48 h study, may re¯ect the fact that cells function properties recovered during culturing, after the aggressive enzymatic isolation process, recovery possibly being at least partly mediated by SCF. In any event, the higher responsiveness together with the high viability of the canine dermal MC primary cultures sustained for up to 5 days, indicates that the method used is an appropriate in vitro model for investigating the effect of modulatory compounds on mature MC activity. Based on
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this observation, we conclude that we did indeed investigate the effect of SCF on our prolonged MC culture system. To investigate the potential role of SCF as a MC survival, growth or regenerating factor, the isolated dermal cells were cultured and studied from days 0 to 5 in medium supplemented with 6 ng/ml of recombinant canine SCF and then studied. The results reported in Fig. 3 show that MC cultured in SCF-supplemented medium, released a higher percentage of histamine at all points in time when compared to cells cultured without exogenous SCF, regardless of whether additional SCF was present or not during the anti-IgE stimulation. Supplementing the culture with SCF probably either improved isolated dermal MC survival and/or further contributed to the restoration of granule mediators. A similar SCF-dependent enhancing effect was observed on MC-mediated TNF-a production and release. TNF-a was shown to be present in skin MC by immunohistochemistry. However, we were not able to detect pre-formed TNF-a in the granules of skin MC (no TNF-a present in dermal MC pellet from non-stimulated cells), suggesting that most of the cytokine released into the medium was newly synthesized upon stimulation. Granule restoration was therefore probably not responsible, in this case, for the increased TNF-a release observed during MC incubation with SCF. In addition, whereas the maximum effect of SCF on histamine release was an 1.5-fold increase, TNF-a release was enhanced up to six times over baseline. The powerful effect of SCF, as a supplementary factor added to the culture medium, on TNF-a production regardless of its presence during stimulation, could re¯ect an overall MC recovery affecting both early and particularly late phase signaling mechanisms. SCF by itself was not capable of triggering MC mediator release in our study. Although Valent et al. demonstrated in 1992 that SCF is an IgE-independent activator of mature tissue MC, no detectable mediator release and cytokine production was observed on either fresh or cultured canine MC challenged with SCF alone. However, when SCF was used as a co-stimulus with anti-IgE, both pre-formed and newly synthesized mediator release was strongly potentiated, and differences were statistically signi®cant at SCF concentrations higher to 20 ng/ml. Again, in our system the effect of SCF used as a costimulator was more potent on TNF-a than on histamine release, suggesting that SCF had a powerful inducing effect (highest 2±6 h post-stimulation) at the gene transcription or protein synthesis levels, in addition to its potential effect on the early signaling cytoplasmic effects. In contrast to our results were those of Lin et al. (1996) who reported a lack of effect of SCF on TNF-a synthesis. Ishuzuka et al. (1998), however, recently showed that the FceRI-mediated TNF-a production is regulated by SCF at the level of cytokine transcription, consistent with our ®ndings. In the same study, the authors investigated the mechanism by which SCF is able to augment FceRI-stimulation of MC, concluding that SCF and anti-IgE induce the activation of similar members of the mitogen-activated protein kinase family. Interestingly, we observed that after culturing the cells in medium supplemented with SCF, the co-stimulatory effect of SCF on MC was not as high as when no SCF was added during culture. This phenomenon was particularly visible when TNF-a production was assessed. We speculate that SCF receptor desensitization on the MC surface may be occurring over the period of culturing, and may be more marked in MC being maintained in medium containing a higher SCF concentration. Consistent with this hypothesis is a
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report by Columbo et al. (1992) showing that 24 h incubation of human skin MC in an SCF-supplemented medium attenuated the potentiating effect of SCF as a MC costimulus. That observation, taken together with ours, implies the existence of interesting new similarities between human and canine MC. Finally, the marked effect of SCF on both MC survival and MC functionality confers to this growth factor a potential role in some pathologic skin conditions (e.g. atopic dermatitis) which are characterized by tissue MC accumulation and increased MC mediator release. Through these mechanisms, an increase in SCF concentration in the skin could contribute to both, MC activation and maintenance of the allergic skin responses through the enhanced release of histamine and TNF-a, one of the most important cytokines in the late phase allergic reactions.
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