- Email: [email protected]
Tumor necrosis factor alpha (TNF-α) modulates rat mast cell reactivity
Immunology Letters 64 (1998) 167 – 171
Tumor necrosis factor alpha (TNF-a) modulates rat mast cell reactivity Alicja K. Olejnik, Ewa Brzezin´ska-Bl*aszczyk * * o´dz, Mazowiecka 11, L * o´dz92 -215, Poland Di6ision of Experimental Immunology, Medical Uni6ersity of L Received 4 August 1998; received in revised form 1 October 1998; accepted 2 October 1998
Abstract Nowadays there is growing evidence that some cytokines regulate biological functions of the mature mast cells. Therefore, we have studied whether TNF-a, the cytokine of multifunctional activities, could directly stimulate rat peritoneal mast cells to histamine secretion and whether it could modulate rat mast cell reactivity in anaphylactic (with ConA) and anaphylactoid (with compound 48/80) reactions. We have established that rat recombinant TNF-a does not activate rat mast cells to histamine release. However, TNF-a-treatment causes the decrease of spontaneous histamine release up to 85% (TNF-a concentration: 2 ×10 − 9 M). Pretreatment of mast cells with TNF-a inhibits ConA-stimulate release of histamine with the percent release decreasing up to 33.7% of the control value (TNF-a concentration: 5 × 10 − 9 M) and this decrease is statistically significant. Pretreatment of mast cells with TNF-a reduces compound 48/80-dependent histamine release as well and the percent release of histamine fell to 64.7% of the control value. We have concluded that TNF-a may play a significant role in regulation of mast cell secretory activity. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Tumor necrosis factor-a; Rat mast cells; Histamine release; Mast cell reactivity
1. Introduction Cytokines are a broad group of low-molecularweight polypeptides or glycoproteins. They are pleiotropic and exert multiple hormone-like actions both within and outside the immune system. In physiological and pathological states cytokines form a complex interacting network that regulates immunological, haematopoietic, and inflammatory processes. In general, cytokines are not expanded constitutively but are rapidly produced after appropriate stimulation [1,2]. Cytokines can be produced by many diverse cells in the body. It is now well documented that also mast cells are an important source of cytokines. These cells are capable of producing and secreting such cytokines as interleukins (IL) 3, 4, 5, 6, 8, 10, 13, tumor necrosis factor
* Corresponding author. Tel.: + 48 42 6783199; fax.: +48 42 6782292
a (TNF-a), transforming growth factor b (TGF-b), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-g) [3,4]. Nowadays it is documented that mast cells are not only a source of cytokines but also they can be an important target for cytokines. It is now clear that most if not all aspects of mast cell development, including growth, proliferation, and the differentiation/maturation of cells in the mast cell lineage, are regulated by cytokines [5–7]. In murines, IL-3, IL-4, IL-9, stem cell factor (SCF) and nerve growth factor (NGF) are well known to induce or promote mast cell proliferation and/or differentiation [8–10]. Also IL-10 is a potent growth factor for mouse mast cells, particularly in the presence of SCF, IL-3, and IL-4 [11]. Hu et al. [12] showed that in mice also IFN-g is crucial for the survival and/or differentiation of splenic mast cell precursor. IFN-g and GM-CSF were reported to be inhibitory on mast cell precursors [13,14]. SCF is the
0165-2478/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00106-0
168
A.K. Olejnik, E. Brzezin´ska-Bl*aszczyk / Immunology Letters 64 (1998) 167–171
major growth factor for human mast cells, which induces the development of mast cells from fetal liver [15], bone marrow, peripheral blood [16], and cord blood [17]. There is growing evidence that cytokines exerted some effects on the mature mast cells. They may regulate the expression of some molecules and receptors on mast cell surface. It was shown that the expression of SCF receptor (SCF-R) on human mast cell line HMC-1 cells is downregulated by SCF and IL-4 [18,19]. It was also established that IL-4 upregulates the expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) [20,21]. Wedi et al. [22] have shown that ICAM-1 expression in HMC-1 mast cells is upregulated not only by IL-4 but also by TNF-a. Love et al. [23] have established that IFN-g significantly increases the expression of MHC class II antigens (HLA-DP and HLA-DQ) by the HMC-1 cells. IL-4 also enhances the expression of some cytokine receptors in the HMC-1 mast cells [24]. It was also demonstrated that cytokines such as SCF, IL-3 and TGF-b1 stimulate mast cell migration and chemotaxis [25 – 28]. At present it is suggested that also the secretory activity of differentiated tissue mast cells is under the control of a wide range of multifunctional cytokines. Therefore we have studied the influence of TNF-a, the cytokine of multidirectional activities, on histamine secretion from rat peritoneal mast cells. We have determined whether the cytokine TNF-a could directly stimulate rat peritoneal mast cells to histamine release and whether it could modulate the response of rat mast cells to anaphylactic and anaphylactoid activation.
2. Materials and methods
2.1. Animals The mast cells were obtained from peritoneal cavities of female albino Wistar rats weighing :200 – 250 g.
2.2. Mast cell isolation The mast cells were collected from the peritoneal cavity by lavage with 10 ml of the medium containing 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES buffer, and 5.6 mM glucose supplemented with 1 mg/ml bovine serum albumin (BSA) (pH of the medium was adjusted to 6.9). After abdominal massage (90 s) the cell suspension was removed from the peritoneal cavity and washed twice by centrifugation (150 g, 5 min, 4°C). Mast cells were purified by recovery of the pelleted fraction after sedimentation through Percoll density gradient after the method of Rossow et al. [29]. Briefly, the cells were
suspended in 1 ml of the medium and layered onto a Percoll solution containing 6.3 ml Percoll and 0.7 ml concentrated salt solution. After centrifugation at 225 g for 20 min at 4°C, the mast cells sedimented. The mast cells on the bottom of the tube were washed twice by centrifugation at 150 g for 5 min. After being washed, the mast cells were counted and resuspended in an appropriate volume of the medium to obtain mast cell concentration 4× 105 cells/ml. The rat peritoneal mast cells were prepared with purity ] 90%, as determined by metachromatic staining with toluidine blue.
2.3. In 6itro experiments on rat peritoneal mast cells The cell suspensions were carefully divided into 90 ml aliquots and incubated for equilibration at 37°C for 5 min. After that, 10 ml of a stimulating agent, i.e. TNF-a, in an appropriate concentration as specified in the results, coumpound 48/80 at final concentration 10 mg/ml, concanavalin A (ConA) at final concentration 100 mg/ml or anti-IgE at final dilution 1:1000, was added. Incubation was carried out in a water bath with constant stirring for different periods of time, as stated in the results. The reaction was stopped by adding 1.9 ml of cold medium. Next, the cell suspensions were centrifuged (150 g, 5 min, 4°C) and the supernatants were decanted into other tubes for histamine determination. A total of 2 ml of distilled water was added to each tube with cell pellet. In every experiment appropriate controls for the determination of spontaneous histamine release in the absence of stimulating agent were included. In some experiments the mast cells were preincubated at 37°C with TNF-a for different periods of time and after washing with the medium were challenged with ConA (in concentration 100 mg/ml) or compound 48/80 (in concentration 10 mg/ml) for 10 min. It should be pointed out that in preliminary experiments we have established that after incubation of mast cells with TNF-a in concentration 10 − 8 M for 120 min \ 95% of these cells were viable, as judged by trypan blue uptakes. The histamine content was determined in both cell pellets (residual histamine) and supernatants (released histamine) by spectrofluorometric method using o-phthalaldehyde (OPT) (the excitation wavelength was 360 nm while the fluorescence wavelength was 450 nm) [30]. Histamine release was expressed as a percentage of the total cellular content of this amine after correction for the spontaneous release found in controls.
2.4. Reagents NaCl, KCl, CaCl2, MgCl2, glucose, N-2-hydroxyethylpiperazine-N%-ethanesulphonic acid (HEPES), concanavalin A (ConA), compound 48/80, bovine serum albumin (BSA), orthophthalaldehyde (OPT)
A.K. Olejnik, E. Brzezin´ska-Bl*aszczyk / Immunology Letters 64 (1998) 167–171
169
were obtained from Sigma. Percoll was obtained from Pharmacia, mouse anti-rat IgE and recombinant rat tumor necrosis factor alpha (rrTNF-a) from Serotec.
2.5. Statistical parameters Statistical parameters included mean value, standard error of the mean (S.E.M.) and Student’s t-test for ‘small groups’. Values of P B0.05 were considered statistically significant.
3. Results The reactivity of rat peritoneal mast cells in anaphylactic reactions with anti-IgE and ConA and in anaphylactoid reaction with compoud 48/80 is shown in Fig. 1. Rat mast cells were activated and released histamine to the challenge with all of these stimulating agents, and the magnitude of histamine release was significant. Anaphylactic histamine release was up to 24.19 4.1% (mean 9 S.E.M.) with ConA at the concentration 100 mg/ml and up to 12.8 9 3.0% with anti-IgE at the dilution 1:1000. Anaphylactoid, compound 48/80-induced histamine release was up to 64.19 2.0% (the concentration of compound 48/80-10 mg/ml). At the same experimental conditions TNF-a did not cause release of histamine from rat mast cells. Moreover, unexpectedly we have noticed that TNF-a treatment resulted in decrease of spontaneous histamine release (Fig. 1 E, F, G). To investigate the effect of TNF-a treatment on rat mast cells releasability more precisely the cells were incubated with different concentrations of TNF-a (from 10 − 11 M to 10 − 8 M) for 30 min. The treatment of mast cells with 10 − 11 M, 5×10 − 11 M or 10 − 10 M reduced
Fig. 1. The sensitivity of rat mast cells to the action of [A] compound 48/80 (concentration: 10 mg/ml, time of reaction: 10 min), [B] Con A (concentration: 100 mg/ml, time of reaction: 10 min), [C] anti-IgE (dilution: 1:1000, time of reaction: 30 min), and [D], [E], [F] TNF-a (concentration: 10-11 M, 5 × 10-10 M, 10-9 M, respectively, time of reaction: 30 min). The results are the means 9 SEM of 5 separate experiments with two replicates in each experiment.
Fig. 2. Effect of pretreatment with TNF-a on spontaneous histamine release from rat mast cells. Mast cells were incubated with different concentrations of TNF-a of buffer alone (control mast cells) for 30 min. Each point represents the mean 9 S.E.M. of five experiments with two replicates in each experiment. *, indicates PB 0.05, **, indicates P B0.01, by comparison with buffer-treated cells. ", % of maximal spontaneous histamine release found in control mast cells.
the spontaneous histamine release up to 78.3, 75.3, and 73.7 of maximal release, respectively. However, the decrease was not statistically significant. The incubation of mast cells with higher concentrations of TNF-a (] 2.5× 10 − 10 M) resulted in concentration-dependent statistically significant inhibition of spontaneous histamine release (Fig. 2). Time-course experiments demonstrated that preincubation of rat mast cells with 10 − 9 M of TNF-a led to decrease of spontaneous histamine release at all time points tested (30, 60, and 120 min) (Table 1). To investigate the effect of TNF-a treatment on mast cell reactivity in anaphylactic and anaphylactoid reactions the cells were preincubated with this cytokine in concentrations 10 − 11 M, 10 − 9 M or 5×10 − 9 M for 60 min, then washed and challenged with ConA or with compound 48/80. We have noticed, that TNF-a pretreatment inhibited ConA-stimulate release of histamine with the percent release decreasing up to 33.7% of the control value (TNF-a concentration 5×10 − 9 M). The decrease was statistically significant. Pretreatment of mast cells with TNF-a resulted in decrease of compound 48/80-dependent histamine release as well. After exposure of mast cells to 5 × 10 − 9 M of TNF-a the percent release of histamine fell to 64.7% of the control value, and this inhibition was statistically significant (Fig. 3).
4. Discussion Several lines of evidence have indicated that some cytokines can influence the secretory activity of mature mast cells. The cytokines have either direct stimulating
A.K. Olejnik, E. Brzezin´ska-Bl*aszczyk / Immunology Letters 64 (1998) 167–171
170
Table 1 Effect of incubation time with TNF-a on spontaneous histamine release from rat mast cells Time of incubation (min)
Spontaneous histamine release (%) 9 S.E.M. Histamine release (%) after incubation with TNF-a9S.E.M. [B] [B]−[A] [A]
30 60 120
14.1 92.7 24.2 93.9 26.9 94.4
5.4 90.5 8.5 9 1.6 10.1 9 1.1
−8.7 −15.7 −16.8
Mast cells were incubated with TNF-a (concentration: 10−9 M) or buffer alone. The results are the means9S.E.M. of five separate experiments with two replicates in each experiment.
or regulatory effects. For example, SCF by itself can induce mast cell degranulation and mediator release [31,32]. IL-1b is a potent stimulus for nitric oxide release by rat peritoneal mast cells [33]. Macrophage inflammatory protein 1 alpha (MIP-1a) stimulates murine peritoneal mast cells to histamine release [34]. SCF, IL-3 and IL-4 enhance the ability of mast cells to release mediators in response to immune challenge [35,36]. Conversely, IFN-g, IFN-a/b, IL-10, and TGFb1 have suppressive effect on mast cells mediator release and cytokine generation [37 – 40]. IFN-g can also overcome the enhancing effects of IL-3 and IL-4 on antigen-induced release of serotonin and arachidonate [41]. TNF-a is now recognized to have a broad range of biological activities with roles in many immunologic and inflammatory reactions. It exerts profound effects on polymorphonuclear neutrophils and eosinophils, causes the production of other cytokines, and also induces an influx of inflammatory cells by increasing
Fig. 3. Effect of pretreatment with TNF-a on rat mast cell reactivity in anaphylactic and anaphylactoid reactions. Mast cells were incubated with 10 − 11 M, 10 − 9 M, 5 × 10 − 9 M of TNF-a or buffer alone (control mast cells) for 60 min, then washed and challenged with Con A in concentration 100 mg/ml (black bars) or with compound 48/80 in concentration 10 mg/ml (white bars) for 10 min. The results are the means9S.E.M. of five experiments with two replicates in each experiment. *, indicates P B0.05, **, indicates PB 0.02, ***, indicates P B0.01, by comparison with buffer-treated cells. ", % of maximal histamine release found in control mast cells.
the expression of adhesion molecules on endothelial cells. Originally, activated monocytes and tissue macrophages were thought to be the principal cellular source of TNF-a [42,43]. More recently, however, different types of cells have been shown to secrete this cytokine, among them mast cells. TNF-a released by mast cells is derived from two distinct pools, a preformed pool which accounts for most of the product released during the first 10 min after stimulation, and a newly synthesized pool, which accounts for the later, sustained release of the cytokine. Thus, TNF-a represents a new type of mast cell-associated mediator which expresses bioactivity through the effects of both preformed and newly synthesized product [44]. In the present study we have investigated the direct as well as the modulating effect of rat recombinant TNF-a on histamine secretion from rat peritoneal mast cells. We have established that rat rTNF-a did not stimulate rat mast cells to histamine release. However, this cytokine can modulate rat mast cell reactivity. Firstly, TNF-a significantly reduced spontaneous histamine release i.e. it decreased mast cell ‘releasability’. Secondly, TNF-a inhibited the secretory activity of rat mast cells both in anaphylactic (with ConA) and anaphylactoid (with compound 48/80) reactions. In vitro pretreatment of mast cells with TNF-a significantly reduced ConA- and compound 48/80-stimulated histamine secretion (the percent release decreasing up to 33.7% and up to 64.7% of the control values, respectively). We have earlier established that human recombinant TNF-a stimulates human adenoidal and cutaneous mast cells to histamine release. HrTNF-a-induced histamine release was concentration- and time-dependent [45]. Van Overveld et al. [46] have found that TNF-a is a stimulus for skin mast cells to degranulate and to release both histamine and tryptase. Hughes et al. [47] have shown however that this cytokine does not activate human pulmonary mast cells to histamine secretion. It was also shown that TNF-a alone and in costimulation with either IL-4 or IFN-g caused selective upregulation of ICAM-1 expression on mast cell surface [22]. Observations that TNF-a can be a modulatory factor for mast cells seem to be of great importance. Firstly,
A.K. Olejnik, E. Brzezin´ska-Bl*aszczyk / Immunology Letters 64 (1998) 167–171
because many cells produce and release this cytokine [42,43]. Secondly, because the mast cells themselves represent a potential source of this cytokine [44]. It has been shown that mast cells constitutively contain large amounts of TNF-a which is localized to the cytoplasmic granules [48,49]. Moreover, it has been demonstrated, that IgE-dependent activation of mast cells induces rapid extracellular release of preformed TNF-a together with histamine and proteoglycans, as well as an increasing mRNA for this cytokine [44]. At present, the biological significance of TNF-a-mast cell interactions cannot be fully interpreted. However, it should be pointed out that mast cells are not only an important source of this cytokine but also an important target of TNF-a. Thus, it may play a significant role in the physiological regulation of mast cell function. Taking into account that mast cells themselves are the powerful source of TNF-a it can be postulated that this cytokine is one of very important autocrine signals modulating the biology of mast cells.
References [1] R. Debets, H.F.J. Savelkoul, Mediat. Inflamm. 5 (1996) 417 – 423. [2] C.O. Elson, K.W. Beagley, in: L.R. Johnson (Ed.), Physiology of the Gastrointestinal Tract, Raven Press, New York, 1994, pp. 243 – 265. [3] J.R. Gordon, P.R. Burd, S.J. Galli, Immunol. Today 11 (1990) 458 – 464. [4] S. Weber, S. Kruger-Krasagakes, J. Grabbe, T. Zuberbier, B.M. Czarnetzki, Internat. J. Dermatol. 14 (1995) 1–10. [5] S.J. Galli, Lab. Invest. 62 (1990) 5–33. [6] T. Ishizaka, H. Mitsui, M. Yanagida, T. Miura, A.M. Dvorak, Curr. Opin. Immunol. 5 (1993) 937–943. [7] Y. Kitamura, T. Kasugai, N. Arizono, H. Matsuda, Am. J. Med. Sci. 306 (1993) 185 –191. [8] H. Matsuda, Y. Kannan, H. Ushio, Y. Kiso, T. Kanemoto, H. Suzuki, Y. Kitamura, J. Exp. Med. 174 (1991) 7–14. [9] M. Tsai, T. Takeishi, H. Thompson, K.E. Langley, K.M. Zsebo, D.D. Metcalfe, E.N. Geissler, S.J. Galli, Proc. Natl. Acad. Sci. USA 88 (1991) 6382–6386. [10] C.A. Smith, D.M. Rennick, Proc. Natl. Acad. Sci. USA 83 (1986) 1857 – 1861. [11] L. Thompson-Snipes, V. Dhar, M.W. Bond, T.R. Mosmann, K.W. Moore, D.M. Rennick, J. Exp. Med. 173 (1991) 507 – 510. [12] Z. Hu, N. Zenda, T. Shimamura, J. Immunol. 156 (1996) 3925 – 3931. [13] J. Nafziger, M. Arock, J.J. Guillosson, J. Wietzerbin, Eur. J. Immunol. 20 (1990) 113–117. [14] R.B. Bressler, H.L. Thompson, J.M. Keffer, D.D. Metcalfe, J. Immunol. 143 (1989) 135–139. [15] A.A. Irani, G. Nilsson, U. Miettinen, S.S. Craig, L.K. Ashman, T. Ishizaka, K.M. Zsebo, L.B. Schwartz, Blood 80 (1992) 3009 – 3021. [16] P. Valent, E. Spanblochl, W.R. Sperr, C. Sillaber, K.M. Zsebo, H. Agis, H. Strobl, K. Geissler, P. Bettelheim, K. Lechner, Blood 80 (1992) 2237–2245. [17] H. Mitsui, T. Furitsu, A.M. Dvorak, A.A. Irani, L.B. Schwartz, N. Inagaki, M. Takei, K. Ishizaka, K.M. Zsebo, S. Gillis, T. Ishizaka, Proc. Natl. Acad. Sci. USA 90 (1993) 735–739.
171
[18] M. Baghestanian, H. Agis, D. Bevec, H.C. Bankl, R. Hofbauer, H.G. Kress, J.H. Butterfield, M.R. Muller, L.K. Ashman, W. Fureder, M. Willheim, K. Lechner, P. Valent, Exp. Hematol. 24 (1996) 1377 – 1386. [19] W.R. Sperr, H. Agis, K. Czerwenka, W. Klepetko, E. Kubista, G. Boltz-Nitulescu, K. Lechner, P. Valent, Ann. Hematol. 65 (1992) 10 – 16. [20] H. Toru, T. Kinashi, C. Ra, S. Nonoyama, J. Yata, T. Nakahata, Blood 89 (1997) 3296 – 3302. [21] P. Valent, D. Bevec, D. Maurer, J. Besemer, F. Di-Padova, J.H. Butterfield, W. Speiser, O. Majdic, K. Lechner, P. Bettelheim, Proc. Natl. Acad. Sci. USA 88 (1991) 3339 – 3342. [22] B. Wedi, J. Elsner, W. Czech, J.H. Butterfield, A. Kapp, Allergy 51 (1996) 676 – 684. [23] K.S. Love, R.R. Lakshmanan, J.H. Butterfield, C.C. Fox, Cell. Immunol. 170 (1996) 85 – 90. [24] J.W. Coleman, M.G. Buckley, A.M. Taylor, E.M.S. Banks, C.M.M. Williams, M.R. Holliday, J. Thompson, Int. Arch. Allergy Immunol. 107 (1995) 154 – 155. [25] B.L. Gruber, M.J. Marchese, R.R. Kew, J. Immunol. 152 (1994) 5860 – 5867. [26] N. Matsuura, B.R. Zetter, J. Exp. Med. 170 (1989) 1421–1426. [27] C.J. Meininger, H. Yano, R. Rottapel, A. Bernstein, K.M. Zsebo, B.R. Zetter, Blood 79 (1992) 958 – 963. [28] G. Nilsson, J.H. Butterfield, K. Nilsson, A. Siegbahn, J. Immunol. 153 (1994) 3717 – 3723. [29] N. Rossow, S.D. Sandy, H. Rephe, Biomed. Biochim. Acta 10 (1986) 1343 – 1347. [30] P.A. Shore, A. Burkhalter, V.H. Cohn, J. Pharmacol. Exp. Ther. 127 (1959) 182 – 186. [31] K. Nakajima, K. Hirai, M. Yamaguchi, T. Takaishi, K. Ohta, Y. Morita, K. Ito, Biochem. Biophys. Res. Commun. 183 (1992) 1076 – 1083. [32] A.M. Taylor, S.J. Galli, J.W. Coleman, Immunology 86 (1995) 427 – 433. [33] C.M. Hogaboam, A.D. Befus, J.L. Wallace, J. Immunol. 151 (1993) 3767 – 3774. [34] R. Alam, P.A. Forsythe, S. Stafford, M.A. Lett-Brown, J.A. Grant, J. Exp. Med. 176 (1992) 781 – 786. [35] J.W. Coleman, M.R. Holliday, I. Kimber, K.M. Zsebo, S.J. Galli, J. Immunol. 150 (1993) 556 – 562. [36] T.J. Lin, E.Y. Bissonnette, A. Hirsh, A.D. Befus, Immunology 89 (1996) 301 – 307. [37] M. Arock, C. Zuany-Amorim, M. Singer, M. Benhamou, M. Pretolani, Eur. J. Immunol. 26 (1996) 166 – 170. [38] E.Y. Bissonnette, B. Chin, A.D. Befus, Immunology 86 (1995) 12 – 17. [39] E.Y. Bissonnette, J.A. Enciso, A.D. Befus, Am. J. Respir. Cell Mol. Biol. 16 (1997) 275 – 282. [40] T.J. Lin, A.D. Befus, J. Immunol. 159 (1997) 4015 – 4023. [41] M.R. Holliday, E.M. Banks, R.J. Dearman, I. Kimber, J.W. Coleman, Immunology 82 (1994) 70 – 74. [42] B.B. Aggarwal, K. Natarajan, Eur. Cytokine Netw. 7 (1996) 93 – 124. [43] L. Rink, H. Kirchner, Int. Arch. Allergy Immunol. 111 (1996) 199 – 209. [44] J.R. Gordon, S.J. Galli, J. Exp. Med. 174 (1991) 103 –107. [45] E. Brzezin´ska-Blaszczyk, A. Pietrzak, Immunol. Lett. 59 (1997) 139 – 143. [46] F.J. Van Overveld, P.G. Jorens, M. Rampart, W. De Backer, P.A. Vermeire, Clin. Exp. Allergy 21 (1991) 711 – 714. [47] J.M. Hughes, R.S. Stringer, J.L. Black, C.L. Armour, Pulm. Pharmacol. 8 (1995) 31 – 36. [48] W.J. Beil, G.R. Login, S.J. Galli, A.M. Dvorak, J. Allergy Clin. Immunol. 94 (1994) 531 – 536. [49] W.J. Beil, G.R. Login, M. Aoki, L.O. Lunardi, E.S. Morgan, S.J. Galli, A.M. Dvorak, Int. Arch. Allergy Immunol. 109 (1996) 383 – 389.
Tumor necrosis factor alpha (TNF-a) modulates rat mast cell reactivity Alicja K. Olejnik, Ewa Brzezin´ska-Bl*aszczyk * * o´dz, Mazowiecka 11, L * o´dz92 -215, Poland Di6ision of Experimental Immunology, Medical Uni6ersity of L Received 4 August 1998; received in revised form 1 October 1998; accepted 2 October 1998
Abstract Nowadays there is growing evidence that some cytokines regulate biological functions of the mature mast cells. Therefore, we have studied whether TNF-a, the cytokine of multifunctional activities, could directly stimulate rat peritoneal mast cells to histamine secretion and whether it could modulate rat mast cell reactivity in anaphylactic (with ConA) and anaphylactoid (with compound 48/80) reactions. We have established that rat recombinant TNF-a does not activate rat mast cells to histamine release. However, TNF-a-treatment causes the decrease of spontaneous histamine release up to 85% (TNF-a concentration: 2 ×10 − 9 M). Pretreatment of mast cells with TNF-a inhibits ConA-stimulate release of histamine with the percent release decreasing up to 33.7% of the control value (TNF-a concentration: 5 × 10 − 9 M) and this decrease is statistically significant. Pretreatment of mast cells with TNF-a reduces compound 48/80-dependent histamine release as well and the percent release of histamine fell to 64.7% of the control value. We have concluded that TNF-a may play a significant role in regulation of mast cell secretory activity. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Tumor necrosis factor-a; Rat mast cells; Histamine release; Mast cell reactivity
1. Introduction Cytokines are a broad group of low-molecularweight polypeptides or glycoproteins. They are pleiotropic and exert multiple hormone-like actions both within and outside the immune system. In physiological and pathological states cytokines form a complex interacting network that regulates immunological, haematopoietic, and inflammatory processes. In general, cytokines are not expanded constitutively but are rapidly produced after appropriate stimulation [1,2]. Cytokines can be produced by many diverse cells in the body. It is now well documented that also mast cells are an important source of cytokines. These cells are capable of producing and secreting such cytokines as interleukins (IL) 3, 4, 5, 6, 8, 10, 13, tumor necrosis factor
* Corresponding author. Tel.: + 48 42 6783199; fax.: +48 42 6782292
a (TNF-a), transforming growth factor b (TGF-b), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-g) [3,4]. Nowadays it is documented that mast cells are not only a source of cytokines but also they can be an important target for cytokines. It is now clear that most if not all aspects of mast cell development, including growth, proliferation, and the differentiation/maturation of cells in the mast cell lineage, are regulated by cytokines [5–7]. In murines, IL-3, IL-4, IL-9, stem cell factor (SCF) and nerve growth factor (NGF) are well known to induce or promote mast cell proliferation and/or differentiation [8–10]. Also IL-10 is a potent growth factor for mouse mast cells, particularly in the presence of SCF, IL-3, and IL-4 [11]. Hu et al. [12] showed that in mice also IFN-g is crucial for the survival and/or differentiation of splenic mast cell precursor. IFN-g and GM-CSF were reported to be inhibitory on mast cell precursors [13,14]. SCF is the
0165-2478/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2478(98)00106-0
168
A.K. Olejnik, E. Brzezin´ska-Bl*aszczyk / Immunology Letters 64 (1998) 167–171
major growth factor for human mast cells, which induces the development of mast cells from fetal liver [15], bone marrow, peripheral blood [16], and cord blood [17]. There is growing evidence that cytokines exerted some effects on the mature mast cells. They may regulate the expression of some molecules and receptors on mast cell surface. It was shown that the expression of SCF receptor (SCF-R) on human mast cell line HMC-1 cells is downregulated by SCF and IL-4 [18,19]. It was also established that IL-4 upregulates the expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) [20,21]. Wedi et al. [22] have shown that ICAM-1 expression in HMC-1 mast cells is upregulated not only by IL-4 but also by TNF-a. Love et al. [23] have established that IFN-g significantly increases the expression of MHC class II antigens (HLA-DP and HLA-DQ) by the HMC-1 cells. IL-4 also enhances the expression of some cytokine receptors in the HMC-1 mast cells [24]. It was also demonstrated that cytokines such as SCF, IL-3 and TGF-b1 stimulate mast cell migration and chemotaxis [25 – 28]. At present it is suggested that also the secretory activity of differentiated tissue mast cells is under the control of a wide range of multifunctional cytokines. Therefore we have studied the influence of TNF-a, the cytokine of multidirectional activities, on histamine secretion from rat peritoneal mast cells. We have determined whether the cytokine TNF-a could directly stimulate rat peritoneal mast cells to histamine release and whether it could modulate the response of rat mast cells to anaphylactic and anaphylactoid activation.
2. Materials and methods
2.1. Animals The mast cells were obtained from peritoneal cavities of female albino Wistar rats weighing :200 – 250 g.
2.2. Mast cell isolation The mast cells were collected from the peritoneal cavity by lavage with 10 ml of the medium containing 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES buffer, and 5.6 mM glucose supplemented with 1 mg/ml bovine serum albumin (BSA) (pH of the medium was adjusted to 6.9). After abdominal massage (90 s) the cell suspension was removed from the peritoneal cavity and washed twice by centrifugation (150 g, 5 min, 4°C). Mast cells were purified by recovery of the pelleted fraction after sedimentation through Percoll density gradient after the method of Rossow et al. [29]. Briefly, the cells were
suspended in 1 ml of the medium and layered onto a Percoll solution containing 6.3 ml Percoll and 0.7 ml concentrated salt solution. After centrifugation at 225 g for 20 min at 4°C, the mast cells sedimented. The mast cells on the bottom of the tube were washed twice by centrifugation at 150 g for 5 min. After being washed, the mast cells were counted and resuspended in an appropriate volume of the medium to obtain mast cell concentration 4× 105 cells/ml. The rat peritoneal mast cells were prepared with purity ] 90%, as determined by metachromatic staining with toluidine blue.
2.3. In 6itro experiments on rat peritoneal mast cells The cell suspensions were carefully divided into 90 ml aliquots and incubated for equilibration at 37°C for 5 min. After that, 10 ml of a stimulating agent, i.e. TNF-a, in an appropriate concentration as specified in the results, coumpound 48/80 at final concentration 10 mg/ml, concanavalin A (ConA) at final concentration 100 mg/ml or anti-IgE at final dilution 1:1000, was added. Incubation was carried out in a water bath with constant stirring for different periods of time, as stated in the results. The reaction was stopped by adding 1.9 ml of cold medium. Next, the cell suspensions were centrifuged (150 g, 5 min, 4°C) and the supernatants were decanted into other tubes for histamine determination. A total of 2 ml of distilled water was added to each tube with cell pellet. In every experiment appropriate controls for the determination of spontaneous histamine release in the absence of stimulating agent were included. In some experiments the mast cells were preincubated at 37°C with TNF-a for different periods of time and after washing with the medium were challenged with ConA (in concentration 100 mg/ml) or compound 48/80 (in concentration 10 mg/ml) for 10 min. It should be pointed out that in preliminary experiments we have established that after incubation of mast cells with TNF-a in concentration 10 − 8 M for 120 min \ 95% of these cells were viable, as judged by trypan blue uptakes. The histamine content was determined in both cell pellets (residual histamine) and supernatants (released histamine) by spectrofluorometric method using o-phthalaldehyde (OPT) (the excitation wavelength was 360 nm while the fluorescence wavelength was 450 nm) [30]. Histamine release was expressed as a percentage of the total cellular content of this amine after correction for the spontaneous release found in controls.
2.4. Reagents NaCl, KCl, CaCl2, MgCl2, glucose, N-2-hydroxyethylpiperazine-N%-ethanesulphonic acid (HEPES), concanavalin A (ConA), compound 48/80, bovine serum albumin (BSA), orthophthalaldehyde (OPT)
A.K. Olejnik, E. Brzezin´ska-Bl*aszczyk / Immunology Letters 64 (1998) 167–171
169
were obtained from Sigma. Percoll was obtained from Pharmacia, mouse anti-rat IgE and recombinant rat tumor necrosis factor alpha (rrTNF-a) from Serotec.
2.5. Statistical parameters Statistical parameters included mean value, standard error of the mean (S.E.M.) and Student’s t-test for ‘small groups’. Values of P B0.05 were considered statistically significant.
3. Results The reactivity of rat peritoneal mast cells in anaphylactic reactions with anti-IgE and ConA and in anaphylactoid reaction with compoud 48/80 is shown in Fig. 1. Rat mast cells were activated and released histamine to the challenge with all of these stimulating agents, and the magnitude of histamine release was significant. Anaphylactic histamine release was up to 24.19 4.1% (mean 9 S.E.M.) with ConA at the concentration 100 mg/ml and up to 12.8 9 3.0% with anti-IgE at the dilution 1:1000. Anaphylactoid, compound 48/80-induced histamine release was up to 64.19 2.0% (the concentration of compound 48/80-10 mg/ml). At the same experimental conditions TNF-a did not cause release of histamine from rat mast cells. Moreover, unexpectedly we have noticed that TNF-a treatment resulted in decrease of spontaneous histamine release (Fig. 1 E, F, G). To investigate the effect of TNF-a treatment on rat mast cells releasability more precisely the cells were incubated with different concentrations of TNF-a (from 10 − 11 M to 10 − 8 M) for 30 min. The treatment of mast cells with 10 − 11 M, 5×10 − 11 M or 10 − 10 M reduced
Fig. 1. The sensitivity of rat mast cells to the action of [A] compound 48/80 (concentration: 10 mg/ml, time of reaction: 10 min), [B] Con A (concentration: 100 mg/ml, time of reaction: 10 min), [C] anti-IgE (dilution: 1:1000, time of reaction: 30 min), and [D], [E], [F] TNF-a (concentration: 10-11 M, 5 × 10-10 M, 10-9 M, respectively, time of reaction: 30 min). The results are the means 9 SEM of 5 separate experiments with two replicates in each experiment.
Fig. 2. Effect of pretreatment with TNF-a on spontaneous histamine release from rat mast cells. Mast cells were incubated with different concentrations of TNF-a of buffer alone (control mast cells) for 30 min. Each point represents the mean 9 S.E.M. of five experiments with two replicates in each experiment. *, indicates PB 0.05, **, indicates P B0.01, by comparison with buffer-treated cells. ", % of maximal spontaneous histamine release found in control mast cells.
the spontaneous histamine release up to 78.3, 75.3, and 73.7 of maximal release, respectively. However, the decrease was not statistically significant. The incubation of mast cells with higher concentrations of TNF-a (] 2.5× 10 − 10 M) resulted in concentration-dependent statistically significant inhibition of spontaneous histamine release (Fig. 2). Time-course experiments demonstrated that preincubation of rat mast cells with 10 − 9 M of TNF-a led to decrease of spontaneous histamine release at all time points tested (30, 60, and 120 min) (Table 1). To investigate the effect of TNF-a treatment on mast cell reactivity in anaphylactic and anaphylactoid reactions the cells were preincubated with this cytokine in concentrations 10 − 11 M, 10 − 9 M or 5×10 − 9 M for 60 min, then washed and challenged with ConA or with compound 48/80. We have noticed, that TNF-a pretreatment inhibited ConA-stimulate release of histamine with the percent release decreasing up to 33.7% of the control value (TNF-a concentration 5×10 − 9 M). The decrease was statistically significant. Pretreatment of mast cells with TNF-a resulted in decrease of compound 48/80-dependent histamine release as well. After exposure of mast cells to 5 × 10 − 9 M of TNF-a the percent release of histamine fell to 64.7% of the control value, and this inhibition was statistically significant (Fig. 3).
4. Discussion Several lines of evidence have indicated that some cytokines can influence the secretory activity of mature mast cells. The cytokines have either direct stimulating
A.K. Olejnik, E. Brzezin´ska-Bl*aszczyk / Immunology Letters 64 (1998) 167–171
170
Table 1 Effect of incubation time with TNF-a on spontaneous histamine release from rat mast cells Time of incubation (min)
Spontaneous histamine release (%) 9 S.E.M. Histamine release (%) after incubation with TNF-a9S.E.M. [B] [B]−[A] [A]
30 60 120
14.1 92.7 24.2 93.9 26.9 94.4
5.4 90.5 8.5 9 1.6 10.1 9 1.1
−8.7 −15.7 −16.8
Mast cells were incubated with TNF-a (concentration: 10−9 M) or buffer alone. The results are the means9S.E.M. of five separate experiments with two replicates in each experiment.
or regulatory effects. For example, SCF by itself can induce mast cell degranulation and mediator release [31,32]. IL-1b is a potent stimulus for nitric oxide release by rat peritoneal mast cells [33]. Macrophage inflammatory protein 1 alpha (MIP-1a) stimulates murine peritoneal mast cells to histamine release [34]. SCF, IL-3 and IL-4 enhance the ability of mast cells to release mediators in response to immune challenge [35,36]. Conversely, IFN-g, IFN-a/b, IL-10, and TGFb1 have suppressive effect on mast cells mediator release and cytokine generation [37 – 40]. IFN-g can also overcome the enhancing effects of IL-3 and IL-4 on antigen-induced release of serotonin and arachidonate [41]. TNF-a is now recognized to have a broad range of biological activities with roles in many immunologic and inflammatory reactions. It exerts profound effects on polymorphonuclear neutrophils and eosinophils, causes the production of other cytokines, and also induces an influx of inflammatory cells by increasing
Fig. 3. Effect of pretreatment with TNF-a on rat mast cell reactivity in anaphylactic and anaphylactoid reactions. Mast cells were incubated with 10 − 11 M, 10 − 9 M, 5 × 10 − 9 M of TNF-a or buffer alone (control mast cells) for 60 min, then washed and challenged with Con A in concentration 100 mg/ml (black bars) or with compound 48/80 in concentration 10 mg/ml (white bars) for 10 min. The results are the means9S.E.M. of five experiments with two replicates in each experiment. *, indicates P B0.05, **, indicates PB 0.02, ***, indicates P B0.01, by comparison with buffer-treated cells. ", % of maximal histamine release found in control mast cells.
the expression of adhesion molecules on endothelial cells. Originally, activated monocytes and tissue macrophages were thought to be the principal cellular source of TNF-a [42,43]. More recently, however, different types of cells have been shown to secrete this cytokine, among them mast cells. TNF-a released by mast cells is derived from two distinct pools, a preformed pool which accounts for most of the product released during the first 10 min after stimulation, and a newly synthesized pool, which accounts for the later, sustained release of the cytokine. Thus, TNF-a represents a new type of mast cell-associated mediator which expresses bioactivity through the effects of both preformed and newly synthesized product [44]. In the present study we have investigated the direct as well as the modulating effect of rat recombinant TNF-a on histamine secretion from rat peritoneal mast cells. We have established that rat rTNF-a did not stimulate rat mast cells to histamine release. However, this cytokine can modulate rat mast cell reactivity. Firstly, TNF-a significantly reduced spontaneous histamine release i.e. it decreased mast cell ‘releasability’. Secondly, TNF-a inhibited the secretory activity of rat mast cells both in anaphylactic (with ConA) and anaphylactoid (with compound 48/80) reactions. In vitro pretreatment of mast cells with TNF-a significantly reduced ConA- and compound 48/80-stimulated histamine secretion (the percent release decreasing up to 33.7% and up to 64.7% of the control values, respectively). We have earlier established that human recombinant TNF-a stimulates human adenoidal and cutaneous mast cells to histamine release. HrTNF-a-induced histamine release was concentration- and time-dependent [45]. Van Overveld et al. [46] have found that TNF-a is a stimulus for skin mast cells to degranulate and to release both histamine and tryptase. Hughes et al. [47] have shown however that this cytokine does not activate human pulmonary mast cells to histamine secretion. It was also shown that TNF-a alone and in costimulation with either IL-4 or IFN-g caused selective upregulation of ICAM-1 expression on mast cell surface [22]. Observations that TNF-a can be a modulatory factor for mast cells seem to be of great importance. Firstly,
A.K. Olejnik, E. Brzezin´ska-Bl*aszczyk / Immunology Letters 64 (1998) 167–171
because many cells produce and release this cytokine [42,43]. Secondly, because the mast cells themselves represent a potential source of this cytokine [44]. It has been shown that mast cells constitutively contain large amounts of TNF-a which is localized to the cytoplasmic granules [48,49]. Moreover, it has been demonstrated, that IgE-dependent activation of mast cells induces rapid extracellular release of preformed TNF-a together with histamine and proteoglycans, as well as an increasing mRNA for this cytokine [44]. At present, the biological significance of TNF-a-mast cell interactions cannot be fully interpreted. However, it should be pointed out that mast cells are not only an important source of this cytokine but also an important target of TNF-a. Thus, it may play a significant role in the physiological regulation of mast cell function. Taking into account that mast cells themselves are the powerful source of TNF-a it can be postulated that this cytokine is one of very important autocrine signals modulating the biology of mast cells.
References [1] R. Debets, H.F.J. Savelkoul, Mediat. Inflamm. 5 (1996) 417 – 423. [2] C.O. Elson, K.W. Beagley, in: L.R. Johnson (Ed.), Physiology of the Gastrointestinal Tract, Raven Press, New York, 1994, pp. 243 – 265. [3] J.R. Gordon, P.R. Burd, S.J. Galli, Immunol. Today 11 (1990) 458 – 464. [4] S. Weber, S. Kruger-Krasagakes, J. Grabbe, T. Zuberbier, B.M. Czarnetzki, Internat. J. Dermatol. 14 (1995) 1–10. [5] S.J. Galli, Lab. Invest. 62 (1990) 5–33. [6] T. Ishizaka, H. Mitsui, M. Yanagida, T. Miura, A.M. Dvorak, Curr. Opin. Immunol. 5 (1993) 937–943. [7] Y. Kitamura, T. Kasugai, N. Arizono, H. Matsuda, Am. J. Med. Sci. 306 (1993) 185 –191. [8] H. Matsuda, Y. Kannan, H. Ushio, Y. Kiso, T. Kanemoto, H. Suzuki, Y. Kitamura, J. Exp. Med. 174 (1991) 7–14. [9] M. Tsai, T. Takeishi, H. Thompson, K.E. Langley, K.M. Zsebo, D.D. Metcalfe, E.N. Geissler, S.J. Galli, Proc. Natl. Acad. Sci. USA 88 (1991) 6382–6386. [10] C.A. Smith, D.M. Rennick, Proc. Natl. Acad. Sci. USA 83 (1986) 1857 – 1861. [11] L. Thompson-Snipes, V. Dhar, M.W. Bond, T.R. Mosmann, K.W. Moore, D.M. Rennick, J. Exp. Med. 173 (1991) 507 – 510. [12] Z. Hu, N. Zenda, T. Shimamura, J. Immunol. 156 (1996) 3925 – 3931. [13] J. Nafziger, M. Arock, J.J. Guillosson, J. Wietzerbin, Eur. J. Immunol. 20 (1990) 113–117. [14] R.B. Bressler, H.L. Thompson, J.M. Keffer, D.D. Metcalfe, J. Immunol. 143 (1989) 135–139. [15] A.A. Irani, G. Nilsson, U. Miettinen, S.S. Craig, L.K. Ashman, T. Ishizaka, K.M. Zsebo, L.B. Schwartz, Blood 80 (1992) 3009 – 3021. [16] P. Valent, E. Spanblochl, W.R. Sperr, C. Sillaber, K.M. Zsebo, H. Agis, H. Strobl, K. Geissler, P. Bettelheim, K. Lechner, Blood 80 (1992) 2237–2245. [17] H. Mitsui, T. Furitsu, A.M. Dvorak, A.A. Irani, L.B. Schwartz, N. Inagaki, M. Takei, K. Ishizaka, K.M. Zsebo, S. Gillis, T. Ishizaka, Proc. Natl. Acad. Sci. USA 90 (1993) 735–739.
171
[18] M. Baghestanian, H. Agis, D. Bevec, H.C. Bankl, R. Hofbauer, H.G. Kress, J.H. Butterfield, M.R. Muller, L.K. Ashman, W. Fureder, M. Willheim, K. Lechner, P. Valent, Exp. Hematol. 24 (1996) 1377 – 1386. [19] W.R. Sperr, H. Agis, K. Czerwenka, W. Klepetko, E. Kubista, G. Boltz-Nitulescu, K. Lechner, P. Valent, Ann. Hematol. 65 (1992) 10 – 16. [20] H. Toru, T. Kinashi, C. Ra, S. Nonoyama, J. Yata, T. Nakahata, Blood 89 (1997) 3296 – 3302. [21] P. Valent, D. Bevec, D. Maurer, J. Besemer, F. Di-Padova, J.H. Butterfield, W. Speiser, O. Majdic, K. Lechner, P. Bettelheim, Proc. Natl. Acad. Sci. USA 88 (1991) 3339 – 3342. [22] B. Wedi, J. Elsner, W. Czech, J.H. Butterfield, A. Kapp, Allergy 51 (1996) 676 – 684. [23] K.S. Love, R.R. Lakshmanan, J.H. Butterfield, C.C. Fox, Cell. Immunol. 170 (1996) 85 – 90. [24] J.W. Coleman, M.G. Buckley, A.M. Taylor, E.M.S. Banks, C.M.M. Williams, M.R. Holliday, J. Thompson, Int. Arch. Allergy Immunol. 107 (1995) 154 – 155. [25] B.L. Gruber, M.J. Marchese, R.R. Kew, J. Immunol. 152 (1994) 5860 – 5867. [26] N. Matsuura, B.R. Zetter, J. Exp. Med. 170 (1989) 1421–1426. [27] C.J. Meininger, H. Yano, R. Rottapel, A. Bernstein, K.M. Zsebo, B.R. Zetter, Blood 79 (1992) 958 – 963. [28] G. Nilsson, J.H. Butterfield, K. Nilsson, A. Siegbahn, J. Immunol. 153 (1994) 3717 – 3723. [29] N. Rossow, S.D. Sandy, H. Rephe, Biomed. Biochim. Acta 10 (1986) 1343 – 1347. [30] P.A. Shore, A. Burkhalter, V.H. Cohn, J. Pharmacol. Exp. Ther. 127 (1959) 182 – 186. [31] K. Nakajima, K. Hirai, M. Yamaguchi, T. Takaishi, K. Ohta, Y. Morita, K. Ito, Biochem. Biophys. Res. Commun. 183 (1992) 1076 – 1083. [32] A.M. Taylor, S.J. Galli, J.W. Coleman, Immunology 86 (1995) 427 – 433. [33] C.M. Hogaboam, A.D. Befus, J.L. Wallace, J. Immunol. 151 (1993) 3767 – 3774. [34] R. Alam, P.A. Forsythe, S. Stafford, M.A. Lett-Brown, J.A. Grant, J. Exp. Med. 176 (1992) 781 – 786. [35] J.W. Coleman, M.R. Holliday, I. Kimber, K.M. Zsebo, S.J. Galli, J. Immunol. 150 (1993) 556 – 562. [36] T.J. Lin, E.Y. Bissonnette, A. Hirsh, A.D. Befus, Immunology 89 (1996) 301 – 307. [37] M. Arock, C. Zuany-Amorim, M. Singer, M. Benhamou, M. Pretolani, Eur. J. Immunol. 26 (1996) 166 – 170. [38] E.Y. Bissonnette, B. Chin, A.D. Befus, Immunology 86 (1995) 12 – 17. [39] E.Y. Bissonnette, J.A. Enciso, A.D. Befus, Am. J. Respir. Cell Mol. Biol. 16 (1997) 275 – 282. [40] T.J. Lin, A.D. Befus, J. Immunol. 159 (1997) 4015 – 4023. [41] M.R. Holliday, E.M. Banks, R.J. Dearman, I. Kimber, J.W. Coleman, Immunology 82 (1994) 70 – 74. [42] B.B. Aggarwal, K. Natarajan, Eur. Cytokine Netw. 7 (1996) 93 – 124. [43] L. Rink, H. Kirchner, Int. Arch. Allergy Immunol. 111 (1996) 199 – 209. [44] J.R. Gordon, S.J. Galli, J. Exp. Med. 174 (1991) 103 –107. [45] E. Brzezin´ska-Blaszczyk, A. Pietrzak, Immunol. Lett. 59 (1997) 139 – 143. [46] F.J. Van Overveld, P.G. Jorens, M. Rampart, W. De Backer, P.A. Vermeire, Clin. Exp. Allergy 21 (1991) 711 – 714. [47] J.M. Hughes, R.S. Stringer, J.L. Black, C.L. Armour, Pulm. Pharmacol. 8 (1995) 31 – 36. [48] W.J. Beil, G.R. Login, S.J. Galli, A.M. Dvorak, J. Allergy Clin. Immunol. 94 (1994) 531 – 536. [49] W.J. Beil, G.R. Login, M. Aoki, L.O. Lunardi, E.S. Morgan, S.J. Galli, A.M. Dvorak, Int. Arch. Allergy Immunol. 109 (1996) 383 – 389.