INTERLEUKIN 8 INHIBITION OF CYTOKINE-INDUCED HISTAMINE RELEASE FROM BOSOPHILS OR MAST CELLS

Abstract: IL-8 inhibited HRF-induced histamine release from basophils obtained from 17 donors of 20 donors at concentrations as low as 10<-9> to 10<-8>M. The inhibition of histamine release by IL-8 was significantly higher in normal subjects than in allergic patients (59 +/- 8 % vs 31 +/- 7 %, p<0.04). IL-8 inhibited only cytokine-induced histamine release, and does not affect histamine release by anti-IgE, FMLP and C5a. A method for inhibiting release of allergic mediators with interleukin 8 is claimed.

Description: INTERLEUKIN 8 INHIBITION OF CYTOKINE-INDUCED HISTAMINE RELEASE FROM BOSOPHILS OR MAST CELLS FUNDING: Development of the present invention was aided in part by finding from NIH grant no. AI27864. Accordingly, the Federal Government may own certain rights. Histamine releasing factors (HRF) represent a group of cyto ines that release mediators from basophils and mast cells. Our prior related applications. Serial No. , 143,094 and its continuation. International Application No. WO 89/06545 (both of which are expressly incorporated herein by reference) , describe cytokines that inhibit histamine release induced with ononuclear cell-derived HRF. We have now discovered that recombinant interleukin 8 (IL-8) is a potent inhibitor of HRF-induced histamine release. Methods for inhibiting HRF-induced release of allergic mediators by exposing proallergic cells, such as mast cells and basophils, to an effective concentration of IL-8 are claimed. Basophils and mast cells have been the subjects of scientific investigation since they were described by Paul Ehrlich in the 1870s. Mast cells are generally found in connective tissue; basophils, in the blood. The two cell types have many similarities. Both contain numerous metachromatically staining granules, both have cell surface receptors that bind IgE with high affinity, and both contain a myriad of diverse allergic mediators that can cause symptoms ranging from itching of the skin to the most life-threatening clinical situation, anaphylaxis. Basophils and mast cells collectively account for virtually all of the total body histamine and are collectively referred to as histamine-containing proallergic cells. There is convincing evidence that mast cells and basophils are essential for induction of allergic hypersensitivity reactions. For example, substantial evidence suggests that mast cells are the primary effector cell of such allergic diseases as asthma, allergic rhinitis, conjunctivitis, urticaria and anaphylaxis. Increased numbers of mast cells have been found in bronchoalveolar lavage fluid, respiratory mucosa, nasal mucosa, and biopsy specimens of urticarial lesions in these disorders. In addition, mast cell mediators, such as histamine and prostaglandin (PG) D 2 , have been recovered from serum, bronchoalveolar lavage fluid, nasal washings, and skin blister fluid during natural and provoked allergic reactions. More recently, the mast cell granule-specific enzyme tryptase (not found in basophils) has been detected in serum from patients with allergic and anaphylactoid reactions. Finally, most mast cell-derived mediators, alone or in combination, can evoke such typical allergic symptoms as bronchospasm, angioedema, cough, mucus secretion, rhinorrhea, sneezing, and wheal-and-flare skin reactions and mast cell-specific degranulating agents can induce allergic reactions in vivo. In addition, histologic evidence suggests that mast cells are involved in the pathogenesis of a number of chronic inflammatory diseases. Increased numbers of mast cells have been detected in biopsy specimens from patients with rheumatoid arthritis, ulcerative colitis, Crohn's disease, sarcoidosis, hypersensitivity pneumonitis, pulmonary fibrosis, nasal polyps, atopic dermatitis, allergic contact dermatitis, bullous pemphigoid, keloid formation, scleroderma and progressive systemic sclerosis, acute and chronic graft vs. host disease, and parasitic infestation. Mast cells also have been implicated in regulation of nerve growth, and regulation of mast cell degranulation has proved useful in neurofibro atosis. Furthermore, increased levels of histamine were detected in the bronchoalveolar lavage fluid from patients with sarcoidosis, hypersensitivity pneumonitis, and idiopathic pulmonary fibrosis. Cromolyn sodium, a putative mast cell stabilizer, has been found beneficial to a subgroup of patients with ulcerative colitis, particularly those with proctitis. Basophils appear to be particularly involved in some forms of allergic contact dermatitis, especially to poison ivy and, experimentally, to dinitrochlorobenzene. These cells represent 5% to 15% of the total infiltrating cells and are present within eight hours after application of the allergen to the skin. Given the number of inflammatory mediators released by mast cells and basophils, it is conceivable that sustained piecemeal degranulation of these cells contributes to the chronic inflammatory nature of the aforementioned disorders. Unfortunately, despite the convincing evidence linking mast cells and basophils to these and other disease states, the precise pathogenesis of mast cell and basophil dependent disorders, including allergic disease, is incompletely understood. For example, it is known that mast cells and basophils are stimulated to release histamine, leukotrienes, and other inflammatory mediators by the bridging of cell surface-bound IgE antibodies by appropriate allergens (or anti-IgE antibodies) , but the severity of a number of allergic diseases, for example bronchial asthma, rhinitis, and conjunctivitis, does not correlate with a patient's IgE level. Moreover, although mast cells are believed to play a role in various other diseases such as inflammatory bowel disease, rheumatoid arthritis, pulmonary fibrosis, and sarcoidosis, in the majority of these diseases, IgE antibody cannot be found. Therefore, it appears that other mechanisms of allergic mediator release play a critical role in pathogenesis of allergic diseases and other disorders mentioned above in which basophils and mast cells have been implicated. Elucidation of such mechanisms has been and remains the goal of many skilled medical scientists. One of the most exciting developments in this area was the discovery of histamine releasing activity (HRA) , cytokines designated herein as histamine releasing factor(s) (HRF) , by investigators working in the laboratories of the present inventors. Thueson, et al., fJ. Immunol.. 123:626 (1979); J. Immunol.. 123:633 (1979)) and Lett-Brown, et al., (Cell Immunol.. 87:434 (1984); Cell Immunol.. 87:445 (1984)) first reported that antigen or mitogen stimulated human mononuclear cells secrete a proteinaceous factor that induces release of histamine from basophils and mast cells. Other laboratories then confirmed the synthesis of HRF by mononuclear cells. It has now been shown that HRF is also synthesized by B- lymphocytes and T-lymphocytes, alveolar macrophages, platelets, neutrophils, and blood monocytes cultured in vitro. The wide variety of cell types reported to secrete HRF suggests that it has considerable biologic importance. In addition to mediating histamine release, HRF has been shown to induce secretion of leukotrienes and to be chemotactic for basophils and monocytes. (For a review, see Grant, et al.. Fed. Proc.. 45:2653 (1986), J. Allergy Clin. Immunol.. 77:407 (1986), and Alam, Insights in Allergy, Vol. 2, no. 6 (1987), CV Mosby, St. Louis, all incorporated herein by reference.) Numerous studies provide data directly supporting the importance of HRF as a mediator of human allergic disease. For example, an HRF-like material has been obtained from skin blister fluid obtained during the late allergic reaction, now considered an important factor in the pathogenesis of chronic asthma and other allergic conditions. HRF has also been recovered in nasal washings. In addition, HRF induces bronchoconstriction on inhalation by asthmatic subjects and a wheal-and-flare reaction in humans and non-human primates. Mononuclear cells from asthmatic patients have been shown to spontaneously produce relatively large amounts of HRF, and HRF production is enhanced on jln vitro incubation with specific allergen. Moreover, the magnitude of spontaneous HRF production correlates with the severity of bronchial hyperreactivity in asthmatic patients [Alam, et al., J. Allergy Clin. Immunol.. 79:103 (1987)]. These findings suggest that in asthmatic patients, increased spontaneous HRF production may cause a sustained release of allergic mediators from mast cells or basophils, resulting in chronic inflammation and ultimately leading to the development of bronchial hyperreactivity. In addition, although immunotherapy is a well accepted modality for treating allergic diseases, the mechanism of its action is still obscure. Changes in serum IgE antibody do not correlate well with the efficacy of immunotherapy. Serum IgG blocking antibody usually rises after prolonged treatment. Although some investigators have shown a correlation between the efficacy of immunotherapy and the level of IgG antibody, the correlation is often too tenuous to imply a casual relationship. Recently, however, one of the present inventors has discovered that immunotherapy abrogates the seasonal rise in HRF production and diminishes spontaneous HRF production in patients with clinical improvements. Thus, there is a high correlation between symptom-medication score and spontaneous HRF production (r=0.92, p=0.0002) . Given the important role played by HRF in the pathogenesis of allergic diseases and other disorders including the release of mediators from basophils and mast cells, it is likely that an agent capable of inhibiting HRF-induced mediator release could provide a valuable tool in treating mast cell/basophil dependent disorders, which include the allergic diseases. Our prior patent applications described a human histamine release inhibitory factor having a molecular weight of about 8,000 - 10,000 daltons. We have now determined that recombinant human Interleukin 8 (IL-8) (m.w. about 8,000 daltons) is an extremely potent inhibitor of HRF-induced histamine release. HRIF and IL-8 specifically antagonize cytokine mediated histamine release. IL-8 is secreted as a 79 amino acid peptide which then undergoes proteolytic cleavages yielding either a 77 or a 72 amino acid peptide both being active as neutrophil activator/attractant. (T. Yoshimura et al. , Proc. Natl. Acad. Sci. USA 84:9233 (1987); Leonard, E.J., and T. Yoshimura, Am. J. Respir. Cell Mo. Biol. 2:479 (1990); M. Baggiolini et al., J. Clin. Invest. 84:1045 (1989)). Leonard and Yoshimura have proposed the terms IL-8/NAP1 (neutrophil activating peptide) alpha, beta and gamma for the 79, 77 and 72 amino acid peptides respectively (T. Yoshimura et al., Proc. Natl. Acad. Sci. USA 84:9233 (1987)), and the generic term IL-8 is adopted herein as covering the each of those species. IL-8 is produced by many cells including monocytes, macrophages, lymphocytes, endothelial cells, fibroblasts and keratinocytes. Recombinant human IL-8 beta, a 77 amino acid peptide with alanine in its N-terminus, was used in the studies described below. According to a general embodiment of the present invention, there is provided a method for inhibiting HRF- induced release of an allergic mediator from pro-allergic cells comprising exposing the pro-allergic cells to interleukin 8. In specific embodiments, the pro-allergic cells comprise mast cells, or basophils, and the allergic mediator is histamine. It is contemplated that the method may be performed either in vitro or in vivo, preferably using human interleukin 8 and human pro-allergic cells. In a preferred embodiment the interleukin 8 is present at a concentration of at least about lO'-tø. Although either the 79, 77 or 72 amino acid species of IL-8 (also known as neutrophil activating peptide or leukocyte adhesion inhibitor) may be employed in accordance with the invention, use of the 77 amino acid species is preferred, as is use of a recombinantly produced IL-8. The invention also includes a method for inhibiting release of allergic mediators from pro-allergic cells comprising exposing the pro-allergic cells to an effective concentration of interleukin 8 to inhibit histamine release. In preferred embodiments, histamine release may be inhibited by at least about 10 to at least about 60 percent, at least about 15 to at least about 60 percent, and more preferably, at least about 30 to at least about 60 percent, when measured according to the protocol set forth here. These and other aspects of the invention will become more apparent from a description of particular embodiments when read in conjunction with the drawings. FIGURE 1. Inhibition of HRF-induced histamine release from basophils by IL-8. Leukocytes from 20 donors (ten allergic and ten normal subjects) were preincubated with various concentrations of IL-8 for 5 min and then challenged with mononuclear cell-derived HRF. The percent inhibition of histamine release from cells incubated with IL-8 as compared to buffer is shown. Mean histamine release by HRF was 50+8% for cells from allergic individuals and 38+7% for cells from non-allergic individuals. The difference in the inhibition of histamine release between the two groups was statistically significant (*) at p<0.04. FIGURE 2. Effect of IL-8 on histamine release from basophils by MNC-HRF, anti-IgE, FMLP and C5a. Leukocytes were preincubated with IL-8 (lO^) for 5 min and then challenged with a predetermined dose of various secretagogues. The number of experiments (N) is 20 for MNC-HRF, 10 for anti-IgE, and 3 for FMLP and C5a. Asterisk indicates statistical difference compared to control release at p<0.04. FIGURE 3. The requirement for preincubation of leukocytes with IL-8 was investigated. Cells were separately preincubated with buffer or with IL-8 for 5 minutes and then challenged with HRF. In another set of experiments, IL-8 and HRF were added simultaneously to the cells or HRF was preincubated with cells first and IL-8 was added 5 min later. Results of one of three experiments are shown. FIGURE 4. The effect of removal of IL-8 after preincubation on the inhibition of histamine release. After the preincubation with IL-8, leukocytes were washed 3x with 30 volumes of buffer. The cells were then challenged with HRF. In control experiments cells were preincubated with buffer and then treated as above. Results of one of three experiments are shown. FIGURE 5. IL-8-induced histamine release from basophils. Leukocytes from 20 donors were incubated with various concentrations of IL-8 for 45 min and the released histamine was measured. Spontaneous histamine release in the presence of buffer was subtracted. Basophils from 6 of twenty donors (two normals and four allergic subjects) responded to IL-8 beta. Results shown are mean+SEM of histamine release by basophils from the six responder donors. FIGURE 6. The effect of neutrophil depletion on IL-8- induced histamine release from basophils. Neutrophil-rich and neutrophil-depleted leukocytes from three donors were separated using sedimentation with hydroxyethyl starch and Ficoll-Hypaque gradient (sp. gr. 1.077) centrifugation respectively. The cells were then incubated with IL-8 and the released histamine was assayed. FIGURE 7. The effect of preincubation with IL-8 on subsequent IL-8-induced histamine release from leukocytes. Leukocytes from four preselected allergic donors were preincubated with different concentrations of IL-8 for 5 min and then challenged with IL-8 (lO-^) . Error bars were omitted for clarity (SD<1%). FIGURE 8. The combined action of IL-3, IL-8 and GM- CSF on basophils and comparison with MNC-HRF. Leukocytes from 7 allergic donors were incubated with IL-3 (1 ug/ml final concentration) , IL-8 (10"^) and GM-CSF (1 ug/ml) added simultaneously to the cells. The histamine release by these three cytokines was compared with that by MNC-HRF. The histamine release by IL-3, IL-8 and GM-CSF were 30%, 7% and 7% in SC, 15%, 6% and 12% in TW and 12%, 6%, and 0% in BW respectively. The release by the cytokines in other donors was negligible. EXAMPLE I. Materials and Methods A. Reagents. RPMI 1640 was obtained from GIBCO Laboratories, Grand Island, NY; human serum albumin, glutamine, Ficoll, Hypaque, Concanavalin A (Con A) , penicillin, streptomycin, FMLP and recombinant C5a from Sigma Chemicals Co. , St. Louis, MO; Hepes from Research Organics, Inc., Cleveland, OH; hydroxyethyl starch (HetaStarch) from American McGaw, Irvine, CA; human recombinant IL-3 and human recombinant endothelial IL-8 (77 amino acid peptide with alanine in its N-terminus) was obtained from Pepro Tech. Inc., Rocky Hills, NJ. A sample of IL3 (δxlO^/mg) and GM-CSF (1.7xl0 7 U/mg) were obtained from the Genetics Institute, Boston, MA, courtesy of Dr. Steven Clark; rabbit and-human IgE serum (460,000 IU/ml) was from Behring Diagnostics, Somervilie, NY. B. Generation of HRF -containing supernatant . Leukocytes were isolated from buffy coats obtained from normal blood bank donors. MNC were isolated by Ficoll-Hypaque gradient centrifugation as previously described (R. Alam et al., J. Clin. Invest. 82:2056 (1988)) and pulsed with Con A (25ug/ml in RPMI 1640 medium) for 4 hr, washed twice with Hanks' balanced salt solution, resuspended in RPMI 1640 medium, and then cultured for 72 hr. Supernatants were harvested and concentrated 5Ox using an Amicon ultrafiltration chamber with YM-5 filters (MW cut-off 5,000) and ultracentrifuged. Recovery of the activity was approximately 50-60%. The ultracentrifuged supernatant was applied to a TSK 2000 gel filtration column. The fractions containing HRF activity (15-40 KD) were pooled, and aliquoted. The aliquots were frozen at - 70 * C and used as a source of HRF. The concentration of protein of this preparation was 8 ug/ml. C. Isolation of peripheral blood leukocytes . Blood donors were selected from a large group of allergic and non-allergic subjects that were screened in our laboratory for histamine release to HRF, anti-IgE and C5a. Allergic status was defined by the presence of clinical symptoms, past allergic history and positive reaction to prick skin testing to a panel of local aeroallergens (32 allergens) . Venous blood from donors was anticoagulated with 10 M EDTA and sedimented with 1.5% hydroxyethyl starch for 30 min at room temperature (R. Alam et al. , J. Clin. Invest. 82:2056 (1988)). The leukocyte-rich buffy coat was collected and washed three times in HA buffer (Hepes buffered-saline, pH 7.4 and 0.03% human serum albumin) in a refrigerated centrifuge (4 * C) at 300 x g. The washed leukocytes were suspended in HACM buffer (Hepes buffered- saline, pH 7.4 0.03% human serum albumin, 2 mM CaCl 2 and 1 mM MgCl 2 ) . D. Histamine release assay. Aliquots of 50 ul of HRF, anti-IgE (1:3,000 of the stock solution, 460,000 IU/ml) or recombinant IL-8 (final concentrations: 10' 11 to 10"*-M) were incubated with 50 ul of leukocyte suspension for 45 min in a shaking water bath at 37 * C. Each experiment was done in duplicate. Four hundred microliters of HA buffer was added to each tube at the end of the incubation. After incubation the supernatants were separated from the cells by centrifugation at 600 x g for 5 min at 4 * C. The histamine content of the supernatants was measured using an automated fluormetric analyzer (R. Alam et al., J. Clin. Invest. 82:2056 (1988)). Spontaneous histamine release was assessed by incubating the cells in HACM buffer alone. The total histamine content of the cells was measured by lysing the cells with 3% perchloric acid. The percentage of histamine release was calculated according to the formula: [(histamine in the supernatant) lOO]/(total histamine in the cells). Spontaneous histamine release from the cells was usually less than 5%. The values of spontaneous histamine release were subtracted from the calculated histamine release. E. Histamine release inhibition assay. For the inhibition assay, 50 ul aliquots of cells were first incubated with various dilutions of IL-8 (10" n to 10" * -M) for 5 min at room temperature and then challenged with 50 ul of HRF, anti-IgE (1:3000 dilution), IL3 (1 ug/ml), C5a (1 ug/ml) or FMLP (1:3000 dilution) separately (all concentrations shown are final) . The cells were then further incubated for 45 min in a water bath at 37 * C and the supernatants were separated by centrifugation. Histamine content of the supernatant and total cellular content were determined as described above. A typical experimental protocol includes: Preincubation Challenge a. leukocytes + buffer + buffer b. leukocytes + buffer + HRF* c. leukocytes + IL-8 + buffer d. leukocytes + IL-8 + HRF* * or other secretagogues The percentage of inhibition of histamine release was calculated according to the formula (R. Alam et al., J. Clin. Invest. 82:2056 (1988)): [ (b-a) ] (d-c) ]X100/(b-a) Results are expressed as mean+SEM. Statistical analyses were done with Wilcoxon's rank sum test. II. Demonstration of histamine release inhibiting activity Of IL-8 In order to assess the inhibitory activity of IL-8, the e fect of preincubation of leukocytes with the cytokine on histamine release by MNC-HRF was studied on cells from 10 allergic and 10 non-allergic donors. IL-8 inhibited HRF-induced histamine release from basophils obtained from 17 of 20 subjects (Fig. 1) . Two of the non-inhibited leukocyte samples were obtained from allergic donors; the other was obtained from a non-allergic donor. As shown in Section III below, however, leukocytes from the two allergic donors released low levels of histamine upon exposure to IL-8 alone, but cells from the normal donor did not. A significant inhibitory activity (>15%) was apparent at 10"^, although in some donors this was apparent at 10" U M. The inhibition of histamine release was significantly higher in normal subjects than in allergic patients ( r >9+9% vs 31+7%, p>0.04. Fig. 1) at 10^. IL-8 does not affect histamine release induced with anti-IgE, FMLP and C5a (Fig. 2). IL-3 also causes histamine release from a subgroup of allergic patients. We have identified three allergic patients that respond to IL-3 among 30 routine blood donors in our laboratory. The following studies were performed to investigate whether IL-8 inhibited IL-3-induced histamine from basophils. Basophils from all three donors were studied for the inhibition of IL-3-induced histamine release by IL-8. IL-8 inhibited IL-3-induced histamine release from two donors. The release by IL-3 (1 ug/ml) from leukocytes preincubated with buffer were 40+1% and 15+0.5% from donor 1 and donor 2 respectively. When leukocytes were preincubated with IL-8 (10" 10 10' 6 M) , the release of by IL-3 were 31+1% and 7+0.3% respectively at the highest concentration of IL-8. The third donor did not show any inhibition. This particular donor is a possible non-responder to IL-8 since there was no inhibition of HRF- induced histamine release by IL-8 from this donor, either. The requirement of preincubation of basophils with IL-8 for optimal inhibition of histamine release was also investigated. In our in vitro system, IL-8 did not show any inhibitory activity when added to the cells simultaneously with MNC-HRF or added five min after MNC-HRF (Fig. 3) . In order to determine whether continued presence of IL-8 is necessary after preincubation with the cells, leukocytes were preincubated with IL-8 for five minutes, washed 3 times with 30 volumes of buffer, and then challenged with MNC-HRF. As shown in Figure 4, that procedure did not abrogate the inhibitory effect of IL-8. Thus, these results have established that IL-8 acts as a strong inhibitor of cytokine-induced histamine release at concentrations as low as 10 ~9 -10" 8 M. III. Studies on histamine releasing activity of IL-8 The results reported above are surprising in view of reports from other investigators (M.V. White et al., Immunol. Lett. 22:151 (1989); C.A. Dahinden et al., J. Exp. Med. 170:1787 (1989)) who have concluded that IL-8 is capable of inducing or potentiating histamine release from pro-allergic cells. Therefore, we investigated the histamine releasing activity of IL-8 using basophils from the same 20 donors described above. Using a concentration range of 10" 11 to 10"^, we found that IL-8 released histamine from basophils obtained from two of ten non-allergic donors and four of ten allergic donors only at the highest concentration tested (10" 6 M) , which was 100 to 1000 fold higher than the concentrations at which histamine release inhibition was observed. The histamine release ranged from 6-16% and the mean was 8.7+0.8% (Fig. 5) . The experiment was repeated on three responder donors on two separate occasions, and the results were reproduced. By increasing the concentration of IL-8 up to 3 x 10"*^, were able to observe a small increase in histamine release in the range of 15-20%. By comparison, anti IgE and MNC-HRF released 34+7% and 51+7% of histamine respectively from the same donors. Neutrophils have high affinity receptors for IL-8 (also described as neutrophil activating peptide 1) (T. Yoshimura et al., Proc. Natl. Acad. Sci. USA 84:9233 (1987)). Since our leukocyte preparation contained neutrophils, we postulated that the low histamine release from basophils by IL-8 might be due to the avid binding of the cytokine to neutrophils. Therefore we have compared IL-8 activity using neutrophil-depleted mononuclear cell preparations that contained 2-3% of basophils with leukocyte preparations containing approximately 60% neutrophils. Neutrophil-depleted mononuclear cells (less than 3% neutrophils) were purified by Ficoll-Hypaque gradient (sp. gr. 1.077). As shown in Figure 6, simultaneous experiments done with neutrophils-depleted and neutrophil-rich preparations did not show any difference in histamine release. Since IL-8 did release a low amount of histamine from cells of selected donors, we performed the following experiment in order to determine whether inhibition of HRF- induced histamine release by IL-8 could be due to specific desensitization (down regulation of receptors) . For that experiment, leukocytes obtained from selected IL-8 responder donors were preincubated with various concentrations of IL-8 and then challenged with 10"^ of IL-8. Results of four experiments are shown in Fig. 7. Although a moderate desensitization was observed in two donors at lower concentrations of IL-8, results were equivocal in the other two subjects. Even among the responders, however, very low histamine release was obtained upon exposure of the cells to IL-8, thus, complicating the interpretation of the result. In other experiments, we studied the priming effect of IL-3 on IL-8-induced histamine release described by Dahinden et al fJ. Exp. Med. 170:1787 (1989)). Leukocytes from 7 donors, six who did not respond to IL-8 and one who did, were preincubated with IL-3 (25 ng/ml) for 5 min, and then challenged with IL-8 at concentrations 10" 9 to 10"^. No histamine release was observed with cells from the six non- responder donors. Cells from the one responder donor demonstrated a synergistic effect of IL-3 and IL-8. The release by IL-8 was 5+0.1% and 8+0.1% at 10" 7 and 10"^ concentrations respectively. IL-3 alone (25 ng/ml) released 8+0.2% of histamine. Preincubation with IL-3 followed by incubation with IL-8 cause 20+0.5% and 32+1% of histamine release respectively. This particular allergic donor is highest "releaser 1 * of histamine among our donors, and he is also one of few donors who responds to both IL-3 and IL-8. We have demonstrated that IL-3 and GM-CSF release histamine from basophils of selected donors at high concentrations (1 ug/ml) (R. Alam et al., J. Immunol. 142:3431 (1989)). We, therefore, asked whether the combined action of IL-3, IL-8 and GM-CSF would mimic the activity of mononuclear cell-derived HRF in cells from those donors. Seven patients were studied using relatively high concentrations of IL-3 (final concentration of 1 ug/ml), IL-δflO-^M) and GM-CSF (1 ug/ml). Three allergic patients released significant amounts of histamine, although less than MNC-derived HRF. Four other allergic subjects did not respond to IL-3, IL-8 and GM-CSF, but did release histamine in response to MNC-derived HRF (Fig. 8) . We did not perform any experiments with basophils from healthy controls since their leukocytes, in general, do not respond to IL-3, GM-CSF(2) and IL-8 as shown above. In contrast, MNC-derived HRF released histamine from basophils obtained from most normal donors. CLINICAL APPLICATIONS Due to precautions necessarily attendant to development of every new pharmaceutical, the present invention has not yet been tested in a clinical setting in human subjects. Therefore, the in vitro activity of Interleukin 8 in inhibiting histamine release has been used to demonstrate the utility of the present invention as a pharmacologic agent since the histamine release assay is accepted by those of skill in the art as a reliable correlate of in vivo histamine release. The following prophetic embodiments represent the best mode contemplated by the present inventors for carrying out the practice of the invention in various clinical settings. First, it is believed that the Interleukin 8 will prove to be useful in treating numerous diseases in which mast cells and basophils are involved, especially the allergic disorders. In particular, these include, but are not limited to, bronchial asthma, allergic rhinitis, conjunctivitis, and urticaria. Although the best mode of administering the factor will depend on the particular clinical situation, it is believed that the factor may be most easily administered by formulating it together with a suitable pharmaceutical excipient and administering the formulation topically. For example, the factor could be formulated as a component of an aerosol for intranasal or intrabronchial administration. These delivery devices might be modified to be powered by freon. This mode of administration may be particularly useful in treating certain allergic diseases, for example, allergic rhinitis and bronchial asthma. The factor could also be administered topically to the skin or eye; this formulation might prove effective for allergic disorders at these sites. Alternatively, the factor could be formulated for intravenous, intramuscular, subcutaneous, intradermal, or intraarticular injection; such injections might be used to treat inflammatory reactions at these sites in which the triggering of mediator release from basophils and mast cells is involved in the pathogenesis of the illness. In all these formulations, suitable excipients, for example, saline or physiologic buffers, are known to those of skill in the art and may be used. Of course, in some cases, it may be desirable to incorporate a preservative into this excipient. Methods for incorporating therapeutic agents into pharmaceutical vehicles are believed to be well within the skill of the art. As stated above, the invention has not yet been used in clinical settings. The inventors have relied upon the published results with other cytokines in predicting the acceptable pharmaceutical dosage for Interleukin 8. The most relevant studies of an aerosol were the use of intranasal alpha 2-interferon to prevent viral upper respiratory infections. Douglas, et al., (New Engl. J. Med.. 314:65 (1986)) and Hayden, et al. , (New Engl. J. Med.. 314:71 (1986)) administered 5 X 10 6 international units (IU) per day for an effective response. This cytokine is currently licensed for treatment of hairy cell leukemia at a dose of 3 X 10 6 IU per day by intramuscular or subcutaneous administration. Nathan, et al., (New Engl. J. Med.. 315:6 (1986)) administered 20,000 to 200,000 U of interferon-gamma intradermally to persons with lepromatous leprosy for a therapeutic response. The development and clinical use of interferons has recently been reviewed by Baron, et al., The Interferon System: A Current Review to 1987. University of Texas Press, Austin. As reported in New Engl. J. Med.. 313:1485 (1985), interleukin-2 has been administered intravenously to patients with cancer at doses of 10 4 to 10 5 units per kg over eight hours and maximal dose of up to 3.3 X 10 6 per kg. Finally, Vakhan-Raj, et al., (New Engl. J. Med.. 317:1545 (1987)) recently injected G/M CSF by continuous infusion at doses of 1.5 to 25 X 10 6 units/M 2 of body surface for an effective response in patients with myelodysplasia. Therefore, the inventors would propose that the effective dose of Interleukin 8 will be from about 10 4 to about 10 7 units. Furthermore, the inventors would define a unit in the traditional manner: 1 unit causes 50% inhibition of near maximal histamine release from HRF-stimulated basophils. The exact doses of Interleukin 8 to be used in a particular clinical application must be determined by accepted pharmaceutical methods known to those skilled in the pharmaceutical arts. * * * * * * * * * The foregoing description of the invention has been directed to particular preferred embodiments in accordance with the requirements of the patent statutes and for purposes of explanation and illustration. It will be apparent, however, to those skilled in the art that many modifications and changes may be made without departing from the scope and the spirit of the invention. REFERENCES This application contains a number of references which may facilitate understanding or practice of certain aspects of the present invention. Inclusion of a reference in this application is not intended to and does not constitute an admission that such reference represents prior art with respect to the present invention.

Claims: -20- Claims : 1. A method for inhibiting HRF-induced release of an allergic mediator from pro-allergic cells comprising exposing the pro-allergic cells to interleukin 8. 2. The method of claim 1 wherein said pro-allergic cells comprise mast cells. 3. The method of claim 1 wherein said pro-allergic cells comprise basophils. 4. The method of claim 1 wherein said allergic mediator is histamine. 5. The method of claim 1 wherein said method is performed in vitro. 6. The method of claim 1 wherein said interleukin 8 is human interleukin 8. 7. The method of claim 1 wherein said interleukin 8 is present at a concentration of at least about 10-9 M. 8. The method of claim 1 wherein said interleukin 8 comprises 77 amino acids. 9. The method of claim 1 wherein said interleukin 8 comprises 79 amino acids. 10. The method of claim 1 wherein said interleukin 8 comprises 72 amino acids. 11. A method for inhibiting release of allergic mediators from pro-allergic cells comprising exposing the pro¬ allergic cells to an effective concentration of interleukin 8 to inhibit histamine release.

Inventors: ALAM RAFEUL, GRANT J ANDREW, LETT-BROWN MICHAEL A, ALAM, RAFEUL, GRANT, J., ANDREW, LETT-BROWN, MICHAEL, A.
Applicants: UNIV TEXAS, BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM
Application year: 1991
Main classification: A61K37
IPC codes: A61K38, A61P37, C07K14, C12P21