(g) 1991 S. K argcr AG, Basel 0020-5915/91/0 944-0127 S 2.75/0
Int Arch Allergy Appl Immunol 1991;94:127-132
Structural Cell-Derived Cytokines in Allergie Inflammation Judah A. Denburg, Jack Gauldie, Jerry Dolovich, Takayuki Ohtoshi, Gerard Cox, Manel Jordana McMaster University, Hamilton, Canada
Introduction The concept of an interaction between cells resi dent in tissues (structural cells) and inflammatory cell progenitors or mature forms, the latter representing the effector component of allergic inflammatory reac tions, has been supported by evidence in experimen tal and clinical models. Both in rodents and man, for example, progenitors of basophils, mast cells or eo sinophils circulate in the blood or lymph, and can be shown in vivo to traffic to tissues where they ap pear to undergo terminal differentiation into mature effector cells [1-4]. Examples include graft-versus-
host disease [4] and nematode infestation leading to mast cell hyperplasia [1, 2]; moreover, there is substantial in vitro evidence for microenvironmen tal influences on cell phenotype or activation state [5-7]. In man, studies by our group over the years, sup ported by observations in vitro from several labora tories, have provided evidence for the circulation, fluctuation and differentiation of inflammatory cell progenitors as an important component of inflam mation in allergy [8-12]. Since the severity of symp toms or activity of an allergic reaction cannot be related directly to IgE levels and therefore the degree
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
Abstract. Based on observations of fluctuations in progenitors for inflammatory cells during allergic re sponses, we have proposed that a primary determinant of allergic inflammation involves microenvironmental influences on hemopoietic cell differentiation and phenotype; in addition, as a corollary of this, inflammatory cell burden is proposed as an important indicator of the severity and pattern of the inflammatory process in al lergy. The studies outlined here focus on the effects of epithelial-cell- and fibroblast-derived cytokines on gran ulocytic and monocytic cell differentiation and activation in models involving allergic reactions in the upper and lower airways. Pure cultures of nasal or bronchial epithelial cells or fibroblasts are observed to give rise to cytokines important in inducing the differentiation of basophils, eosinophils, neutrophils and monocyte/macrophages. Gene expression, production and secretion of granulocyte/macrophage-colony-stimulating factor, interleukin-6 (1L-6) and IL-8 can be demonstrated in vitro and in vivo. Up-regulation of gene expression and production of these cytokines, which are important in inducing basophil, eosinophil and neutrophil/macrophage differentiation in several assays, is seen with IL-1 and the neuropeptide substance P; conversely, inhibi tion of cytokine production by structural cells is observed after pretreatment with corticosteroids in vitro, par alleling in vivo effects. Other modulatory effects also examined include: antiallergic compounds, which may affect posttranscriptional events in cytokine production, and heavy metal ions, which can also induce changes in gene expression. Structural-cell-derived extracellular matrices appear also to be important both in mast cell differentiation and in macrophage cytokine gene expression, both of which potentially feedback upon chronic allergic inflammatory processes, leading to their perpetuation. On the basis of these studies, it is proposed that microenvironmental controls of the inflammatory process involve the initiation and perpetuation of allergic inflammation through effects on cell differentiation by altered structural cells resident in the tissue. In vivo models to directly test this hypothesis are currently under exploration.
Denburg/G auldie/D olovich/O htoshi/Cox/Jordana
128
Structural Cells Derived from Allergic and Related Tissues Epithelial Scrapings Initially, we performed studies on scrapings de rived from nasal mucosa in both allergic rhinitis and nasal polyposis, which demonstrated the elaboration, from tissue cultures of nasal mucosal scrapings (con sisting of mixed cell populations), of hemopoietic growth factor activity as measured in colony-forming assays in methylcellulose. Specifically, allergic rhini tis nasal mucosal epithelial scrapings were shown to produce a higher colony-stimulating activity in vitro than normal nasal mucosal epithelial scrapings; this activity was most pronounced when supernatants of epithelial cultures from individuals with allergic rhi nitis were used to stimulate circulating (activated?) progenitor cell populations from this group of pa tients, as opposed to those from nonatopic subjects [14-17], Moreover, colony-stimulating activity was elabo rated in high amounts into supernatants of nasal mu cosal epithelial scrapings taken from excised polyp tissue prepared ex vivo [14, 17]. In the context of doc umentation that both allergic rhinitis and nasal polyp tissues contain, throughout the epithelial and subepithelial-stromal levels, significant numbers of muco sal mast cells as well as (in the situation of polyps) activated eosinophils [18, 19], these initial culture studies suggested that immature, inflammatory cell progenitors may be influenced locally by epitheliumand subepithelium-derived factors, among which were the hemopoietic colony-stimulating factors. Moreover, T cell subpopulations present within the peripheral-blood compartment of polyp individuals also appeared to enhance the effect of epithelium-de rived colony-stimulating factors [14]. Lastly, in one important study, a highly enriched, low-frequency metachromatic cell progenitor could be shown to be resident within nasal polyp mononuclear cell popula
tions [17]. These preliminary studies led to the formu lation of the hypothesis of ‘in situ hemopoiesis’ as an active mechanism in inflammatory cell recruitment [ 20 ,
21 ],
Pure Cultures of Structural Cells from Airway Tissues It was important to us to isolate and identify those structural cells which were giving rise to the colonystimulating factors and other presumably important activities related to inflammatory cell differentiation, activation and recruitment. Using modifications of methods previously described for the isolation of ep ithelial and fibroblast cell lines, we derived pure cul tures of nasal or bronchial epithelial cells and fibro blasts from individual patients with nasal polyposis, allergic rhinitis, pulmonary fibrosis and nonatopic, normal controls [21-23]. The culture methods for the derivation of epithelial or fibroblast lines, including pulmonary fibroblasts, are detailed elsewhere [24-27]; fundamentally, these involve variations in the pres ence of fetal calf serum as well as of digestion proce dures for tissues.
Cytokines Derived from Structural Cells Detection o f Cytokine Activities in Bioassays The presence of specific, hemopoietic cytokines in supernatants from airway structural cells was first de tected in bioassay systems, using standard hemopoiet ic assays in methylcellulose (which detect progeni tors) and leukemic cell lines (e.g. HL-60) inducible to differentiation along several of the granulocytic line ages, including basophilic [27]. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) was the principle hemopoietic activity elaborated by nasal or bronchial epithelial cells and fibroblasts; this could be demonstrated formally by abrogation of all colonystimulating or differentiation-inducing activities elab orated by these pure structural cell cultures through the use of monoclonal, specific, neutralizing antibod ies to GM-CSF [21, 22]. The presence of GM-CSF ac counted fully for differentiation of progenitors into eosinophils and neutrophils; GM-CSF and other, as yet unidentified, factors were important in basophilic and monocytic differentiation [21-27], depending on culture conditions (using the HL-60 leukemic cell line; table 1). The metachromatic cells differentiating in the
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
of sensitization per se [13], the effector component involving progenitors and their corresponding, ma ture inflammatory cell types has received much wider attention in recent years. The purpose of the present paper is to provide information extending our initial observations on the contribution of structural cells, through the elaboration of specific cytokines, to aller gic inflammation.
Cytokines in Allergie Inflammation
Immunoassay of Cytokines To confirm and quantitate the presence of the cyto kines mentioned above, immunoassays were per formed for GM-CSF, IL-6 and IL-8 in epithelial cell and Fibroblast supernatants derived from individual patient cell lines. The levels of these cytokines were found to be elevated in nasal polyp-derived structural cell culture supernatants, into which they were constitutively produced; levels were intermediate but still elevated from allergic rhinitis structural cells, and were either undetectable or very low when derived from normal nasal mucosal cells [21, 22, 25, 26], The levels of GM-CSF, for example, correlated well with the bioassay results showing the same hierarchical re lationship among polyposis, allergic rhinitis and nor mals (table 2). Evidence o f Gene Activation of Cytokines in Structural Cells Relevant to the above, evidence for increased ex pression of genes for GM-CSF, IL-6 and IL-8 was provided by studies of messenger RNA using oligo nucleotide and full-length cDNA probes; constitutive GM-CSF, IL-6 and IL-8 gene expression was highest in structural cells derived from nasal polyp tissues [22, 23] (fig. 1). Other Assays To further confirm the presence of GM-CSF, the effect of airway structural-cell-derived supernatants on eosinophil survival was measured (fig. 2); both fibroblast- and epithelial-cell-derived GM-CSF ac counted for all of the eosinophil survival-promoting activities elaborated by these cells [30,311.
Table 1. Effects of human airway structural cell supernatants on inflammatory cell differentiation Cell source
Conditions
Differentiation
Cytokine
Epithelial cell
pH 7.4
neutrophilic monocytic1
GM-CSF G-GSF other
Epithelial cell
pH 7.8
basophilic eosinophilic11
GM-CSF
Fibroblast
pH 7.4
neutrophilic
GM-CSF
Fibroblast
pH 7.8
basophilic eosinophilic6
other
a Predominant monocyte/macrophage differentiation; not rec onstituted with rGM-CSF alone or in combination with other known hemopoietic cytokines [24]. 6 Constitutive eosinophilic differentiation after alkaline passage in the presence of 0.3 mM sodium butyrate; the addition of GMCSF enhances basophilic differentiation, in concert with eosino philic lineage expression [27].
Table 2. GM-CSF gene expression and production by structural cells from different patient populations Method of assessment“
Clinical correlations
mRNA Bioassay Immunoassay
N P>A R > NN NP = AR> NN NP> AR> NN
NP = Nasal polyposis; AR = allergic rhinitis; NN = normal nasal mucosa. a mRNA: slot blot or Northern blot, using oligonucleotide probe for GM-CSF [22, 31]; bioassav: HL-60 or methylcellulose assays; abrogation by 1:50 to 1:200 dilution of neutralizing, monoclonal anti-GM-CSFantibody [25,27]; immunoassay: ELISA [31].
Regulation o f Cytokine Production and Expression by Structural Cells IL-1. IL-1 up-regulated gene expression and pro duction of GM-CSF as well as IL-6 and IL-8 from air way fibroblasts but not from epithelial cells [21, 22, 24, 25]. This accords with observations on endothelial cells and fibroblasts derived from other sources, which suggest a cascade of effects from initial inflam matory signals such as IL-1 and tumor necrosis factor-a to the elaboration of cytokines such as GM-CSF and IL-6, leading to further stages of inflammation.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
HL-60 and colony assays appeared to resemble baso phils, not mast cells, although certain phenotypic fea tures suggest the possible presence of an early, imma ture cell type intermediate between the two [28]. In methylcellulose cultures, the effect of GM-CSF de rived from airway structural cells was most pro nounced on the basophil/eosinophil progenitor pop ulation [21]. In other bioassays both interleukin-6 (IL-6) and IL-8 activities could be shown to also be present in structural-cell-derived supernatants [21,22, 27]. These activities included hepatocyte-stimulating as well as neutrophil chemotactic activities [29], both of which could be abrogated by specific, neutralizing antibod ies (to IL-6 or IL-8, respectively) in the bioassays.
129
130
Denburg/G auldie/D olovich/O htoshi/Cox/Jordana
1L-6
IL-8
2 3 4 5
6 Calf liver
Fig. 1. Slot blot demonstrating mRNA expression of 1L-6, IL-8 and GM-CSF by human upper airway epithelial cells derived from nasal polyps.
100 80 |
c 15
60 -
I
40-
w 200
i----------1--------- 1----------1--------- 1--------- 1--------- 1--------- ;----------1
0.1
0.5
1
2.5
5
10
50
100
Conditioned medium, %
Fig. 2. Eosinophil survival at day 3 in vitro in the presence of bronchial epithelial cell ( • ) and fibroblast (O) supernatants. In the absence of conditioned medium there is negligible eosinophil sur vival. The active moiety in these conditioned media which is re sponsible for this effect can be shown to be GM-CSF [30],
Neuropeptides. The neuropeptide substance P could also be shown to up-regulate the expression of IL-6 and IL-8 derived from airway structural cells; the production of GM-CSF, however, in the situation of nasal polyp structural cell lines, was constitutively high and could not be further up-regulated by sub stance P. These studies form a basis for further explo ration of neuropeptides in inflammatory cell recruit ment. Modulation by Antiallergic Compounds. Corticoste roids were shown to diminish or even abrogate gene expression or production of GM-CSF, IL-6 and IL-8 by airway epithelial cells and fibroblasts [21, 22, 24, 25, 30, 31]; an antihistaminic compound, ketotifen, decreased cytokine production at a posttranscrip
tional level. In the case of corticosteroids, a topically active compound, budesonide, when given by inhala tion in vivo had effects on the circulating numbers of mature basophils, eosinophils as well as their pro genitors, and was a potent suppressor of GM-CSF production by structural cells [32]. This effect may be of major importance in the clinical activity of inhaled steroids in allergic rhinitis and asthma, and suggests that the airway epithelium in vivo may secrete GMCSF and other progenitor-influencing cytokines into the circulation, mimicking effects seen when the pure cytokine is given in vivo to primates [33,34], Structural-Cell-Derived Extracellular Matrices Not only are structural-cell-derived cytokines ap parently important in the regulation of inflammatory cell recruitment and activation as well as differentia tion, but the extracellular matrices laid down by these cells may be pivotal in determining the biologic con sequences of the cytokines elaborated at any given time. Studies still in progress reveal that both for mac rophages and mast cells upper airway structural-cellderived extracellular matrices have a profound effect on activation and, possibly, phenotype switch of these inflammatory cells, which may be relevant and complementary to those effects we have found in cell differentiation assays.
Conclusions The studies described above form the basis for the microenvironmental differentiation hypothesis of aller gic inflammation in man[22]. Events occurring in or at mucosal surfaces, involving a complex network of structural cells, cytokines (including neuropeptides) and inflammatory cells in immature or mature forms are part of a process leading to the perpetuation of in flammation of allergic and related types. Thus, whether or not sensitization occurs via IgE, and not withstanding the intensity of the initiating signal in an allergic reaction, the maintenance and chronicity of inflammatory processes at tissue sites is hypothe sized to be under control by structural cell popula tions. Whether or not structural cells in atopy and re lated disorders such as polyposis and asthma are con stitutionally different from normal, and how they have developed their capacity for continual elabora tion of proinflammatory and hemopoietic cytokines remains to be elucidated. In vivo models to test the
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
G M -C S F
hypothesis of structural-cell-derived cytokines are currently being pursued in parallel to in vitro studies. Detection of the clinically relevant presence of struc tural-cell-derived cytokines in natural or provoked allergic reactions can now be assessed.
References 1 Denburg JA, Befus AD, Bienenstock J: Growth and differentia tion in vitro of mast cells from mesenteric lymph nodes of Nipposlrongylus brasiliensis-infected rats. Immunology 1982;41: 195-202. 2 Haig DM. McKee TA. Jarrett EEE, Woodbury R, Miller HPR: Generation of mucosal mast cells is stimulated by in vitro fac tors derived from T cells of helminth-infected rats. Nature 1982: 300:180. 3 Crapper RM, Schrader JW: Frequency of mast cell precursors in normal tissues determined by an in vitro assay: Antigen in duces parallel increases in the frequency of P-cell precursors and mast cells. J Immunol 1983;131:923-928. 4 Guy-Grand D, Dy M, Luffau L, Vassalli P: Gut mucosal mast cells: Origin, traffic and differentiation. J Exp Med 1984:160: 12-28. 5 Owen WF Jr, Rothenberg ME, Silberstein DS, Gasson JC, Ste vens RL, Austen KF, Soberman RJ: Regulation of human eosinophil viability, density and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fi broblasts. J Exp Med 1987;166:129-141. 6 Rothenberg ME, Owen WF Jr, Silberstein DS, Soberman RJ, Austen KF, Stevens RL: Eosinophils co-cultured with endo thelial cells have increased survival and functional properties. Science 1987;237:645-647. 7 Levi-Schaffer F, Austen KF, Caulfield J. Hein A, Bloes WF, Stevens RL: Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro. J Immunol 1985:135:3454-3462. 8 Denburg JA, Telizyn S, Belda A, Dolovich J, Bienenstock J: In creased numbers of circulating basophil/mast cell progenitors in atopic patients. J Allergy Clin Immunol 1985;76:466-472. 9 Gibson P, Dolovich J, Hargreave FH, Denburg J A: The inflam matory response in asthma exacerbation: Changes in circulat ing eosinophils, basophils, and their progenitors. Clin Exp Al lergy 1990:20:661-668. 10 Otsuka H, Dolovich J, Befus AD, Telizyn S, Bienenstock J, Denburg JA: Basophilic cell progenitors, nasal metachromatic cells and peripheral blood basophils in ragweed allergic patients.J Allergy Clin Immunol 1986;78:365-371. 11 Otsuka H, Dolovich J, Befus AD, Bienenstock J, Denburg J: Basophilic cell progenitors, nasal metachromatic cells in pa tients with allergic rhinitis. Am Rev Respir Dis 1986:133: 757-762. 12 Otsuka H, Dolovich J, Bienenstock J, Denburg JA: Metachro matic cell progenitors and specific growth and differentiation factors in human nasal mucosa and polyps. Am Rev Respir Dis 1987:136:710-717. 13 Nickelsen JA, Georgitis JW, Reisman RE: Lack of correlation between titers of serum allergen-specific IgE and symptoms in
131
untreated patients with seasonal allergic rhinitis. J Allergy Clin Immunol 1986:77:43-48. 14 Ohnishi M, Ruhno J, Bienenstock J, Milner R, Dolovich J, Denburg JA: Human nasal polyp epithelial basophil/mast cell and eosinophil colony-stimulating activity. The effect is T-celldependent. Am Rev Respir Dis 1988:138:560-564. 15 Ohnishi M, Ruhno J, Dolovich J, Denburg J A: Allergic rhinitis nasal mucosal conditioned medium stimulates growth and dif ferentiation of basophil/mast cell and eosinophil progenitors from atopic blood. J Allergy Clin Immunol 1988:81:1149-1154. 16 Ohnishi M, Ruhno J, Bienenstock J, Dolovich J, Denburg JA: Hemopoietic growth factor production by human nasal polyp epithelial scrapings: kinetics, cell source and relationship to clinical status. J Allergy Clin Immunol 1989;83:1091-1099. 17 Otsuka H, Denburg JA, Dolovich J, Hitch D, Lapp P, Rajan RS, Bienenstock J, Befus AD: Heterogeneity of metachromatic cells in human nose: Significance of mucosal mast cells. J Al lergy Clin Immunol 1985:76:695-702. 18 Ruhno J, Howie K, Anderson M, Anderssen B, Vanzieleghem M, Hitch D, Lapp P, Denburg J, Dolovich J: Rhinitis of nasal polyps: The increased numbers of epithelial mast cells in nasal polyps and adjacent turbinates are not related to allergy. Al lergy 1990;45:1-5. 19 Kawabori S, Denburg JA, Schwartz LB, Irani A-M, Wong D, Jordana G, Evans S, Dolovich J: Histochemical and immuno histochemical studies of nasal polyp mast cells. J Allergy Clin Immunol, submitted. 20 Denburg JA, Dolovich J: In situ hemopoietic mechanisms un derlying allergic tissue inflammation. Pharmacia Allergy Re search Foundation Award Book. Allergy I988;(suppl):20-25. 21 Denburg JA, Jordana M, Vancheri C, Gauldie J, Harnish D, Ohtoshi T, Tsuda T, Gibson P, Ruhno J, Bienenstock J, Har greave FE, Dolovich J: Locally derived hemopoietic cytokines for human basophils, mast cells and eosinophils in respiratory disease; in Galli S, Austen KF (eds): Human Mast Cell and Ba sophil Functional Differentiation in Health and Disease. New York, Raven Press, 1988, pp 59-69. 22 Denburg JA, Dolovich J, Ohtoshi T, Cox G, Gauldie J. Jordana M: The microenvironmental differentiation hypothesis of air way inflammation. Am J Rhinol 1990;4:29-34. 23 Dolovich J, Ohtoshi T, Jordana M, Gauldie J, Denburg JA: Na sal polyps: Local inductive microenvironment in the pathogen esis of the inflammation: in Pipkorn U (ed): Rhinitis and Asthma - Similarities and Differences. Copenhagen, Munksgaard, 1990, pp 233-241. 24 Ohtoshi T, Vancheri C, Cox G, Gauldie J, Dolovich J, Denburg JA, Jordana M: Monocyte-macrophage differentiation induced by human upper airway epithelial cells. Am J Respir Cell Mol Biol, in press. 25 Ohtoshi T, Tsuda T, Vancheri C, Abrams JS, Gauldie J, Dolo vich J, Denburg JA, Jordana M: Human upper airway epithel ial cell-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) induces histamine-containing cell differentia tion of human progenitor cells. Submitted. 26 Vancheri C, Ohtoshi T, Cox G, Abrams JS, Xaubet A, Gauldie J, Dolovich J, Denburg JA, Jordana M: Neutrophilic differen tiation induced by human upper airway fibroblast-derived granulocyte-macrophage colony-stimulating factor (GM-CSF). Am J Respir Cell Mol Biol 1991 ;4:11-17. 27 Hutt-Taylor SR, Harnish D, Richardson M, Ishizaka T, Den-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
Cytokines in Allergie Inflammation
28
29
30
31
burg JA: Sodium butyrate and T-lymphocyte cell line-derived differentiation factor induce basophilic differentiation of the human promyelocytic leukemia cell line HL-60. Blood 1988;71: 209-215. Wong Da, Kawabori S, Switzer J, Valent P, Bettelheim P, Dolovich J, Ishizaka T, Jordana M, Denburg J: Characterization of histamine-containing basophilic cells after in vitro induction of HL-60 cell line (abstract). J Allergy Clin Immunol 1990:85:173. Gauldie J, Richards C, Harnish D, Landsdorp P, Baumann H: Interferon p,/B-cel! stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regu lates the major acute phase protein response in liver cells. Proc Natl Acad Sci USA 1987;84:76251-7255. Cox G, Vancheri C, Ohtoshi T, Gauldie J, Dolovich J, Jordana M, Denburg JA: Human bronchial epithelial cell-derived gran ulocyte-macrophage colony stimulating factor (GM-CSF) pro longs survival of human eosinophils (abstract). J Allergy Clin Immunol 1990:85:233. Vancheri C, Gauldie J, Bienenstock J, Cox G, Scicchitano R, Stanisz A, Jordana M: Human lung fibroblast-derived granulo cyte-macrophage colony-stimulating factor (GM-CSF) medi ates eosinophil survival in vitro. Am J Respir Cell Mol Biol 1989;1:289-295.
Denburg/G auldie/D olovich/O htoshi/Cox/Jordana
32 Ohtoshi T, Xaubet A, Andersson B, Vanzieleghem M, Dolovich J, Jordana M, Denburg JA: Nasal inflammation mediated by human structural cell-derived GM-CSF: Effect of budesonide (abstract). J Allergy Clin Immunol 1990:84:297. 33 Mayer P, Valent P, Schmidt G, Liehl E, Bettelheim P: The in vivo effects of recombinant human IL-3: Demonstration of ba sophil differentiation factor, histamine-producing activity, and priming of GM-CSF-responsive progenitors in non-human pri mates. Blood 1989;74:613-621. 34 Valent P, Schmidt G, Besemer J, Mayer P, Zeuke G, Liehl E, Hinterberger W, Lechner K, Bettleheim P: lnterleukin-3 is a differentiation factor for human basophils. Blood 1989;73: 1763-1769.
Correspondence to: Dr. Judah A. Denburg McMaster University Hamilton, Ont. (Canada)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
132
Int Arch Allergy Appl Immunol 1991;94:127-132
Structural Cell-Derived Cytokines in Allergie Inflammation Judah A. Denburg, Jack Gauldie, Jerry Dolovich, Takayuki Ohtoshi, Gerard Cox, Manel Jordana McMaster University, Hamilton, Canada
Introduction The concept of an interaction between cells resi dent in tissues (structural cells) and inflammatory cell progenitors or mature forms, the latter representing the effector component of allergic inflammatory reac tions, has been supported by evidence in experimen tal and clinical models. Both in rodents and man, for example, progenitors of basophils, mast cells or eo sinophils circulate in the blood or lymph, and can be shown in vivo to traffic to tissues where they ap pear to undergo terminal differentiation into mature effector cells [1-4]. Examples include graft-versus-
host disease [4] and nematode infestation leading to mast cell hyperplasia [1, 2]; moreover, there is substantial in vitro evidence for microenvironmen tal influences on cell phenotype or activation state [5-7]. In man, studies by our group over the years, sup ported by observations in vitro from several labora tories, have provided evidence for the circulation, fluctuation and differentiation of inflammatory cell progenitors as an important component of inflam mation in allergy [8-12]. Since the severity of symp toms or activity of an allergic reaction cannot be related directly to IgE levels and therefore the degree
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
Abstract. Based on observations of fluctuations in progenitors for inflammatory cells during allergic re sponses, we have proposed that a primary determinant of allergic inflammation involves microenvironmental influences on hemopoietic cell differentiation and phenotype; in addition, as a corollary of this, inflammatory cell burden is proposed as an important indicator of the severity and pattern of the inflammatory process in al lergy. The studies outlined here focus on the effects of epithelial-cell- and fibroblast-derived cytokines on gran ulocytic and monocytic cell differentiation and activation in models involving allergic reactions in the upper and lower airways. Pure cultures of nasal or bronchial epithelial cells or fibroblasts are observed to give rise to cytokines important in inducing the differentiation of basophils, eosinophils, neutrophils and monocyte/macrophages. Gene expression, production and secretion of granulocyte/macrophage-colony-stimulating factor, interleukin-6 (1L-6) and IL-8 can be demonstrated in vitro and in vivo. Up-regulation of gene expression and production of these cytokines, which are important in inducing basophil, eosinophil and neutrophil/macrophage differentiation in several assays, is seen with IL-1 and the neuropeptide substance P; conversely, inhibi tion of cytokine production by structural cells is observed after pretreatment with corticosteroids in vitro, par alleling in vivo effects. Other modulatory effects also examined include: antiallergic compounds, which may affect posttranscriptional events in cytokine production, and heavy metal ions, which can also induce changes in gene expression. Structural-cell-derived extracellular matrices appear also to be important both in mast cell differentiation and in macrophage cytokine gene expression, both of which potentially feedback upon chronic allergic inflammatory processes, leading to their perpetuation. On the basis of these studies, it is proposed that microenvironmental controls of the inflammatory process involve the initiation and perpetuation of allergic inflammation through effects on cell differentiation by altered structural cells resident in the tissue. In vivo models to directly test this hypothesis are currently under exploration.
Denburg/G auldie/D olovich/O htoshi/Cox/Jordana
128
Structural Cells Derived from Allergic and Related Tissues Epithelial Scrapings Initially, we performed studies on scrapings de rived from nasal mucosa in both allergic rhinitis and nasal polyposis, which demonstrated the elaboration, from tissue cultures of nasal mucosal scrapings (con sisting of mixed cell populations), of hemopoietic growth factor activity as measured in colony-forming assays in methylcellulose. Specifically, allergic rhini tis nasal mucosal epithelial scrapings were shown to produce a higher colony-stimulating activity in vitro than normal nasal mucosal epithelial scrapings; this activity was most pronounced when supernatants of epithelial cultures from individuals with allergic rhi nitis were used to stimulate circulating (activated?) progenitor cell populations from this group of pa tients, as opposed to those from nonatopic subjects [14-17], Moreover, colony-stimulating activity was elabo rated in high amounts into supernatants of nasal mu cosal epithelial scrapings taken from excised polyp tissue prepared ex vivo [14, 17]. In the context of doc umentation that both allergic rhinitis and nasal polyp tissues contain, throughout the epithelial and subepithelial-stromal levels, significant numbers of muco sal mast cells as well as (in the situation of polyps) activated eosinophils [18, 19], these initial culture studies suggested that immature, inflammatory cell progenitors may be influenced locally by epitheliumand subepithelium-derived factors, among which were the hemopoietic colony-stimulating factors. Moreover, T cell subpopulations present within the peripheral-blood compartment of polyp individuals also appeared to enhance the effect of epithelium-de rived colony-stimulating factors [14]. Lastly, in one important study, a highly enriched, low-frequency metachromatic cell progenitor could be shown to be resident within nasal polyp mononuclear cell popula
tions [17]. These preliminary studies led to the formu lation of the hypothesis of ‘in situ hemopoiesis’ as an active mechanism in inflammatory cell recruitment [ 20 ,
21 ],
Pure Cultures of Structural Cells from Airway Tissues It was important to us to isolate and identify those structural cells which were giving rise to the colonystimulating factors and other presumably important activities related to inflammatory cell differentiation, activation and recruitment. Using modifications of methods previously described for the isolation of ep ithelial and fibroblast cell lines, we derived pure cul tures of nasal or bronchial epithelial cells and fibro blasts from individual patients with nasal polyposis, allergic rhinitis, pulmonary fibrosis and nonatopic, normal controls [21-23]. The culture methods for the derivation of epithelial or fibroblast lines, including pulmonary fibroblasts, are detailed elsewhere [24-27]; fundamentally, these involve variations in the pres ence of fetal calf serum as well as of digestion proce dures for tissues.
Cytokines Derived from Structural Cells Detection o f Cytokine Activities in Bioassays The presence of specific, hemopoietic cytokines in supernatants from airway structural cells was first de tected in bioassay systems, using standard hemopoiet ic assays in methylcellulose (which detect progeni tors) and leukemic cell lines (e.g. HL-60) inducible to differentiation along several of the granulocytic line ages, including basophilic [27]. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) was the principle hemopoietic activity elaborated by nasal or bronchial epithelial cells and fibroblasts; this could be demonstrated formally by abrogation of all colonystimulating or differentiation-inducing activities elab orated by these pure structural cell cultures through the use of monoclonal, specific, neutralizing antibod ies to GM-CSF [21, 22]. The presence of GM-CSF ac counted fully for differentiation of progenitors into eosinophils and neutrophils; GM-CSF and other, as yet unidentified, factors were important in basophilic and monocytic differentiation [21-27], depending on culture conditions (using the HL-60 leukemic cell line; table 1). The metachromatic cells differentiating in the
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
of sensitization per se [13], the effector component involving progenitors and their corresponding, ma ture inflammatory cell types has received much wider attention in recent years. The purpose of the present paper is to provide information extending our initial observations on the contribution of structural cells, through the elaboration of specific cytokines, to aller gic inflammation.
Cytokines in Allergie Inflammation
Immunoassay of Cytokines To confirm and quantitate the presence of the cyto kines mentioned above, immunoassays were per formed for GM-CSF, IL-6 and IL-8 in epithelial cell and Fibroblast supernatants derived from individual patient cell lines. The levels of these cytokines were found to be elevated in nasal polyp-derived structural cell culture supernatants, into which they were constitutively produced; levels were intermediate but still elevated from allergic rhinitis structural cells, and were either undetectable or very low when derived from normal nasal mucosal cells [21, 22, 25, 26], The levels of GM-CSF, for example, correlated well with the bioassay results showing the same hierarchical re lationship among polyposis, allergic rhinitis and nor mals (table 2). Evidence o f Gene Activation of Cytokines in Structural Cells Relevant to the above, evidence for increased ex pression of genes for GM-CSF, IL-6 and IL-8 was provided by studies of messenger RNA using oligo nucleotide and full-length cDNA probes; constitutive GM-CSF, IL-6 and IL-8 gene expression was highest in structural cells derived from nasal polyp tissues [22, 23] (fig. 1). Other Assays To further confirm the presence of GM-CSF, the effect of airway structural-cell-derived supernatants on eosinophil survival was measured (fig. 2); both fibroblast- and epithelial-cell-derived GM-CSF ac counted for all of the eosinophil survival-promoting activities elaborated by these cells [30,311.
Table 1. Effects of human airway structural cell supernatants on inflammatory cell differentiation Cell source
Conditions
Differentiation
Cytokine
Epithelial cell
pH 7.4
neutrophilic monocytic1
GM-CSF G-GSF other
Epithelial cell
pH 7.8
basophilic eosinophilic11
GM-CSF
Fibroblast
pH 7.4
neutrophilic
GM-CSF
Fibroblast
pH 7.8
basophilic eosinophilic6
other
a Predominant monocyte/macrophage differentiation; not rec onstituted with rGM-CSF alone or in combination with other known hemopoietic cytokines [24]. 6 Constitutive eosinophilic differentiation after alkaline passage in the presence of 0.3 mM sodium butyrate; the addition of GMCSF enhances basophilic differentiation, in concert with eosino philic lineage expression [27].
Table 2. GM-CSF gene expression and production by structural cells from different patient populations Method of assessment“
Clinical correlations
mRNA Bioassay Immunoassay
N P>A R > NN NP = AR> NN NP> AR> NN
NP = Nasal polyposis; AR = allergic rhinitis; NN = normal nasal mucosa. a mRNA: slot blot or Northern blot, using oligonucleotide probe for GM-CSF [22, 31]; bioassav: HL-60 or methylcellulose assays; abrogation by 1:50 to 1:200 dilution of neutralizing, monoclonal anti-GM-CSFantibody [25,27]; immunoassay: ELISA [31].
Regulation o f Cytokine Production and Expression by Structural Cells IL-1. IL-1 up-regulated gene expression and pro duction of GM-CSF as well as IL-6 and IL-8 from air way fibroblasts but not from epithelial cells [21, 22, 24, 25]. This accords with observations on endothelial cells and fibroblasts derived from other sources, which suggest a cascade of effects from initial inflam matory signals such as IL-1 and tumor necrosis factor-a to the elaboration of cytokines such as GM-CSF and IL-6, leading to further stages of inflammation.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
HL-60 and colony assays appeared to resemble baso phils, not mast cells, although certain phenotypic fea tures suggest the possible presence of an early, imma ture cell type intermediate between the two [28]. In methylcellulose cultures, the effect of GM-CSF de rived from airway structural cells was most pro nounced on the basophil/eosinophil progenitor pop ulation [21]. In other bioassays both interleukin-6 (IL-6) and IL-8 activities could be shown to also be present in structural-cell-derived supernatants [21,22, 27]. These activities included hepatocyte-stimulating as well as neutrophil chemotactic activities [29], both of which could be abrogated by specific, neutralizing antibod ies (to IL-6 or IL-8, respectively) in the bioassays.
129
130
Denburg/G auldie/D olovich/O htoshi/Cox/Jordana
1L-6
IL-8
2 3 4 5
6 Calf liver
Fig. 1. Slot blot demonstrating mRNA expression of 1L-6, IL-8 and GM-CSF by human upper airway epithelial cells derived from nasal polyps.
100 80 |
c 15
60 -
I
40-
w 200
i----------1--------- 1----------1--------- 1--------- 1--------- 1--------- ;----------1
0.1
0.5
1
2.5
5
10
50
100
Conditioned medium, %
Fig. 2. Eosinophil survival at day 3 in vitro in the presence of bronchial epithelial cell ( • ) and fibroblast (O) supernatants. In the absence of conditioned medium there is negligible eosinophil sur vival. The active moiety in these conditioned media which is re sponsible for this effect can be shown to be GM-CSF [30],
Neuropeptides. The neuropeptide substance P could also be shown to up-regulate the expression of IL-6 and IL-8 derived from airway structural cells; the production of GM-CSF, however, in the situation of nasal polyp structural cell lines, was constitutively high and could not be further up-regulated by sub stance P. These studies form a basis for further explo ration of neuropeptides in inflammatory cell recruit ment. Modulation by Antiallergic Compounds. Corticoste roids were shown to diminish or even abrogate gene expression or production of GM-CSF, IL-6 and IL-8 by airway epithelial cells and fibroblasts [21, 22, 24, 25, 30, 31]; an antihistaminic compound, ketotifen, decreased cytokine production at a posttranscrip
tional level. In the case of corticosteroids, a topically active compound, budesonide, when given by inhala tion in vivo had effects on the circulating numbers of mature basophils, eosinophils as well as their pro genitors, and was a potent suppressor of GM-CSF production by structural cells [32]. This effect may be of major importance in the clinical activity of inhaled steroids in allergic rhinitis and asthma, and suggests that the airway epithelium in vivo may secrete GMCSF and other progenitor-influencing cytokines into the circulation, mimicking effects seen when the pure cytokine is given in vivo to primates [33,34], Structural-Cell-Derived Extracellular Matrices Not only are structural-cell-derived cytokines ap parently important in the regulation of inflammatory cell recruitment and activation as well as differentia tion, but the extracellular matrices laid down by these cells may be pivotal in determining the biologic con sequences of the cytokines elaborated at any given time. Studies still in progress reveal that both for mac rophages and mast cells upper airway structural-cellderived extracellular matrices have a profound effect on activation and, possibly, phenotype switch of these inflammatory cells, which may be relevant and complementary to those effects we have found in cell differentiation assays.
Conclusions The studies described above form the basis for the microenvironmental differentiation hypothesis of aller gic inflammation in man[22]. Events occurring in or at mucosal surfaces, involving a complex network of structural cells, cytokines (including neuropeptides) and inflammatory cells in immature or mature forms are part of a process leading to the perpetuation of in flammation of allergic and related types. Thus, whether or not sensitization occurs via IgE, and not withstanding the intensity of the initiating signal in an allergic reaction, the maintenance and chronicity of inflammatory processes at tissue sites is hypothe sized to be under control by structural cell popula tions. Whether or not structural cells in atopy and re lated disorders such as polyposis and asthma are con stitutionally different from normal, and how they have developed their capacity for continual elabora tion of proinflammatory and hemopoietic cytokines remains to be elucidated. In vivo models to test the
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
G M -C S F
hypothesis of structural-cell-derived cytokines are currently being pursued in parallel to in vitro studies. Detection of the clinically relevant presence of struc tural-cell-derived cytokines in natural or provoked allergic reactions can now be assessed.
References 1 Denburg JA, Befus AD, Bienenstock J: Growth and differentia tion in vitro of mast cells from mesenteric lymph nodes of Nipposlrongylus brasiliensis-infected rats. Immunology 1982;41: 195-202. 2 Haig DM. McKee TA. Jarrett EEE, Woodbury R, Miller HPR: Generation of mucosal mast cells is stimulated by in vitro fac tors derived from T cells of helminth-infected rats. Nature 1982: 300:180. 3 Crapper RM, Schrader JW: Frequency of mast cell precursors in normal tissues determined by an in vitro assay: Antigen in duces parallel increases in the frequency of P-cell precursors and mast cells. J Immunol 1983;131:923-928. 4 Guy-Grand D, Dy M, Luffau L, Vassalli P: Gut mucosal mast cells: Origin, traffic and differentiation. J Exp Med 1984:160: 12-28. 5 Owen WF Jr, Rothenberg ME, Silberstein DS, Gasson JC, Ste vens RL, Austen KF, Soberman RJ: Regulation of human eosinophil viability, density and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fi broblasts. J Exp Med 1987;166:129-141. 6 Rothenberg ME, Owen WF Jr, Silberstein DS, Soberman RJ, Austen KF, Stevens RL: Eosinophils co-cultured with endo thelial cells have increased survival and functional properties. Science 1987;237:645-647. 7 Levi-Schaffer F, Austen KF, Caulfield J. Hein A, Bloes WF, Stevens RL: Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro. J Immunol 1985:135:3454-3462. 8 Denburg JA, Telizyn S, Belda A, Dolovich J, Bienenstock J: In creased numbers of circulating basophil/mast cell progenitors in atopic patients. J Allergy Clin Immunol 1985;76:466-472. 9 Gibson P, Dolovich J, Hargreave FH, Denburg J A: The inflam matory response in asthma exacerbation: Changes in circulat ing eosinophils, basophils, and their progenitors. Clin Exp Al lergy 1990:20:661-668. 10 Otsuka H, Dolovich J, Befus AD, Telizyn S, Bienenstock J, Denburg JA: Basophilic cell progenitors, nasal metachromatic cells and peripheral blood basophils in ragweed allergic patients.J Allergy Clin Immunol 1986;78:365-371. 11 Otsuka H, Dolovich J, Befus AD, Bienenstock J, Denburg J: Basophilic cell progenitors, nasal metachromatic cells in pa tients with allergic rhinitis. Am Rev Respir Dis 1986:133: 757-762. 12 Otsuka H, Dolovich J, Bienenstock J, Denburg JA: Metachro matic cell progenitors and specific growth and differentiation factors in human nasal mucosa and polyps. Am Rev Respir Dis 1987:136:710-717. 13 Nickelsen JA, Georgitis JW, Reisman RE: Lack of correlation between titers of serum allergen-specific IgE and symptoms in
131
untreated patients with seasonal allergic rhinitis. J Allergy Clin Immunol 1986:77:43-48. 14 Ohnishi M, Ruhno J, Bienenstock J, Milner R, Dolovich J, Denburg JA: Human nasal polyp epithelial basophil/mast cell and eosinophil colony-stimulating activity. The effect is T-celldependent. Am Rev Respir Dis 1988:138:560-564. 15 Ohnishi M, Ruhno J, Dolovich J, Denburg J A: Allergic rhinitis nasal mucosal conditioned medium stimulates growth and dif ferentiation of basophil/mast cell and eosinophil progenitors from atopic blood. J Allergy Clin Immunol 1988:81:1149-1154. 16 Ohnishi M, Ruhno J, Bienenstock J, Dolovich J, Denburg JA: Hemopoietic growth factor production by human nasal polyp epithelial scrapings: kinetics, cell source and relationship to clinical status. J Allergy Clin Immunol 1989;83:1091-1099. 17 Otsuka H, Denburg JA, Dolovich J, Hitch D, Lapp P, Rajan RS, Bienenstock J, Befus AD: Heterogeneity of metachromatic cells in human nose: Significance of mucosal mast cells. J Al lergy Clin Immunol 1985:76:695-702. 18 Ruhno J, Howie K, Anderson M, Anderssen B, Vanzieleghem M, Hitch D, Lapp P, Denburg J, Dolovich J: Rhinitis of nasal polyps: The increased numbers of epithelial mast cells in nasal polyps and adjacent turbinates are not related to allergy. Al lergy 1990;45:1-5. 19 Kawabori S, Denburg JA, Schwartz LB, Irani A-M, Wong D, Jordana G, Evans S, Dolovich J: Histochemical and immuno histochemical studies of nasal polyp mast cells. J Allergy Clin Immunol, submitted. 20 Denburg JA, Dolovich J: In situ hemopoietic mechanisms un derlying allergic tissue inflammation. Pharmacia Allergy Re search Foundation Award Book. Allergy I988;(suppl):20-25. 21 Denburg JA, Jordana M, Vancheri C, Gauldie J, Harnish D, Ohtoshi T, Tsuda T, Gibson P, Ruhno J, Bienenstock J, Har greave FE, Dolovich J: Locally derived hemopoietic cytokines for human basophils, mast cells and eosinophils in respiratory disease; in Galli S, Austen KF (eds): Human Mast Cell and Ba sophil Functional Differentiation in Health and Disease. New York, Raven Press, 1988, pp 59-69. 22 Denburg JA, Dolovich J, Ohtoshi T, Cox G, Gauldie J. Jordana M: The microenvironmental differentiation hypothesis of air way inflammation. Am J Rhinol 1990;4:29-34. 23 Dolovich J, Ohtoshi T, Jordana M, Gauldie J, Denburg JA: Na sal polyps: Local inductive microenvironment in the pathogen esis of the inflammation: in Pipkorn U (ed): Rhinitis and Asthma - Similarities and Differences. Copenhagen, Munksgaard, 1990, pp 233-241. 24 Ohtoshi T, Vancheri C, Cox G, Gauldie J, Dolovich J, Denburg JA, Jordana M: Monocyte-macrophage differentiation induced by human upper airway epithelial cells. Am J Respir Cell Mol Biol, in press. 25 Ohtoshi T, Tsuda T, Vancheri C, Abrams JS, Gauldie J, Dolo vich J, Denburg JA, Jordana M: Human upper airway epithel ial cell-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) induces histamine-containing cell differentia tion of human progenitor cells. Submitted. 26 Vancheri C, Ohtoshi T, Cox G, Abrams JS, Xaubet A, Gauldie J, Dolovich J, Denburg JA, Jordana M: Neutrophilic differen tiation induced by human upper airway fibroblast-derived granulocyte-macrophage colony-stimulating factor (GM-CSF). Am J Respir Cell Mol Biol 1991 ;4:11-17. 27 Hutt-Taylor SR, Harnish D, Richardson M, Ishizaka T, Den-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
Cytokines in Allergie Inflammation
28
29
30
31
burg JA: Sodium butyrate and T-lymphocyte cell line-derived differentiation factor induce basophilic differentiation of the human promyelocytic leukemia cell line HL-60. Blood 1988;71: 209-215. Wong Da, Kawabori S, Switzer J, Valent P, Bettelheim P, Dolovich J, Ishizaka T, Jordana M, Denburg J: Characterization of histamine-containing basophilic cells after in vitro induction of HL-60 cell line (abstract). J Allergy Clin Immunol 1990:85:173. Gauldie J, Richards C, Harnish D, Landsdorp P, Baumann H: Interferon p,/B-cel! stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regu lates the major acute phase protein response in liver cells. Proc Natl Acad Sci USA 1987;84:76251-7255. Cox G, Vancheri C, Ohtoshi T, Gauldie J, Dolovich J, Jordana M, Denburg JA: Human bronchial epithelial cell-derived gran ulocyte-macrophage colony stimulating factor (GM-CSF) pro longs survival of human eosinophils (abstract). J Allergy Clin Immunol 1990:85:233. Vancheri C, Gauldie J, Bienenstock J, Cox G, Scicchitano R, Stanisz A, Jordana M: Human lung fibroblast-derived granulo cyte-macrophage colony-stimulating factor (GM-CSF) medi ates eosinophil survival in vitro. Am J Respir Cell Mol Biol 1989;1:289-295.
Denburg/G auldie/D olovich/O htoshi/Cox/Jordana
32 Ohtoshi T, Xaubet A, Andersson B, Vanzieleghem M, Dolovich J, Jordana M, Denburg JA: Nasal inflammation mediated by human structural cell-derived GM-CSF: Effect of budesonide (abstract). J Allergy Clin Immunol 1990:84:297. 33 Mayer P, Valent P, Schmidt G, Liehl E, Bettelheim P: The in vivo effects of recombinant human IL-3: Demonstration of ba sophil differentiation factor, histamine-producing activity, and priming of GM-CSF-responsive progenitors in non-human pri mates. Blood 1989;74:613-621. 34 Valent P, Schmidt G, Besemer J, Mayer P, Zeuke G, Liehl E, Hinterberger W, Lechner K, Bettleheim P: lnterleukin-3 is a differentiation factor for human basophils. Blood 1989;73: 1763-1769.
Correspondence to: Dr. Judah A. Denburg McMaster University Hamilton, Ont. (Canada)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 3:18:43 PM
132
