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Fungal β-glucans modulate macrophage release of tumor necrosis factor-α in response to bacterial lipopolysaccharide
lmmunoh~gy Letters, 37 (1993) 19 25 0165 2478 , 93 ' $ 6.00 ~ 1993 Elsevier Science Publishers B.V. All rights reserved IMLET 01982
Fungal fl-glucans modulate macrophage release of tumor necrosis factor- in response to bacterial lipopolysaccharide O r l e e n A. H o f f m a n , Eric J. O l s o n a n d A n d r e w H. L i m p e r Thoracic Disease Research Unit, Division of Thoracic Diseases and Internal Medicine. Mayo Clinic and Foundation, Rochester. MN 55905, USA (Received 11 February 1993; revision received 28 April 1993; accepted 3 May 1993)
1.
Summary
Tumor necrosis factor-alpha (TNF:0 is a potent cytokine believed to participate in the development of endotoxin-induced shock and the adult respiratory distress syndrome [1,2]. Treatment of animals with fl-glucan prior to bacterial challenge reduces TNF~ release and prevents death [3]. We therefore hypothesized that fl-glucan might regulate TNF~ secretion from macrophages in response to lipopolysaccharide (LPS). Rat alveolar macrophages were cultured in the presence of fl-glucan alone and the TNF~ secretion quantified using an L929 cytotoxicity assay. Concentrations of fl-glucan less than 500 ~g/ml were found to stimulate TNFct release from macrophages. However, concentrations of fl-glucan greater than 500 #g/ml resulted in suppression of the TNF2 activity released. This reduction in TNF~ release was not mediated by a toxic effect of fl-glucan, as large concentrations of flglucan had no effect on macrophage viability. We further observed that the incubation of macrophages with large concentrations of fl-glucan (500 #g/ml) also inhibited the secretion of TNF~ induced by bacterial LPS. Furthermore, interferon-7 (IFNT), a potent activator of TNF~ exKey words: TNF:c fl-Glucan: Alveolar macrophage: LPS: Interferon-), Correspondence to." Andrew H. Limper, Thoracic Diseases, E-18B Mayo Clinic, Rochester, MN 55905, USA.
pression, failed to overcome the inhibition of TNFct caused by fl-glucan. These data suggest an immunomodulatory role for fl-glucan which may explain both the TNFc~-stimulating and -inhibiting effects of fungal fl-glucans during infection. 2.
Introduction
The host response to infection involves the release of multiple inflammatory mediators from macrophages interacting with bacterial and fungal components. Tumor necrosis factor-alpha (TNF~) is a proinflammatory cytokine released from macrophages in response to bacterial lipopolysaccharide (LPS) and other agents. TNF~ possesses a number of activities which promote inflammatory cell recruitment, increase endothelial permeability, and initiate tissue healing and fibrosis [4,5]. The myriad biological effects of TNF~ play important roles in the development of endotoxin-induced shock and the adult respiratory distress syndrome [1,2]. Recent work suggests that alveolar macrophages also release TNF~ during certain fungal infections via recognition of cell surface fl-glucans [6]. fl-Glucans are polymers of d-glucose containing fl-l,3 and fl-l,6 linkages which comprise the major structural component of fungal cell walls [7]. fl-Glucans interact with specific receptors present on mononuclear phagocytes which mediate cell activation and phagocytosis [8,9]. However, in addition to promoting macrophage release of TNF~, other studies suggest that fl-glu19
cans might also be capable of downregulating the expression of TNF~. Treatment of animals with particulate fl-glucan derivatives prior to bacterial challenge reduces the release of TNF~ from macrophages and prevents death from infection [3]. The basis of these apparently contrasting activities of/%glucans has not been previously investigated. The following study was therefore undertaken to determine the effects of /%glucans on TNF~ release from alveolar macrophages in vitro. In particular, we sought to establish which concentrations of particulate /~-glucan stimulate the release of TNF~. from macrophages and which inhibit its liberation. We further investigated whether macrophage challenged with fungal/%glucans would exhibit an impaired response to bacterial LPS. We hypothesized that under conditions of excess fl-glucan, macrophages might become refractory to stimulation by other agonists. Finally, we evaluated whether IFNT, a cytokine which augments the macrophage release of TNF~, would modify the effects of/%glucan on the macrophage release of TNF~ in response to bacterial LPS. 3.
3.1.
Materials and Methods
Stimulation of ah,eolar macrophages with flglucan
The dose-response characteristics of /3-glucan stimulation of macrophages were determined as follows. Alveolar macrophages were obtained from pathogen-free Harlan Sprague-Dawley rats by whole lung lavage performed with 50 ml of HBSS (without calcium or magnesium) as described previously [6]. The lavage was centrifuged at 2 0 0 × g for 10 rain at 4°C and the cells resuspended in mixed medium (Medium 199:RPMI at a ratio of 1:1, containing 2 mM glutamine, 10 000 U penicillin per liter, 1 mg streptomycin per liter, and 25 ~g amphotericin per liter). Cytopreparation smears routinely demonstrated greater than 95% alveolar macrophages in these isolates. Alveolar macrophages (2 x 105 per well) were plated on 96-well tissue culture dishes. The macrophages were allowed to adhere for 60 min 20
and were gently washed to remove any unattached cells. Typically, 95% or more of the macrophages were firmly adherent after this initial incubation [6]. Subsequently, //-glucan derived from Saccharomyces cerevisiae with a mean size of 4.2 x 101° glucose residues per particle (Sigma) was suspended in mixed medium and added to the wells at the indicated concentrations. These/~-glucan preparations were found to contain less than 0.125 U of endotoxin per liter using a sensitive kimulus amebocyte lysate assay (Whittaker M.A. Bioproducts, Walkersville, MD). The /]-glucanstimulated macrophages were incubated for 16 h at 37':C in 5% CO2. Subsequently, the medium was removed and centrifuged (10 0 0 0 x g for 4 rain) to remove cell debris and particulate glucan, and the supernatants frozen at - 7 0 - C until assayed.
3.2. Quantification o/ TNF~ Sensitive immunoassays for rat TNF~, are not yet widely available. Therefore, the T N F 7 activity released into the culture medium was assayed using a sensitive and specific L929 cytotoxicity bioassay as described by Kramer and Carver [10]. L929, a murine connective tissue cell line exquisitely sensitive to TNF~, was obtained from the American Type Culture Collection and grown in Eagle's minimum essential medium (EMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 10 000 U penicillin per liter, 1 mg streptomycin per liter, and 25 tlg amphotericin per liter. Cell monolayers were detached using trypsin (2 /~g/ml) and EDTA4Na (0.2 mg/ml: Sigma). Cells (4 x 104) were seeded into 96-well plates and incubated for 24 h at 37°C in 5% CO2, resulting in confluent monolayers. Spent medium was replaced with EMEM containing actinomycin D at a final concentration of 1 l~g/ml. Serial 1:2 dilutions of standards or sample were performed, with a final volume of 100 /~1 per well. Additional cells were cultured in EMEM alone to serve as 100% viability controls. Recombinant murine TNF~. (Genzyme, Cambridge, MA) was used as a relative standard for this bioassay. Plates were incubated at 37:C in 5% CO2 for 18 h, medium removed, and the cells stained with 0.5% crystal violet in 20% methanol.
Subsequently, the absorbance was determined at 490 nm (Vmax Dynamic Microplate Reader; Molecular Devices, Menlo Park, CA). A standard concentration curve was prepared from the O.D. of the standards and used to determine the relative TNF~ concentrations of the samples.
3.3.
Assessment of macrophage viability during ~glucan stimulation
Additional experiments were performed to determine whether /3-glucan exerted a toxic effect on macrophage viability. Alveolar macrophages were obtained and plated on 96-well tissue culture dishes (200 000 cells/well) in mixed medium as described above. The macrophages were allowed to adhere for 60 min, were gently washed to remove any unattached cells, and were subsequently cultured with /3-glucan (0, 100, 500, or 1000 lLg/ml) in mixed medium for 16 h in a manner identical to that described previously for the TNF~ determination experiments. After incubation, macrophage viability was assessed by vital dye exclusion. The medium was removed and replaced with 0.2% trypan blue solution in 0.81% sodium chloride and 0.06% potassium phosphate dibasic. The percentage of viable cells which excluded trypan blue was determined by counting 200 cells for each culture condition. Viability assays were performed in triplicate.
3.4.
Effect of fungal [3-glucan on macrophage release o[" TNF~t in response to bacterial LPS
During our initial studies we determined that macrophages challenged with large concentrations of/~-glucan exhibited suppression of TNF:~ release. To determine whether macrophages challenged with large concentrations of /%glucan would also be refractory to stimulation by bacterial LPS, alveolar macrophages were stimulated with LPS in the presence of inhibiting concentrations of/3-glucan. LPS derived from Escheriehia coli strain 0127:B8 (Sigma) was added to macrophages at the indicated concentrations in the presence of 500 Ftg/ml/~-glucan. After 16 h of incubation, the supernatants were removed and centrifuged, and the TNF~ activity assayed as described above.
3.5.
Effect of interferon-'; on ~-glucan-induced inhibition of macrophage TNFc~ release
Interferon-~' (IFN``,) is known to enhance the production of TNFz~ by macrophages significantly in response to LPS [I 1]. We therefore investigated whether IFN7 would ameliorate the impairment of macrophage TNF~ release induced by high concentrations of /%glucan. Accordingly, macrophages were incubated with recombinant murine IFN7 (1 or 10 ng/ml; Genzyme) for 16 h of incubation with LPS (0 10 pg;' ml) and /~-glucan (500 Izg/ml). TNF~ activity released into the medium was assayed as described above. We concurrently assessed the effect of IFN), alone (1-I0 ng/ml) on the macrophage generation of TNF~ as well as the effect of IFN7 (110 ng/ml) on the LPS-induced release of TNFz~ from macrophages in the absence of/~-glucan.
3.6.
Statistical analysis
All data are expressed as m e a n + S E M . We have observed that the absolute amounts of TNF~ released from alveolar macrophages in response to LPS or fl-glucan varied somewhat in magnitude when using macrophages isolated on different occasions. However, the general concentration characterisitcs of the macrophage TNF~ response to/%glucan and the induction of refractoriness during stimulation with high concentrations of fl-glucan were consistently present in all experiments. Accordingly, nonparametric methods were employed to test for differences between groups of data [12]. Differences between experimental and control data groups were determined using the Mann-Whitney U-test for nonparametric statistics performed on the Statview II statistical package (Abacus Conscepts, Berkeley, CA) using a Macintosh lIci personal computer. Differences were considered to statistically significant if P < 0.05 for two-sided alternatives. 4.
Results
Alveolar macrophages stimulated with fungal fl-glucans at concentrations less than 500 /Lg/ml exhibited an enhancement of TNFc~ release (Fig, 1). Maximal release of TNF~ activity occurred 21
1,000
Z I'-
4O0
0
0
1
10
100
5oo
1,000
B-glue, an (/.a:j/mL) *Denotes P<0.05 compared to control
Fig. 1. fl-Glucan stimulates the release of TNF~ from alveolar macrophages in a dose-dependent fashion. Alveolar macrophages (2 x l0 t) were incubated with increasing amounts of [Jgtucan for 16 h and TNF:~ release determined using an L929 cytotoxicity assay. While concentrations of/l-glucan less than 500 pg.ml stimulated TNF:< release from alveolar macrophages, concentrations /> 500/~g,;ml caused minimal release of TNFzc. Each data point represents the mean + SEM of 6 deterininations.
when alveolar macrophages were cultured with 100 pg/ml of ]~-glucan (P = 0.0003 compared with control). Interestingly, however, concentrations of //-glucan greater than or equal to 500 itg/ml caused a minimal release of T N F e activity. Indeed, macrophage release of TNF~ in the presence of 1000/tg/ml of/l-glucan was significantly lower than from macrophages cultured in the absence of/?-glucan (P=0.002). These data suggest that larger concentrations of fungal /~-glucan (>~ 500 /~g/ml) may actually inhibit the release of TNF:~ activity from alveolar macrophages. We next questioned whether the reduction of macrophage TNF~ release was due to an adverse effect of fungal/~-glucans on macrophage viability. Immediately upon isolation, macrophage viability was assessed to be 95.3_+0.2% by trypan blue exclusion. After 16 h of incubation in the absence of /?-glucan, macrophage viability was found to 93.7_+0.4%. Increasing concentrations of/~-glucan failed to cause any adverse effect on macrophage viability. Even after 16 h of incubation in the presence of 1000 /~g/ml of/~-glucan, macrophage viability remained at 93.3_+0.3% (not significantly different). These experiments document that the reduction of macrophage T N F 7 release induced by /~-glucan was not due 22
to impairment of macrophage viability. Instead, ¢/-glucan appears to modulate macrophage release of TNFc~ activity into the culture medium. To veri~' that /~-glucans were not interfering with the TNFc~ assay, we pertbrmed the following experiment. Supernatants from macrophages stimulated with low concentrations of /3-glucan (100 llg/ml) contained readily detectable quantities of TNF:~. These supernatants were removed from the macrophages and treated with either large concentrations of/~-glucan (1000 izg/ml) or no ~-glucan for an additional hour. Subsequently, the supernatants were centrifuged and the T N F 7 level determined using the L929 assay. We found the levels of TNF:~ to be equivalent in the supernatants treated with high concentrations of/~-glucan compared to those that remained untreated (P=0.19; not significantly different). It is therefore unlikely that high concentrations of []glucan interfered with the TNFc~ assay. Instead, it appeared that contact of the fl-glucan particles with the macrophages was required to modulate the release of TNFc~ activity. Next, we determined that macrophages challenged with large concentrations of fungal //-glucan were also refractory to stimulation by bacterial LPS, an extremely potent agonist for TNFT.
loo.ooo r
• ,8-glucan (500/J.g/mL)
Pj,I,I 1110 10 1
.
0
10
100
1,000
10,000
LPS concentration(ng/mL) Fig. 2. Large concentrations of fungaI //-glucan inhibit macrophage release of TNF:< in response to bacterial LPS. Alveolar macrophages (2 × 10-s) were preincubated with ,q-glucan (500 /~g/ml) for 16 h in the presence of increasing amounts of LPS. TNFc~ release into the medium was determined using an L929 cytotoxicity assay. Large concentrations of/~-glucan ( ~>500 ,ug' ml) caused suppression of the amount of TNF:< released into the medium. Each data point represents the mean + SEM of 6 determinations.
100.000 10,000
uE
1,000
ZQ. I-- " "
o,=
.9°=
100
10
1
• LPS alone LPS + IFN m LPS + IFN + .8-glucan
/' 0
1,000
10,000
LPS concentration (ng/mL) Fig. 3. Interferon-,' does not reverse the/3-glucan-induced inhibition of macrophage T N F x release in response to LPS. Alveolar macrophages (2 × 105) were incubated with IFN-,' (I ng/ml) and increasing concentrations of LPS in the presence or absence of/~-glucan (500/~g,'ml) for 16 h. TNF:~ release into the medium was determined using an L929 cytotoxicity assay. In the absence of fl-glucan, IFN-'; significantly increased macrophage TNFc~ release in response to LPS. However, in the presence of 500 pg,,ml/~-glucan, macrophage release of TNF~ into the medium was not increased above baseline. Each point represents the mean +_SEM of 3 separate determinations.
generation. Interestingly, large concentrations of /#glucan (500 pg/ml) consistently suppressed the macrophage release of TNF:~ activity even following stimulation with progressively larger concentrations of LPS (Fig. 2). Macrophages cultured with LPS alone released substantial quantities of TNF:~ into the medium. However, macrophages incubated with LPS in the presenceof /3-glucan (500/~g/ml) exhibited a significant suppression of TNF~ activity (P=0.0001, comparing macrophages cultured with LPS in the presence or absence of/3-glucan). These findings confirmed our hypothesis that higher concentrations of/~-glucan rendered macrophages refractory to TNF:~ release. Interferon-7 (IFN~') is a potent cytokine derived from T lymphocytes which significantly enhances TNF~ release from mononuclear phagocytes. It has been previously reported that TNFz~ can reverse the endotoxin-resistant phenotype of macrophages and restore their ability to release TNFc~ in response to LPS [11]. Full activation of macrophage TNF~ biosynthesis occurs at an IFN;, concentration as low as 1 ng/ml [11]. Accordingly, we investigated whether IFN7 would
enhance the release of TNF~ from alveolar macrophages stimulated with LPS in the absence of/3-glucan and whether IFN7 would restore the ability of macrophages to release TNF~ in the presence of inhibitory concentrations of/?-glucan (Fig. 3). As anticipated, alveolar macrophages cultured without /~-glucan exhibited a substantial enhancement of TNFc~ release following LPS stimulation in the presence of IFN),. While macrophages challenged with 1000 ng/ml of LPS alone released 628.0_+259.4 pg/ml of TNF~, macrophages stimulated with the same concentration of LPS in the presence of IFN (1 ng/ml) liberated 4503.1_+1194.8 pg/ml of TNF~ (P = 0.0007). However, in the presence of 500 pg/ml of/3-glucan, macrophage release of TNF~ uniformly remained at baseline levels with or without the addition IFN'~, (Fig. 3). Macrophages cultured with IFN7 (1 ng/ml) and challenged with up to 10 000 ng/ml of LPS released only 8.3_+0.1 pg/ml of TNF:~ in the presence of 500 /tg/ml of/%glucan (not significantly different to TNF~ release from unstimulated macrophages). In contrast, macrophages cultured under identical conditions (IFNT, 1 ng/ml: LPS, 10 000 ng/ml) released 20 870.4 _+497.9 pg/ml of TNF~. in the absence of/3-glucan. Even in the presence of tenfold greater concentrations of IFN), (10 ng/ml), J~-glucan still caused a substantial inhibition of macrophage TNFc~ release in response to LPS. Macrophages cultured with ~-glucan (500/~g/ml) and stimulated with up to 10 000 ng/ml of LPS in the presence of 10 ng/ ml IFN7 still released 70% less TNFc~ than macrophages cultured under identical conditions without J~-glucan. These data demonstrate that fungal j%glucans can render macrophages refractory to LPS stimulation and that IFN?' is not effective in reversing the inhibition of TNFc~ release caused by J?-glucan. 5.
Discussion
These studies for the first time demonstrate several important features of the interaction of fungal /~-glucans with alveolar macrophages. /3Glucans exhibit an interesting dose-response relationship for the macrophage stimulation of TNFc~ release. While lower doses of /~-glucan enhance 23
macrophage TNFc~ release, higher concentrations (~> 500/~g/ml) lead to minimal TNF,~ release and in fact appear to inhibit its liberation. Indeed, macrophages cultured in the presence of large concentrations of fungal fl-glucan are refractory to stimulation by bacterial LPS even in the presence of IFNT, an agent which substantially enhances macrophage TNF:~ release. The observed response of macrophages to stimulation with increasing concentrations of fungal fi-glucan may further explain the contrasting activities of fl-glucan which have been previously reported. The existing literature suggests that under differing conditions, fi-glucans can either stimulate or suppress macrophage activation [3,6,1318]. Published observations by ourselves and others document that fungal fl-glucans can activate alveolar macrophages [13 15], enhancing the release of TNF:~ [6] and arachidonic acid metabolites [16]. Others have similarly shown that particulate fl-glucans can stimulate the macrophage release of IL-I [13 15]. In contrast, Rasmussen and Seljelid's work suggests that particulate fl-l,3polyglucose (glucan) may under other circumstances modulate macrophage activity and suppress TNF:~ release [3]. In their study, the intraperitoneal administration of glucan-derivatized microbeads inhibited the lethal TNF~. release from peritoneal macrophages in response to E. coli and prevented death in these animals [3]. These observations are consistent with our data which demonstrate that macrophages exposed to high local concentrations of fl-glucan exhibit an impaired TNF~ release even in response to bacterial LPS. Although macrophages exhibit an impaired release of TNFc~ activity following exposure to large concentrations of fungal /%glucans, other macrophage responses appear to remain intact. In particular, Daum and Rohrbach have recently reported that alveolar macrophages continue to exhibit alterations of eicosanoid metabolism and release arachidonic acid even in the face of large concentrations of fl-glucan, strongly suggesting that macrophages are not totally anergic during fl-glucan stimulation and can continue to exhibit other cellular responses [16]. Furthermore, alveolar macrophages stimulated with viable fungal organisms such as Pneumocvstis carinii in the pre24
sence of large concentrations of fi-glucan ( >~500 llg/ml) exhibit an impairment of TNF:~ release but continue to liberate arachidonic acid in response to the organisms [6,17]. Taken together, these findings suggest that the inhibition of macrophage TNF:~ release induced by high concentrations of fl-glucan does not result in a generalized inactivation of the macrophage. The cellular mechanisms by which fi-glucans render the macrophage refractory to LPS-induced TNF~ release are presently unknown. Stimulation of macrophage with fi-glucans does not necessarily require particle phagocytosis but does require that macrophages recognize a fixed steric arrangement of glucan epitopes [13]. For instance, although particulate fl-glucans and glucan-derivatized plastics both cause macrophage stimulation, soluble glucans do not usually possess these activities [13]. This may suggest that a rearrangement or clustering of glucan receptors is necessary for macrophage activation. It is possible that when presented with large concentrations of fl-glucan, the cognate receptors are scattered, and clustering is prevented, fi-Glucans possess other activities which may regulate TNF~. expression as well. For instance, fl-glucans induce the macrophage release of arachidonic acid and PGE2 [16]. PGE2 has been demonstrated to suppress TNFu, release in vitro [18]. It is also possible that large concentrations of fungal fl-glucans may enhance the macrophage uptake and degradation of TNFzc Clearly, additional studies will be required to define the exact mechanisms by which fl-glucans can both stimulate and inhibit the macrophage release of TNFzc The clinical relevance of our observations remains to be determined. Modulation of TNF:~ release from mononuclear phagocytes in response to LPS is of potential therapeutic importance in a number of conditions. TNF~ has established activity in the pathogenesis of endotoxin-induced shock and the adult respiratory distress syndrome [1,2]. fl-Glucans and related sugars may provide additional means to modify TNF~. activity during such disorders. However, extreme caution is advised in rapidly extrapolating these in vitro observations to the living host. Additional basic and preclinical investigations will be required to confirm these activities in the whole organism.
Acknowledgements This work was supported by funds from the American Heart Association (Clinician Scientist Award No. 91004230), an American Lung Association Research Grant (No. RG-011-N), and funds from the Mayo Foundation to A.H.L. We wish to thank Drs. Michael Rohrbach and Zvezdana Vuk-Pavlovic for many helpful discussions, Mr. Edward Mansfield for assistance with the Limulus amebocyte lysate for endotoxin, and Ms. Kathy Streich for her assistance in preparation of the final manuscript.
References [1] Jaattela, M. (1991) Lab. Invest. 64, 724. [2] H3,ers. T.M., Tricomi, S.M.. Dettenmeier, P.A. and Fowler, A.A. (1991) Am. Rev. Respir. Dis. 144, 268. [3] Rasmussen L.T. and Seljelid, R. (19921 J. Cell. Biochem. 46, 60. [4] Le, J. and Vilcek, J. ~1987) Lab. Invest. 56, 234. [5] Beutler. B. and Cerami, A. (1988) Annu. Rev. Biochem. 57, 505. [6] Hoffinan. O.A., Standing, J.E. and Limper, A.H. (1993) J.
[mmunol. 150, 3932. [7] Manners, D.J., Masson, A.J. and Patterson, J.C. 11974) J. Gen. Microbiol. 80, 41 I. [8] Kadish, J.L., Choi, C.C. and Czop, J.K. (1986) Fed. Proc. 45, 1115. [9] Janusz, M.J., Austen, K.F. and Czop, J.K. ~1986) J. Immunol. Methods 93, 201. [10] Kramer, S.M. and Carver, M.E. (1986) J. lmmunol. Methods 93, 201. [11] Beutler, B., Tkacenko, V., Milsark. I.. Korchin, N. and Cerami, A. (1986) J. Exp. Med. 164, 1791. [12] Rosner, B. (1986) Fundamentals of Biostatistics, Duxbury Press, Boston. [13] Bogwe[l, J., Gouda, I., Hoffmam 1.. Larm, O., Larsson, R. and Seljelid, R. (1986) Scan& J. lmmunol. 20. 355. [14] Seljelid. R., Fingenschau, Y., Bogwell, J., Rasmussen. L.T. and Austgu[en. J. (1989) Scand. J. lmmunol. 30, 687. [15] Seljelid, R., Bogwell, J., Rasmussen, L.T., Larm, O.. Hoffman, I., Berge, A. and Austgulen, J. (1985) Scand. J. lmmunol. 21,601. [16] Daum, T. and Rohrbach, M.S. (1992) FEBS Lett. 309. 119. [17] Castro, M., Morgenthaler, T.I., Hoffman, O.A., Standing, J.E., Rohrbach, M.S. and Limper, A.H. (1993) Am. J. Respir. Cell. Mol. Biol. 9, in press. [18] Kunkel, S i . , Wiggins, R.C., Chensue, S.W. and Larrick, J. (1986) Biochem. Biophys. Res. Commun. 137, 404.
25
Fungal fl-glucans modulate macrophage release of tumor necrosis factor- in response to bacterial lipopolysaccharide O r l e e n A. H o f f m a n , Eric J. O l s o n a n d A n d r e w H. L i m p e r Thoracic Disease Research Unit, Division of Thoracic Diseases and Internal Medicine. Mayo Clinic and Foundation, Rochester. MN 55905, USA (Received 11 February 1993; revision received 28 April 1993; accepted 3 May 1993)
1.
Summary
Tumor necrosis factor-alpha (TNF:0 is a potent cytokine believed to participate in the development of endotoxin-induced shock and the adult respiratory distress syndrome [1,2]. Treatment of animals with fl-glucan prior to bacterial challenge reduces TNF~ release and prevents death [3]. We therefore hypothesized that fl-glucan might regulate TNF~ secretion from macrophages in response to lipopolysaccharide (LPS). Rat alveolar macrophages were cultured in the presence of fl-glucan alone and the TNF~ secretion quantified using an L929 cytotoxicity assay. Concentrations of fl-glucan less than 500 ~g/ml were found to stimulate TNFct release from macrophages. However, concentrations of fl-glucan greater than 500 #g/ml resulted in suppression of the TNF2 activity released. This reduction in TNF~ release was not mediated by a toxic effect of fl-glucan, as large concentrations of flglucan had no effect on macrophage viability. We further observed that the incubation of macrophages with large concentrations of fl-glucan (500 #g/ml) also inhibited the secretion of TNF~ induced by bacterial LPS. Furthermore, interferon-7 (IFNT), a potent activator of TNF~ exKey words: TNF:c fl-Glucan: Alveolar macrophage: LPS: Interferon-), Correspondence to." Andrew H. Limper, Thoracic Diseases, E-18B Mayo Clinic, Rochester, MN 55905, USA.
pression, failed to overcome the inhibition of TNFct caused by fl-glucan. These data suggest an immunomodulatory role for fl-glucan which may explain both the TNFc~-stimulating and -inhibiting effects of fungal fl-glucans during infection. 2.
Introduction
The host response to infection involves the release of multiple inflammatory mediators from macrophages interacting with bacterial and fungal components. Tumor necrosis factor-alpha (TNF~) is a proinflammatory cytokine released from macrophages in response to bacterial lipopolysaccharide (LPS) and other agents. TNF~ possesses a number of activities which promote inflammatory cell recruitment, increase endothelial permeability, and initiate tissue healing and fibrosis [4,5]. The myriad biological effects of TNF~ play important roles in the development of endotoxin-induced shock and the adult respiratory distress syndrome [1,2]. Recent work suggests that alveolar macrophages also release TNF~ during certain fungal infections via recognition of cell surface fl-glucans [6]. fl-Glucans are polymers of d-glucose containing fl-l,3 and fl-l,6 linkages which comprise the major structural component of fungal cell walls [7]. fl-Glucans interact with specific receptors present on mononuclear phagocytes which mediate cell activation and phagocytosis [8,9]. However, in addition to promoting macrophage release of TNF~, other studies suggest that fl-glu19
cans might also be capable of downregulating the expression of TNF~. Treatment of animals with particulate fl-glucan derivatives prior to bacterial challenge reduces the release of TNF~ from macrophages and prevents death from infection [3]. The basis of these apparently contrasting activities of/%glucans has not been previously investigated. The following study was therefore undertaken to determine the effects of /%glucans on TNF~ release from alveolar macrophages in vitro. In particular, we sought to establish which concentrations of particulate /~-glucan stimulate the release of TNF~. from macrophages and which inhibit its liberation. We further investigated whether macrophage challenged with fungal/%glucans would exhibit an impaired response to bacterial LPS. We hypothesized that under conditions of excess fl-glucan, macrophages might become refractory to stimulation by other agonists. Finally, we evaluated whether IFNT, a cytokine which augments the macrophage release of TNF~, would modify the effects of/%glucan on the macrophage release of TNF~ in response to bacterial LPS. 3.
3.1.
Materials and Methods
Stimulation of ah,eolar macrophages with flglucan
The dose-response characteristics of /3-glucan stimulation of macrophages were determined as follows. Alveolar macrophages were obtained from pathogen-free Harlan Sprague-Dawley rats by whole lung lavage performed with 50 ml of HBSS (without calcium or magnesium) as described previously [6]. The lavage was centrifuged at 2 0 0 × g for 10 rain at 4°C and the cells resuspended in mixed medium (Medium 199:RPMI at a ratio of 1:1, containing 2 mM glutamine, 10 000 U penicillin per liter, 1 mg streptomycin per liter, and 25 ~g amphotericin per liter). Cytopreparation smears routinely demonstrated greater than 95% alveolar macrophages in these isolates. Alveolar macrophages (2 x 105 per well) were plated on 96-well tissue culture dishes. The macrophages were allowed to adhere for 60 min 20
and were gently washed to remove any unattached cells. Typically, 95% or more of the macrophages were firmly adherent after this initial incubation [6]. Subsequently, //-glucan derived from Saccharomyces cerevisiae with a mean size of 4.2 x 101° glucose residues per particle (Sigma) was suspended in mixed medium and added to the wells at the indicated concentrations. These/~-glucan preparations were found to contain less than 0.125 U of endotoxin per liter using a sensitive kimulus amebocyte lysate assay (Whittaker M.A. Bioproducts, Walkersville, MD). The /]-glucanstimulated macrophages were incubated for 16 h at 37':C in 5% CO2. Subsequently, the medium was removed and centrifuged (10 0 0 0 x g for 4 rain) to remove cell debris and particulate glucan, and the supernatants frozen at - 7 0 - C until assayed.
3.2. Quantification o/ TNF~ Sensitive immunoassays for rat TNF~, are not yet widely available. Therefore, the T N F 7 activity released into the culture medium was assayed using a sensitive and specific L929 cytotoxicity bioassay as described by Kramer and Carver [10]. L929, a murine connective tissue cell line exquisitely sensitive to TNF~, was obtained from the American Type Culture Collection and grown in Eagle's minimum essential medium (EMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 10 000 U penicillin per liter, 1 mg streptomycin per liter, and 25 tlg amphotericin per liter. Cell monolayers were detached using trypsin (2 /~g/ml) and EDTA4Na (0.2 mg/ml: Sigma). Cells (4 x 104) were seeded into 96-well plates and incubated for 24 h at 37°C in 5% CO2, resulting in confluent monolayers. Spent medium was replaced with EMEM containing actinomycin D at a final concentration of 1 l~g/ml. Serial 1:2 dilutions of standards or sample were performed, with a final volume of 100 /~1 per well. Additional cells were cultured in EMEM alone to serve as 100% viability controls. Recombinant murine TNF~. (Genzyme, Cambridge, MA) was used as a relative standard for this bioassay. Plates were incubated at 37:C in 5% CO2 for 18 h, medium removed, and the cells stained with 0.5% crystal violet in 20% methanol.
Subsequently, the absorbance was determined at 490 nm (Vmax Dynamic Microplate Reader; Molecular Devices, Menlo Park, CA). A standard concentration curve was prepared from the O.D. of the standards and used to determine the relative TNF~ concentrations of the samples.
3.3.
Assessment of macrophage viability during ~glucan stimulation
Additional experiments were performed to determine whether /3-glucan exerted a toxic effect on macrophage viability. Alveolar macrophages were obtained and plated on 96-well tissue culture dishes (200 000 cells/well) in mixed medium as described above. The macrophages were allowed to adhere for 60 min, were gently washed to remove any unattached cells, and were subsequently cultured with /3-glucan (0, 100, 500, or 1000 lLg/ml) in mixed medium for 16 h in a manner identical to that described previously for the TNF~ determination experiments. After incubation, macrophage viability was assessed by vital dye exclusion. The medium was removed and replaced with 0.2% trypan blue solution in 0.81% sodium chloride and 0.06% potassium phosphate dibasic. The percentage of viable cells which excluded trypan blue was determined by counting 200 cells for each culture condition. Viability assays were performed in triplicate.
3.4.
Effect of fungal [3-glucan on macrophage release o[" TNF~t in response to bacterial LPS
During our initial studies we determined that macrophages challenged with large concentrations of/~-glucan exhibited suppression of TNF:~ release. To determine whether macrophages challenged with large concentrations of /%glucan would also be refractory to stimulation by bacterial LPS, alveolar macrophages were stimulated with LPS in the presence of inhibiting concentrations of/3-glucan. LPS derived from Escheriehia coli strain 0127:B8 (Sigma) was added to macrophages at the indicated concentrations in the presence of 500 Ftg/ml/~-glucan. After 16 h of incubation, the supernatants were removed and centrifuged, and the TNF~ activity assayed as described above.
3.5.
Effect of interferon-'; on ~-glucan-induced inhibition of macrophage TNFc~ release
Interferon-~' (IFN``,) is known to enhance the production of TNFz~ by macrophages significantly in response to LPS [I 1]. We therefore investigated whether IFN7 would ameliorate the impairment of macrophage TNF~ release induced by high concentrations of /%glucan. Accordingly, macrophages were incubated with recombinant murine IFN7 (1 or 10 ng/ml; Genzyme) for 16 h of incubation with LPS (0 10 pg;' ml) and /~-glucan (500 Izg/ml). TNF~ activity released into the medium was assayed as described above. We concurrently assessed the effect of IFN), alone (1-I0 ng/ml) on the macrophage generation of TNF~ as well as the effect of IFN7 (110 ng/ml) on the LPS-induced release of TNFz~ from macrophages in the absence of/~-glucan.
3.6.
Statistical analysis
All data are expressed as m e a n + S E M . We have observed that the absolute amounts of TNF~ released from alveolar macrophages in response to LPS or fl-glucan varied somewhat in magnitude when using macrophages isolated on different occasions. However, the general concentration characterisitcs of the macrophage TNF~ response to/%glucan and the induction of refractoriness during stimulation with high concentrations of fl-glucan were consistently present in all experiments. Accordingly, nonparametric methods were employed to test for differences between groups of data [12]. Differences between experimental and control data groups were determined using the Mann-Whitney U-test for nonparametric statistics performed on the Statview II statistical package (Abacus Conscepts, Berkeley, CA) using a Macintosh lIci personal computer. Differences were considered to statistically significant if P < 0.05 for two-sided alternatives. 4.
Results
Alveolar macrophages stimulated with fungal fl-glucans at concentrations less than 500 /Lg/ml exhibited an enhancement of TNFc~ release (Fig, 1). Maximal release of TNF~ activity occurred 21
1,000
Z I'-
4O0
0
0
1
10
100
5oo
1,000
B-glue, an (/.a:j/mL) *Denotes P<0.05 compared to control
Fig. 1. fl-Glucan stimulates the release of TNF~ from alveolar macrophages in a dose-dependent fashion. Alveolar macrophages (2 x l0 t) were incubated with increasing amounts of [Jgtucan for 16 h and TNF:~ release determined using an L929 cytotoxicity assay. While concentrations of/l-glucan less than 500 pg.ml stimulated TNF:< release from alveolar macrophages, concentrations /> 500/~g,;ml caused minimal release of TNFzc. Each data point represents the mean + SEM of 6 deterininations.
when alveolar macrophages were cultured with 100 pg/ml of ]~-glucan (P = 0.0003 compared with control). Interestingly, however, concentrations of //-glucan greater than or equal to 500 itg/ml caused a minimal release of T N F e activity. Indeed, macrophage release of TNF~ in the presence of 1000/tg/ml of/l-glucan was significantly lower than from macrophages cultured in the absence of/?-glucan (P=0.002). These data suggest that larger concentrations of fungal /~-glucan (>~ 500 /~g/ml) may actually inhibit the release of TNF:~ activity from alveolar macrophages. We next questioned whether the reduction of macrophage TNF~ release was due to an adverse effect of fungal/~-glucans on macrophage viability. Immediately upon isolation, macrophage viability was assessed to be 95.3_+0.2% by trypan blue exclusion. After 16 h of incubation in the absence of /?-glucan, macrophage viability was found to 93.7_+0.4%. Increasing concentrations of/~-glucan failed to cause any adverse effect on macrophage viability. Even after 16 h of incubation in the presence of 1000 /~g/ml of/~-glucan, macrophage viability remained at 93.3_+0.3% (not significantly different). These experiments document that the reduction of macrophage T N F 7 release induced by /~-glucan was not due 22
to impairment of macrophage viability. Instead, ¢/-glucan appears to modulate macrophage release of TNFc~ activity into the culture medium. To veri~' that /~-glucans were not interfering with the TNFc~ assay, we pertbrmed the following experiment. Supernatants from macrophages stimulated with low concentrations of /3-glucan (100 llg/ml) contained readily detectable quantities of TNF:~. These supernatants were removed from the macrophages and treated with either large concentrations of/~-glucan (1000 izg/ml) or no ~-glucan for an additional hour. Subsequently, the supernatants were centrifuged and the T N F 7 level determined using the L929 assay. We found the levels of TNF:~ to be equivalent in the supernatants treated with high concentrations of/~-glucan compared to those that remained untreated (P=0.19; not significantly different). It is therefore unlikely that high concentrations of []glucan interfered with the TNFc~ assay. Instead, it appeared that contact of the fl-glucan particles with the macrophages was required to modulate the release of TNFc~ activity. Next, we determined that macrophages challenged with large concentrations of fungal //-glucan were also refractory to stimulation by bacterial LPS, an extremely potent agonist for TNFT.
loo.ooo r
• ,8-glucan (500/J.g/mL)
Pj,I,I 1110 10 1
.
0
10
100
1,000
10,000
LPS concentration(ng/mL) Fig. 2. Large concentrations of fungaI //-glucan inhibit macrophage release of TNF:< in response to bacterial LPS. Alveolar macrophages (2 × 10-s) were preincubated with ,q-glucan (500 /~g/ml) for 16 h in the presence of increasing amounts of LPS. TNFc~ release into the medium was determined using an L929 cytotoxicity assay. Large concentrations of/~-glucan ( ~>500 ,ug' ml) caused suppression of the amount of TNF:< released into the medium. Each data point represents the mean + SEM of 6 determinations.
100.000 10,000
uE
1,000
ZQ. I-- " "
o,=
.9°=
100
10
1
• LPS alone LPS + IFN m LPS + IFN + .8-glucan
/' 0
1,000
10,000
LPS concentration (ng/mL) Fig. 3. Interferon-,' does not reverse the/3-glucan-induced inhibition of macrophage T N F x release in response to LPS. Alveolar macrophages (2 × 105) were incubated with IFN-,' (I ng/ml) and increasing concentrations of LPS in the presence or absence of/~-glucan (500/~g,'ml) for 16 h. TNF:~ release into the medium was determined using an L929 cytotoxicity assay. In the absence of fl-glucan, IFN-'; significantly increased macrophage TNFc~ release in response to LPS. However, in the presence of 500 pg,,ml/~-glucan, macrophage release of TNF~ into the medium was not increased above baseline. Each point represents the mean +_SEM of 3 separate determinations.
generation. Interestingly, large concentrations of /#glucan (500 pg/ml) consistently suppressed the macrophage release of TNF:~ activity even following stimulation with progressively larger concentrations of LPS (Fig. 2). Macrophages cultured with LPS alone released substantial quantities of TNF:~ into the medium. However, macrophages incubated with LPS in the presenceof /3-glucan (500/~g/ml) exhibited a significant suppression of TNF~ activity (P=0.0001, comparing macrophages cultured with LPS in the presence or absence of/3-glucan). These findings confirmed our hypothesis that higher concentrations of/~-glucan rendered macrophages refractory to TNF:~ release. Interferon-7 (IFN~') is a potent cytokine derived from T lymphocytes which significantly enhances TNF~ release from mononuclear phagocytes. It has been previously reported that TNFz~ can reverse the endotoxin-resistant phenotype of macrophages and restore their ability to release TNFc~ in response to LPS [11]. Full activation of macrophage TNF~ biosynthesis occurs at an IFN;, concentration as low as 1 ng/ml [11]. Accordingly, we investigated whether IFN7 would
enhance the release of TNF~ from alveolar macrophages stimulated with LPS in the absence of/3-glucan and whether IFN7 would restore the ability of macrophages to release TNF~ in the presence of inhibitory concentrations of/?-glucan (Fig. 3). As anticipated, alveolar macrophages cultured without /~-glucan exhibited a substantial enhancement of TNFc~ release following LPS stimulation in the presence of IFN),. While macrophages challenged with 1000 ng/ml of LPS alone released 628.0_+259.4 pg/ml of TNF~, macrophages stimulated with the same concentration of LPS in the presence of IFN (1 ng/ml) liberated 4503.1_+1194.8 pg/ml of TNF~ (P = 0.0007). However, in the presence of 500 pg/ml of/3-glucan, macrophage release of TNF~ uniformly remained at baseline levels with or without the addition IFN'~, (Fig. 3). Macrophages cultured with IFN7 (1 ng/ml) and challenged with up to 10 000 ng/ml of LPS released only 8.3_+0.1 pg/ml of TNF:~ in the presence of 500 /tg/ml of/%glucan (not significantly different to TNF~ release from unstimulated macrophages). In contrast, macrophages cultured under identical conditions (IFNT, 1 ng/ml: LPS, 10 000 ng/ml) released 20 870.4 _+497.9 pg/ml of TNF~. in the absence of/3-glucan. Even in the presence of tenfold greater concentrations of IFN), (10 ng/ml), J~-glucan still caused a substantial inhibition of macrophage TNFc~ release in response to LPS. Macrophages cultured with ~-glucan (500/~g/ml) and stimulated with up to 10 000 ng/ml of LPS in the presence of 10 ng/ ml IFN7 still released 70% less TNFc~ than macrophages cultured under identical conditions without J~-glucan. These data demonstrate that fungal j%glucans can render macrophages refractory to LPS stimulation and that IFN?' is not effective in reversing the inhibition of TNFc~ release caused by J?-glucan. 5.
Discussion
These studies for the first time demonstrate several important features of the interaction of fungal /~-glucans with alveolar macrophages. /3Glucans exhibit an interesting dose-response relationship for the macrophage stimulation of TNFc~ release. While lower doses of /~-glucan enhance 23
macrophage TNFc~ release, higher concentrations (~> 500/~g/ml) lead to minimal TNF,~ release and in fact appear to inhibit its liberation. Indeed, macrophages cultured in the presence of large concentrations of fungal fl-glucan are refractory to stimulation by bacterial LPS even in the presence of IFNT, an agent which substantially enhances macrophage TNF:~ release. The observed response of macrophages to stimulation with increasing concentrations of fungal fi-glucan may further explain the contrasting activities of fl-glucan which have been previously reported. The existing literature suggests that under differing conditions, fi-glucans can either stimulate or suppress macrophage activation [3,6,1318]. Published observations by ourselves and others document that fungal fl-glucans can activate alveolar macrophages [13 15], enhancing the release of TNF:~ [6] and arachidonic acid metabolites [16]. Others have similarly shown that particulate fl-glucans can stimulate the macrophage release of IL-I [13 15]. In contrast, Rasmussen and Seljelid's work suggests that particulate fl-l,3polyglucose (glucan) may under other circumstances modulate macrophage activity and suppress TNF:~ release [3]. In their study, the intraperitoneal administration of glucan-derivatized microbeads inhibited the lethal TNF~. release from peritoneal macrophages in response to E. coli and prevented death in these animals [3]. These observations are consistent with our data which demonstrate that macrophages exposed to high local concentrations of fl-glucan exhibit an impaired TNF~ release even in response to bacterial LPS. Although macrophages exhibit an impaired release of TNFc~ activity following exposure to large concentrations of fungal /%glucans, other macrophage responses appear to remain intact. In particular, Daum and Rohrbach have recently reported that alveolar macrophages continue to exhibit alterations of eicosanoid metabolism and release arachidonic acid even in the face of large concentrations of fl-glucan, strongly suggesting that macrophages are not totally anergic during fl-glucan stimulation and can continue to exhibit other cellular responses [16]. Furthermore, alveolar macrophages stimulated with viable fungal organisms such as Pneumocvstis carinii in the pre24
sence of large concentrations of fi-glucan ( >~500 llg/ml) exhibit an impairment of TNF:~ release but continue to liberate arachidonic acid in response to the organisms [6,17]. Taken together, these findings suggest that the inhibition of macrophage TNF:~ release induced by high concentrations of fl-glucan does not result in a generalized inactivation of the macrophage. The cellular mechanisms by which fi-glucans render the macrophage refractory to LPS-induced TNF~ release are presently unknown. Stimulation of macrophage with fi-glucans does not necessarily require particle phagocytosis but does require that macrophages recognize a fixed steric arrangement of glucan epitopes [13]. For instance, although particulate fl-glucans and glucan-derivatized plastics both cause macrophage stimulation, soluble glucans do not usually possess these activities [13]. This may suggest that a rearrangement or clustering of glucan receptors is necessary for macrophage activation. It is possible that when presented with large concentrations of fl-glucan, the cognate receptors are scattered, and clustering is prevented, fi-Glucans possess other activities which may regulate TNF~. expression as well. For instance, fl-glucans induce the macrophage release of arachidonic acid and PGE2 [16]. PGE2 has been demonstrated to suppress TNFu, release in vitro [18]. It is also possible that large concentrations of fungal fl-glucans may enhance the macrophage uptake and degradation of TNFzc Clearly, additional studies will be required to define the exact mechanisms by which fl-glucans can both stimulate and inhibit the macrophage release of TNFzc The clinical relevance of our observations remains to be determined. Modulation of TNF:~ release from mononuclear phagocytes in response to LPS is of potential therapeutic importance in a number of conditions. TNF~ has established activity in the pathogenesis of endotoxin-induced shock and the adult respiratory distress syndrome [1,2]. fl-Glucans and related sugars may provide additional means to modify TNF~. activity during such disorders. However, extreme caution is advised in rapidly extrapolating these in vitro observations to the living host. Additional basic and preclinical investigations will be required to confirm these activities in the whole organism.
Acknowledgements This work was supported by funds from the American Heart Association (Clinician Scientist Award No. 91004230), an American Lung Association Research Grant (No. RG-011-N), and funds from the Mayo Foundation to A.H.L. We wish to thank Drs. Michael Rohrbach and Zvezdana Vuk-Pavlovic for many helpful discussions, Mr. Edward Mansfield for assistance with the Limulus amebocyte lysate for endotoxin, and Ms. Kathy Streich for her assistance in preparation of the final manuscript.
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[mmunol. 150, 3932. [7] Manners, D.J., Masson, A.J. and Patterson, J.C. 11974) J. Gen. Microbiol. 80, 41 I. [8] Kadish, J.L., Choi, C.C. and Czop, J.K. (1986) Fed. Proc. 45, 1115. [9] Janusz, M.J., Austen, K.F. and Czop, J.K. ~1986) J. Immunol. Methods 93, 201. [10] Kramer, S.M. and Carver, M.E. (1986) J. lmmunol. Methods 93, 201. [11] Beutler, B., Tkacenko, V., Milsark. I.. Korchin, N. and Cerami, A. (1986) J. Exp. Med. 164, 1791. [12] Rosner, B. (1986) Fundamentals of Biostatistics, Duxbury Press, Boston. [13] Bogwe[l, J., Gouda, I., Hoffmam 1.. Larm, O., Larsson, R. and Seljelid, R. (1986) Scan& J. lmmunol. 20. 355. [14] Seljelid. R., Fingenschau, Y., Bogwell, J., Rasmussen. L.T. and Austgu[en. J. (1989) Scand. J. lmmunol. 30, 687. [15] Seljelid, R., Bogwell, J., Rasmussen, L.T., Larm, O.. Hoffman, I., Berge, A. and Austgulen, J. (1985) Scand. J. lmmunol. 21,601. [16] Daum, T. and Rohrbach, M.S. (1992) FEBS Lett. 309. 119. [17] Castro, M., Morgenthaler, T.I., Hoffman, O.A., Standing, J.E., Rohrbach, M.S. and Limper, A.H. (1993) Am. J. Respir. Cell. Mol. Biol. 9, in press. [18] Kunkel, S i . , Wiggins, R.C., Chensue, S.W. and Larrick, J. (1986) Biochem. Biophys. Res. Commun. 137, 404.
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