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This document is also available as a PDF. Induced production of nitric oxide, tumor necrosis factor, and
interleukin-6 in RAW 264.7 macrophages by streptomycetes from
indoor air ofmoldy houses. INDOOR BIOAEROSOLS are important health factors, and they can cause
asthma, bronchitis, repetitive respiratory tract infection, and rhinitis.[1,
2] The tip of the iceberg is seen in moldy houses in which a variety
of fungi and bacteria, especially actinomycetes, are grown; this growth
occurs from the excess moisture in structures after accidental leakage
or from gradual condensation of water.[3] Investigators have not described
satisfactorily the most important causative microbes among the mixed
population. In addition, almost nothing is known about the cellular
mechanisms, even if the clinical entity associated with exposure has
been described and the exposure levels to the most prevalent bacteria
and fungi present in moldy houses have been characterized.[4,5] We
hypothesized that a likely contributing factor in the etiological
mechanism of respiratory symptoms could be an inflammatory response
toward specific organic materials in the microbes. This mechanism
would include production of inflammatory mediators, such as cytokines
and nitric oxide (NO), in immunologically active cells. Cluzel et al.[6] reported that cytokines are well-known mediators,
which play an important physiological role in the functions of the
immune system. Activated macrophages secrete a variety of biologically
active substances (e.g., eicosanoids, oxygen radicals, cytokines [including
tumor necrosis factor alpha {TNF[Alpha]} and interleukin-6 {IL-6}]).
Moreover, it is now evident that the highly reactive radical, nitric
oxide, is an important mediator in nonspecific host defense against
microbes and tumors.[7,8] Recently, investigators have focused much
interest on the possible role of NO as an immune defense molecule.[9]
Researchers have suggested that NO is produced by activated macrophages
via a nonspecific immune response directed against invading microorganisms.
The biological source of NO is L-arginine, which is converted to L-
Citrulline and NO in macrophages by inducible NO synthase (iNOS).[10]
Under normal steady-state conditions, iNOS is not detectable, but
it is induced within a few hours after stimulation by lipopolysaccharide
(LPS) and various cytokines (e.g., interferon-gamma [INF[Gamma]],
TNF[Alpha]).[10] Thus, macrophage-derived NO and cytokines function
as cytotoxic molecules, which can kill invading microorganisms,[11]
but they also have important roles in the functions of the immune
system and in the pathophysiology of inflammatory diseases[12] and
asthma.[13] In this study, we investigated whether any of the microbial strains
typically present in the indoor air of moldy houses (i.e., Candida
sp., Stachybotrys sp., and Streptomyces sp.), or strains belonging
to the normal flora of indoor air (i.e., Penicillium sp., Cladosporium
sp., and Aspergillus sp.), can induce the production of proinflammatory
cytokines TNF[Alpha] and IL-6 or activate iNOS with subsequent NO
production in RAW 264.7 macrophage cell line. Material and Method Materials. We obtained mouse macrophage cell line RAW 264.7 (RAW)
from American Type Tissue Collection (Rockville, Maryland). We grew
it on six well plates at 37 [degrees] c (5% [co.SUB.2]) IN rpmi MEDIUM
1640 (gIBCO lAB [Grand Island, New York]), which was supplemented
with 10% fetal bovine serum (Hyclone Lab [Logan, Utah]), 1% L-glutamine,
and 1% PNS antibiotic mixture (both from Gibco Lab). We obtained
sulfanilamide, naphthylethylenediamine di hydrochloride, and 5-bromo- 1 of 8 7/25/99 3:43 PM Printable Document-Electric Library Personal Edition http://business.elibrary.com/s/elbe/getd...docid=2175305@library_k&dtype=0~0&dinst= 4-chloro-3-idolylphosphate/nitrobluetetrazolium (BCIP/NBT) from Sigma
Chemical Co. (St. Louis, Missouri). In addition, we obtained rabbit
monoclonal 4195 antibody against mouse macrophage iNOS from Burroughs
Welcome (North Carolina) and antirabbit IgG from Jackson Immune Research
Lab (West Grove, Pennsylvania). Isolation, identification, and preparation of fungi and bacteria.
We used five strains of fungi (i.e., Candida sp., Aspergillus sp.,
Cladosporium sp., Penicillium sp., and Stachybotrys sp.) and two
strains of mesophilic gram-positive bacteria (i.e., actinomycetes
A4 and A91, representing the genus Streptomyces). We isolated the
strains from indoor samples of mold problem houses, as described by
Hyvarinen et al. in 1993.[3] We used light microscopy[14,15] to identify
the four strains of fungi (Aspergillus sp., Cladosporium sp., Penicillium
sp., and Stachybotrys sp.) morphologically. One of the fungal strains
was identified as a yeast, Candida albicans, by use of the API ID32C
test kit. The actinomycete strains were identified in Deutsche Sammlung
von Micro-organismen und Zellkulturen GmbH as Streptomyces anulatus
(A91) and Streptomyces californicus M). We cultured the fungal strains on malt extract agar (MEA; Blakeslee)
and the strains of actinomycetes on Trypton yeast-glucose agar (Bacto
Plate Count agar, Oxoid). The cultures were incubated in the dark
at 20-23 [degrees] C for 7 d. The spore suspensions from these cultures
were prepared in a phosphate buffered saline (PBS) medium that contained
0.0001% of Triton X-100. Treatment of RAW 264.7 cells. We incubated the RAW 264.7 macrophages
for 24 h in a fresh medium containing 0, 1 x [10.sup.5], 5 x [10.sup.5],
1 x [10.sup.6], 5 x [10.sup.6], or 1 x [10.sup.7] spores/[10.sup.6]
cells of Candida sp., Aspergillus sp., Cladosporium sp., Penicillium
sp., Stachybotrys sp., or Streptomyces sp. A4 or A91. We studied the
effects of spores on cell viability, nitrite production, expression
of iNOS, and production of TNF[Alpha] and IL-6. Nitrite analysis. Nitrite produces a chromophore with the Griess
reagent, with an absorbance maximum at 543 nm, which can then be quantitated
spectrophotometrically[16] with an automated colorimetric procedure.
Briefly, we added 50 [micro]l of cell culture medium to each well
of a 96-well plate. We added 50 Jai of Griess reagent (1% sulfanilamide
and 0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric
acid) and left the plate shaking for 10 min at room temperature. We
used a microplate reader (Labsystems iEMS Reader MF) to measure the
[OD.sub.540]nm. To calculate nitrite concentrations, we compared them
with standard solutions of sodium nitrite produced in the culture
medium. Western blot analysis. The cells were washed with cold PBS, lysed
in lysis buffer (50 mM Tris-hydrochloric acid (Tris-HCI), 1 mM ethylenediaminetetraacetic
acid (EDTA), 10 [micro]mol PMSF, and 10 [micro]l/ml leupeptin), and
resuspended on ice for 10 min. The cell suspension was lysed further
via ultrasound sonicator, and it was centrifuged at 1 400 rpm for
15 min. At the completion of this procedure, 4x sample buffer was
added to the supernatant and heated at 95 [degrees] C for 10 min.
Lysates (20-[micro]g protein) were subjected to electrophoresis via
7.5% SDS-PAGE, and proteins were transferred electrophoretically to
a nitrocellulose filter. The filters were incubated overnight at 4
[degrees] C in blocking buffer (50 mM Tris-HCI, 150 mM sodium chloride
[NaCI], 1 mg/ml PEG 2000, 3% BSA). The filters were then incubated
for 2 h at room temperature in a washing buffer (50 mM Tri-HCI, 150
mM NaCI, 1 mg/ml PEG 2000, 1 mg/ml BSA) that contained a dilution
of 1:500 of rabbit monoclonal antibody 4195 obtained against the N-
terminal fragment of mouse macrophage iNOS.[17] Three 10-min washes
in washing buffer were completed, after which the filters were incubated 2 of 8 7/25/99 3:43 PM Printable Document-Electric Library Personal Edition http://business.elibrary.com/s/elbe/getd...docid=2175305@library_k&dtype=0~0&dinst= for 1 h at room temperature in a washing buffer containing a 1:2 000
dilution of alkaline-phosphatase-conjugated anti-rabbit IgG. Three
10-min washes in a washing buffer were completed and were followed
by exposure of the filters to alkaline phosphatase developing buffer
(100 mM NaCI, 100 mM Tris-Base, 5 mM magnesium chloride [MgCI], pH
= 9.5) for 2 min; the filters were developed with BCIP/NBT. Analysis of TNF[Alpha] and IL-6. We used 100 [micro]l/well of antibody
solution in 0.1 M sodium carbonate buffer (0.1 M Na[HCO.sup.3], pH
adjusted to 9.6 with 0.1 M [Na.sub.2][CO.sub.3]) to coat the microtiter
strips with a monoclonal antibody for IL-6 (1 [micro]g/ml) or TNF[Alpha]
(2 [micro]g/ml). The strips were shaken at 200 rpm for 2 h at room
temperature. Three washings were completed, after which we blocked
nonspecific binding by incubating the wells with 200 [micro]l of 1%
BSA in 50 mM Tris-HCI buffer containing 150 mM NaCI (pH = 7.5) at
room temperature for 1 h; the strips were again washed three times.
The samples and standards, diluted in the [DELFIA.sup.R] assay buffer,
were added to the strips (200 [micro]l/well) and incubated overnight
at +4 [degrees] C. The strips were washed three times, and the biotinylated
second antibody (250 ng/ml of IL-6 and 500 ng/ml of TNF[Alpha] was
added to the wells in 200 [micro]l of [DELFIA.sup.R] assay buffer.
The strips were incubated for 1 h at room temperature, after which
they were washed once, and 200 [micro]l of Europium-labelled streptavidin
(100 ng/ml) in [DELFIA.sup.K] assay buffer was dispensed to the wells.
The strips were incubated at room temperature at 30 rpm shaking and
were washed six times. Europium was rendered fluorescent by releasing
it from the streptavidin with [DELFIA.sup.K] enhancement solution
(200 [micro]l/well) and by shaking the strips gently for 5 min. After
10-15 min of equilibration, a LKB Wallac 1230 Arcus fluorometer[18]
used the spline smoothed algorithm of the RIA Calc software to measure
fluorescence. Cell viability. We calculated the percentage of macrophages alive,
after dying the cells with Trypan Blue solution, to measure culture
viability. Statistical analysis. We used one-way analysis of variance and
Duncan's multiple-range test to analyze the data statistically. The
accepted level of statistical significance was p [is less than] .05. Results Nitrite production and expression of iNOS. Nitric oxide production
in RAW 264.7 macrophages was assayed in the culture medium as the
stable NO oxidation product, nitrite, and it was significantly and
close dependently increased at 24 h, by all the tested doses of Streptomyces
anulatus (A91) and Streptomyces californicus (M). However, only the
two highest doses of Stachybotrys sp., Candida albicans sp., and Cladosporium
sp. slightly increased the nitrite levels in the culture medium, whereas
Aspergillus sp. and Penicillium sp. did not affect NO production (compared
with controls [Fig. 1]). [Figure 1 ILLUSTRATION OMITTED] To rule out NO production induced by endotoxin contamination in
the two strains of actinomycetes (Streptomyces sp. A4 and A91), we
also used a spectrophotometer method to test spore samples with the
Limulus Amebocyte Lysate test. Both strains were free of endotoxin
contamination (i.e, [is less than] 0.005 ng/[10.sup.6] cells). To definitely demonstrate the different ability of the tested microorganisms
to induced NO production in RAW 264.7 macrophages, we also analyzed
expression of iNOS. Consistent with the nitrite levels in the culture
medium, Western Blot analysis (with antibody against iNOS [1 30 kDa]) 3 of 8 7/25/99 3:43 PM Printable Document-Electric Library Personal Edition http://business.elibrary.com/s/elbe/getd...docid=2175305@library_k&dtype=0~0&dinst= revealed that the expression of iNOS was induced only by Streptomyces
A91 and A4, but not by any of the tested fungal strains (Fig. 2). [Figure 2 ILLUSTRATION OMITTED] Production of TNF[Alpha] and IL-6. The RAW 264.7 macrophages stimulated
by Streptomyces A91 or A4 produced dose-dependent high amounts of
TNF[Alpha] at 24 h. in addition, the production of TNF[Alpha] was
increased by the highest dose of Penicillium sp. and Cladosporium
sp., compared with controls (Fig. 3A). [Figure 3A ILLUSTRATION OMITTED] Incubation of the cells with the graded concentrations of spores
of Streptomyces A4 or A91 induced a massive dose-dependent accumulation
of IL-6 in the culture medium of RAW 264.7 macrophages. All the other
microorganisms tested had only a minor effect on IL-6 production,
compared with controls (Fig. 3B). [Figure 3B ILLUSTRATION OMITTED] Cell viability. We explored the viability of RAW 264.7 macrophages
after they were stimulated for 24 h with the tested microorganisms
to determine whether the alterations in endogenous NO, TNF[Alpha],
or IL-6 production were associated with a loss of cell viability.
Only Stachybotrys sp. induced a significant dose-dependent decrease
in cell viability (i.e., down to 8%) in 24 h, whereas the viability
among control cells was 97%. The other strains of fungi and both strains
of actinomycetes (Streptomyces A4 and A91) either did not appreciably
affect cell viability in the culture or decreased viability only slightly
at the highest dose (Fig. 4). [Figure 4 ILLUSTRATION OMITTED] Discussion The results of the present study indicate that gram positive bacteria
Streptomyces californicus (M) and Streptomyces anulatus (A91), isolated
from the indoor air of moldy buildings, stimulate macrophages, which
produce significant amounts of TNF[Alpha] and IL6, and induce the
expression of iNOS with subsequent production of NO. However, under
the present experimental situation, NO-producing macrophages did not
induce cytotoxicity. Interestingly, the other microorganisms tested,
also typical to moldy buildings, were without any effect or, at the
most, caused only a slight increase in the production of inflammatory
mediators. Streptomyces-induced production of NO in macrophages is of particular
interest because recently Barnes et al.[19] reported that NO was produced
by a variety of cells in the airways, suggesting that respiratory
tract infections may induce iNOS expression.[20] Consistent with this
notion, a marked increase in the concentration of exhaled NO was described
in asthmatic patients[21,22]; perhaps this resulted from iNOS induction
in airway epithelial cells and inflammatory cells (e.g., macrophages).
Sustained production of high amounts of NO by iNOS may exert proinflammatory
effects, including vasodilatation, edema, cytotoxicity, and induction
of cytokine-dependent processes.[10] In this study, we showed that gram-positive bacteria Streptomyces
sp. A4 and A91 induced the expression of iNOS and subsequent production
of NO in the same manner as was reported by Moncada et al.[23] and
Hirvonen et a.[26] for exposure to interferon-gamma ([INF.sub.[Gamma]])
and endotoxin LPS, which is an important factor in the cytotoxicity
induced by gram-negative bacteria. Streptomyces ap. A4 and A91 induced 4 of 8 7/25/99 3:43 PM Printable Document-Electric Library Personal Edition http://business.elibrary.com/s/elbe/getd...docid=2175305@library_k&dtype=0~0&dinst= expression of iNOS, without prior priming of the macrophages, and
this is contrary to the general assumption that iNOS-inducing agents
do not act alone but, rather, in combination with [INF.sub.[Gamma]].[24]
14 Moreover, neither of the strains of Streptomyces sp. was cytotoxic
to NO-producing macrophages themselves, whereas in earlier studies
by others[25] and us,[26] it was suggested that the life span of macrophages,
stimulated by LPS and/or [iNF.sub.[Gamma]], correlated negatively
with their NO production. Only Stachybotrys sp. induced a significant
dose-dependent decrease in cell viability, but it was not related
to NO production of the cells; therefore, perhaps the cellular mechanisms
behind the health effects induced by Stachybotrys sp. are different
from those induced by Streptomyces sp. These results are very interesting,
because researchers have speculated that NO is one of the cytotoxic
agents by which activated macrophages kill bacteria, tumor cells,
and a variety of other pathogens--even normal tissue cells during
autoimmune reactions.[27] Therefore, whereas NO plays an important
immunological, defensive role in the killing of foreign organisms,
in acute inflammation its excessive production may cause tissue damage.[28] During our search of the plausible cascade of events that lead
to iNOS expression and NO production in macrophages stimulated by
gram-positive bacteria A4 and A91 Streptomyces sp., we also studied
the production of proinflammatory cytokines TNF[Alpha] and IL-6. Moncada
et al.[23] reported that these cytokines are released by other iNOS
expression-inducing agents (e.g., LPS). Both of the strains we tested
induced significant release of TNF[Alpha] and IL-6 in cell culture
medium, thereby suggesting that some components of gram-positive bacteria
may also be effective inducers of these mediators. All the other microorganisms
that we isolated from moldy buildings were without effect or, at most,
caused only a slight increase in IL-6 and TNF[Alpha] production.
Increased levels of TNF[Alpha] were associated with elevated levels
of IL-6 in macrophages activated by Streptomyces sp. This finding
is supported by the results of Bauman et al.,[29] who speculated that
TNF[Alpha] is important in the initiation of events that cause the
release of other cytokines (e.g., IL-6). Moreover, these results are
consistent with the assumptions that (a) cytokines are powerful stimulators
of the iNOS pathway in many cell types,[30-32] and (b) lysis of target
cells by activated macrophages can be explained by the actions of
cytokines and NO. The present findings also shed new light on previous results from
our laboratory (i.e., A4 and A91 Streptomyces sp. induced a marked
increase in the production of reactive oxygen metabolites [ROM] in
human polymorphonuclear leukocytes [PMNLs[[33]). Reactive oxygen metabolites
may significantly increase the cytotoxicity of NO, because NO readily
reacts with superoxide to generate a stable peroxynitrite anion, which,
once protonated, decomposes to form an extremely reactive hydroxyl
radical.[34] Both of these radicals are strong oxidants, which may
be involved in tissue damage and in the cytotoxicity attributable
to NO.[34] Investigators have assumed that the reaction of NO with
superoxide anion plays a pivotal role in acute and chronic lung disease,
because proxynitrite induces lipid peroxidation and interferes with
several enzymatic pathways.[35,36] Moreover, given that IL-6 primes
respiratory burst in neutrophils and monocytes,[37] our present observation
of massive production of IL-6 upon stimulation of macrophages by gram-
positive bacteria is consistent with the finding of Ruotsalainen et
al.,[33] who reported increased production of ROM in PMNLs. Therefore, it appears that spores of Streptomyces sp., which are
present in the indoor air of moldy buildings and are sufficiently
small (1 [micro]m) to reach the alveoli, can be added to the list
of stimuli that induce production of cytokines and NO in murine macrophages.
If such production occurs in vivo, cytokines and NO may indeed play
a role in the responses evoked by exposure to these microbes. 5 of 8 7/25/99 3:43 PM Printable Document-Electric Library Personal Edition http://business.elibrary.com/s/elbe/getd...docid=2175305@library_k&dtype=0~0&dinst= The authors thank Ms. Virpi Jokinen and Ms. Tuula Wallenius for
their excellent technical assistance. We also thank Professor Jouko
Tuomisto for reading and commenting on the manuscript. This study
was supported by The Research Council for the Environ mental Sciences
and by The Medical Research Council, The Academy of Finland. Submitted for publication October 11, 1996; revised; accepted for
publication March 31, 1997. Requests for reprints should be sent to
Dr. Maiija-Riitta Hirvonen, Division of Environmental Health, National
Public Health Institute, P.O. Box 95, FIN-70701 Kuopio, Finland. References [1.] Brunekreef B. Damp housing and adult respiratory symptoms. Allergy
1992; 47:498-503. [2.] Spengler J, Neas L, Nakai S, et al. Respiratory symptoms and
housing characteristics. Indoor Air 1994; 4:72-84. [3.] Miller JD. Fungi as contaminants in indoor air. Atmos Environ
1992; 26AI 6:2163-72. [4.] Pasanen AL. Airborne mesophilic fungal spores in various residential
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problem buildings-concentrations and flora of viable fungi. Indoor
Air 1993; 3:337-41. [6.] Cluzel M, Lee TK. Cytokines. In: Asthma: Basic Mechanisms and
Clinical Management. London, Great Britain: Academic Press, 1992;
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activated macrophage effector molecule. Biochem Biophys Res Commun
1988; 157:87-94. [9.] Liew FY, Cox FE. Nonspecific defense mechanism: the role of nitric
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1988; 157:88-94. [12.] Chantry D, Feldman M. The role of cytokines in autoimmunity.
Biotechnol Therap 1990; 1:361-409. [13.] Cluzel M, Lee TK. Asthma: Basic Mechanisms and Clinical Management.
London, Great Britain: Academic Press, 1992; pp 315-31. [14.] Barnett HL, Hunter BB. Illustrated genera of imperfect fungi.
Minneapolis, MN: Burges Publishing Co., 1972; ISBN 8087-0266-1.241. [15.] Barron GIL. The genera of Hyohomycetes from soil. Malaba, FL:
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RAW264 cell line and time-resolved fluoroimmunoassay (TRFIA) as tools
in screening drug effects on cytokine secretion. Int J Immunopharmacol
1995; 17:475-540. [19.] Barnes PJ, Belvisi MG. Nitric oxide and lung disease. Thorax
1993; 48:1034-43. [20.] Heiss LN, Lancaster RJ, Corbett JA, et al. Epithelial autotoxicity
of nitric oxide: role in the respiratory cytopathology of pertussis.
Proc Natl Acad Sci 1994; 91:267-70. [21.] Kharitonov SA, Yates D, Robbins RA, et al. Increased nitric
oxide in exhaled air of asthmatic patients. Lancet 1994; 343:133-35. [22.] Peterson MG, Zetterstrom O, Argenius V, et al. Single-breath
nitric oxide measurements in asthmatic patients and smokers. Lancet
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Engl J Med 1993; 329:2002-12. [24.] Sheffler LA, Wink DA, Melillo G, et al. Exogenous nitric oxide
regulates INF-y plus lipopolysaccharide-induced nitric oxide synthase
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macrophage resistance to the toxic effects of nitric oxide. Biochem
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in autoimmunity. Immunol Today 1992; 13:157-62. [28.] Laskin JD, Heck DE, Laskin DL. Multifunctional role of nitric
oxide in inflammation. Trends Endocrinol Metab 1994; 5:377-82. [29.] Bauman H, Gauldie J. The acute phase response immunology Today
1994; 15:74-80. [30.] Stuehr D, Marietta MA. Induction of nitrite/nitrate synthesis
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gamma. J Immunol 1987; 139:518-23. [31.] Drapier JC, Wietzerbin J, Hibbs JBJ. Interferon-gamma and tumor
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inhibitory factor induces nitric oxide synthase in murine macrophages.
J Immunol 1993; 150:1908-12. [33.] Ruotsalainen M, Hyvarinen A, Nevalainen A, et al. Indoor bioaerosol-
induced production of reactive oxygen metabolites and their interactions 7 of 8 7/25/99 3:43 PM Printable Document-Electric Library Personal Edition http://business.elibrary.com/s/elbe/getd...docid=2175305@library_k&dtype=0~0&dinst= with fMLP and PMA. Environ Res 1995; 69:122-31. [34.] Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl radical
production by peroxynitrite: implications for enclothelial injury
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24. [35.] Dipersio JF, Billing P, Williams R. Human granulocyte-macrophage
colony-stimulating factor and other cytokines prime human neutrophils
for enhanced rachidonic acid release and leukotriene B4 synthesis.
J Immunol 1988; 140:4315-22. [36.] Kooy NW, Royall JA, Ye YZ, et al. Evidence for in vivo peroxynitrite
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151:1250-54. [37.] DeRojas-Walker TS, Tamir S, Ji H, et al. Nitric oxide induces
oxidative damage in addition to deamination in macrophage DNA. Chem
Res Toxicol 1995; 8:473-77. MAIJA-RIITTA HIRVONEN AINO NEVALAINEN Division of Environmental Health
National Public Health Institute Kuopio, Finland NIINA MAKKONEN JUKKA MONKKONEN Department of Pharmaceutics KAI SAVOLAINEN
Department of Pharmacology and Toxicology University of Kuopio Kuopio,
Finland COPYRIGHT 1997 Helen Dwight Reid Educational Foundation Hirvonen, Maija-Riitta; Navalainen, Aino; Makkonen, Niina; Monkkonen, Jukka; Savolainen, Kai,
Induced production of nitric oxide, tumor necrosis factor, and interleukin-6 in RAW 264.7 macrophages
by streptomycetes from indoor air ofmoldy houses.. Vol. 52, Archives of Environmental Health,
11-01-1997, pp 426(7). Copyright © 1998 Infonautics Corporation. All rights reserved. - Terms and Conditions 8 of 8 7/25/99 3:43 PM |
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