 Production by
Human Alveolar Macrophages
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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Tumor necrosis factor alpha (TNF-
), a major product of alveolar macrophages (AM), has been implicated in many pulmonary diseases. Histamine, a mediator important in pulmonary inflammation, has been
demonstrated to regulate the production of TNF- by monocytes. In this study, we show that human AM
and monocytes differ in their responses to histamine. Whereas histamine suppressed lipopolysaccharide (LPS)-stimulated TNF- production by monocytes through a cAMP-dependent mechanism, it had no effect on either cAMP levels or TNF- production by AM. In contrast, both PGE2 and IL-10 suppressed
LPS-stimulated TNF- production by AM and monocytes. The lack of response of AM to histamine appears unique, as histamine suppressed LPS-stimulated TNF- production by mononuclear cells isolated
from sites of acute and chronic inflammation, as well as from noninflammatory tissues, and by macrophages differentiated in vitro. In the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-
methylxanthine, histamine increased cAMP levels in AM. Freshly isolated monocytes and AM did not
differ in PDE activity. However, PDE activity in AM, but not in monocytes, was increased 15 min after
culture with histamine and may, in part, be responsible for the inability of histamine to suppress TNF-
production by AM. However, this increase was small and we hypothesize that additional mechanisms
may contribute to the unresponsiveness of AM to histamine. We suggest that the lack of response of AM
to histamine may be important in the host defense function of AM in the distal lung.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The lungs represent the largest interface between the body and the environment. Consequently, alveolar macrophages (AM), through their ability to produce a wide range of mediators, play a crucial role in host defense against inhaled aerosols in the lower respiratory tract (1).
In addition to stimulating tumor necrosis factor-alpha
(TNF-) production (2), many inhaled pathogens can
trigger mast cell degranulation. Pseudomonas aeruginosa
hemolysins are potent stimulators of histamine release
from mast cells in vitro (6). Interestingly, histamine has
been shown to suppress lipopolysaccharide (LPS)-stimulated TNF- production by human monocytes (7, 8). This
suppression occurs at the level of transcription (7) via an
initial rise in intracellular cAMP, and is inhibited by the histamine type 2 (H2) receptor antagonist cimetidine (8).
Because both histamine and TNF- may be produced in
the lung following inhalation of pathogens, and because
TNF- may play an important role in the host defense capacity of AM, we have examined the effect of histamine
on TNF- production by AM, and compared their response with that of monocytes/macrophages isolated from
other sites.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Reagents
The following reagents were obtained as gifts: recombinant macrophage-colony stimulating factor (M-CSF) (Dr.
J. Schreurs, Chiron Corporation, Cetus Oncology Division,
Emeryville, CA); recombinant granulocyte-macrophage
colony stimulating factor (GM-CSF) (batch 9AO1-NO4O;
Genetics Institute, Cambridge, MA); and recombinant human IL-10 (Dr. S. Narula, Schering-Plough, Kenilworth,
NJ). The mAb to TNF- for ELISA was obtained from
Professor A. C. Allison (Syntex, Palo Alto, CA).
Subjects
Human AM were isolated from the bronchoalveolar lavage (BAL) fluid of healthy, nonsmoking, male volunteers (mean age 25 yr, range 19-30 yr) or patients undergoing investigative bronchoscopies for ailments such as persistent cough. The subjects were premedicated with intravenous atropine (0.3 mg; Astra, Sydney, Australia) and midazolam (3 mg; Roche Products, Sydney, Australia). The nose and pharynx were anesthetized with topical 4% lignocaine (Astra), and 4% lignocaine was gargled. Additional lignocaine (2%) was applied to the vocal cords, trachea, and bronchi via the flexible bronchoscope which was introduced nasally with the subject supine. The tip of the bronchoscope was wedged in the right middle lobe segmental bronchus (for healthy volunteers) or in an unaffected lobe (for patients), and a 10-ml volume of sterile 0.9% NaCl (37°C) was instilled and immediately withdrawn and discarded. Sequential 20-ml volumes were then instilled and withdrawn. Peripheral blood was obtained from healthy volunteers immediately prior to BAL.
Isolation of AM and PBMC
AM were isolated from BAL fluid by twice washing in RPMI 1640 medium (Cytosystems, Castle Hill, NSW, Australia) supplemented with 2 mM MOPS, 50 µM 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Cytosystems) (subsequently referred to as complete-RPMI) by centrifugation (300 × g, 7 min, 4°C). Viability was 94 ± 4% with an AM purity of 78 ± 11% (mean ± SD, n = 14; healthy volunteers) or 90 ± 10% (n = 7; patients), as assessed by morphology on Papanicolaou- and Giemsa-stained cytocentrifuge smears.
Peripheral blood was diluted 1 vol:2 vol with 0.9% NaCl. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation of diluted peripheral blood on a pyrogen-free Lymphoprep density gradient (Nycomed, Oslo, Norway) (470 × g, 30 min, 18°C). PBMC were washed twice in complete-RPMI by centrifugation (300 × g, 7 min, 4°C). Purity of monocyte preparations was 32 ± 5% (n = 18) as assessed by morphology on Giemsa-stained cytocentrifuge smears.
Isolation of Mononuclear Cells from CAPD Fluid
Mononuclear cells were isolated from the peritoneal effluent of patients with peritonitis while on a program of continuous ambulatory peritoneal dialysis (CAPD) as previously described (9). Briefly, peritoneal effluents were centrifuged (400 × g, 10 min), and cell pellets resuspended in complete-RPMI. Mononuclear cells were isolated by centrifugation on a pyrogen-free Lymphoprep density gradient. For cells isolated from 3 patients, 51 ± 3% of the total cell population were monocytes/macrophages as assessed by morphology on Giemsa-stained cytocentrifuge smears, with the rest being lymphocytes.
Isolation of Mononuclear Cells from Synovial Fluid
As previously published (10), synovial fluids were diluted 1 vol:3 vol with 0.9% NaCl within 2 h of joint aspiration. Mononuclear cells were isolated by centrifugation on a Lymphoprep density gradient as discussed above. Monocytes/macrophages comprised 68 ± 8% of the cells isolated from 3 patients with rheumatoid arthritis, the rest being lymphocytes.
Isolation of Mononuclear Cells from Breast Milk
Human breast milk was expressed between 4 and 13 days postpartum. Using a modification of a published technique (11), the milk was diluted to 50% with 0.9% NaCl, and washed by centrifugation 3 times (300 × g, 7 min) within 3 h of collection. For cells isolated from 3 donors, macrophage purity was 49 ± 6%, with the majority of the contaminating cells being lymphocytes.
Differentiation In Vitro of Human Monocytes
Human peripheral blood monocytes were obtained by countercurrent centrifugal elutriation of buffy coats kindly provided by the Adelaide Red Cross Blood Transfusion Service, as previously described (8). Monocyte-enriched fractions (95 ± 3%) were cultured in 60-ml Teflon pots (Savillex, Minnetonka, MN) in complete-RPMI supplemented with 10% (vol/vol) heat-inactivated FCS in the presence of M-CSF (200 ng/ml) or GM-CSF (100 U/ml, 10 ng/ml) (12). After 1 wk, cells were washed by centrifugation (300 × g, 7 min, 4°C), with cell recovery being 51 ± 14% (GM-CSF, n = 3) and 47 ± 12% (M-CSF, n = 3).
Cell Culture for Cytokine Measurements
PBMC (1.25 × 106 cells/500 µl) or AM, CAPD fluid-derived
mononuclear cells, breast milk-derived mononuclear cells,
synovial fluid-derived mononuclear cells, and in vitro
monocyte-derived macrophages (5 × 105 cells/500 µl)
were cultured in complete-RPMI supplemented with 1%
FCS in 48-well tissue culture plates (Falcon, Becton Dickinson, Lindon Park, NJ). The following reagents were added
to give final concentrations as indicated: histamine (histamine diphosphate, Sigma), 10
5 M; PGE2, 100 ng/ml (Advanced Magnetics Inc., Cambridge, MA); IL-10, 100 U/ml,
10 ng/ml; 3-isobutyl-1-methylxanthine (IBMX), 400 µM
(Sigma); and LPS (Escherichia coli 0111:B4), 500 ng/ml (Sigma). Triplicate cultures for each test variable were incubated at 37°C in 5% CO2 for 22 h. Cultures were terminated by removing supernatants and centrifuging (300 × g,
7 min, 4°C) to remove nonadherent cells. After removal of
supernatants, cell lysates were prepared by the addition of
10 mM HEPES in 0.9% NaCl (500 µl/well), followed by 2 freeze-thaw cycles.
Assay of TNF-
Immunoreactive TNF- was assayed by a sandwich
ELISA, using a mouse monoclonal anti-TNF- plate binding antibody, followed by a rabbit anti-TNF- polyclonal
antibody. A biotinylated antirabbit monoclonal antibody
was used, followed by extrAvidin peroxidase conjugate
(Sigma). The TNF- assay was sensitive to TNF- levels of 
40 pg/ml. Bioactive TNF- was measured by determining cytotoxicity to the TNF- sensitive cell line L929, as
previously described (13). One unit of TNF- was defined
as the amount of TNF- resulting in 50% lysis of L929
cells.
3H-Histamine Uptake
3H-histamine uptake by AM was assayed using a modification of the procedure of Mitra and colleagues (14). Briefly, AM (5 × 106 cells/ml) were cultured for 2 h at 37°C in complete-RPMI supplemented with 20% FCS with 3H-histamine dihydrochloride (Amersham, Sydney, Australia; specific activity 56 Ci/mmol; 0.5 µM) and unlabelled histamine (0 to 500 µM). Cultures were terminated by cooling on ice and washing 3 times by centrifugation in complete-RPMI. The cell pellet was solubilized with 0.25 M NaOH and radioactivity was determined. To examine the effect of IBMX on histamine uptake by AM, cells were cultured for 1 or 15 min at 37°C with 3H-histamine (0.5 µM) either alone or together with IBMX (400 µM), and the radioactivity was measured.
cAMP Studies
AM or PBMC, 1.25 × 106, in 125 µl complete-RPMI supplemented with 10% FCS were cultured with LPS (500 ng/
ml) either alone or in the presence of histamine (105 M)
or PGE2 (100 ng/ml). Cultures were established in duplicate (for each variable) in cell nonadherent tubes (Nunc
minisorp, Roskilde, Denmark). Cultures were terminated
after 1 min (8) or 15 min (15) if cultured in the presence of
IBMX (400 µM), by the addition of 375 µl ice-cold ethanol. Samples were sonicated for 30 s, cell debris removed
by centrifugation (300 × g, 7 min, 4°C) and supernatants
dried in a rotary vacuum overnight. cAMP was measured
using an enzyme immunoassay kit (Cayman Chemical Co.,
Ann Arbor, MI) according to the manufacturer's instructions. The assay was sensitive to cAMP levels > 2 pmol/ml.
cAMP PDE Assay
AM or PBMC were resuspended (106 cells/ml) in complete-RPMI supplemented with 1% FCS. Cells were immediately frozen or cultured for 1 min or 15 min with LPS (500 ng/ml) in the presence or absence of histamine (105 M), followed by 3 washes in complete-RPMI. All samples were
frozen and thawed 3 times and sonicated for 30 s. Phosphodiesterase (PDE) activity in cell homogenates was assayed using a modification of a previously published
method (16). Briefly, homogenate (200 µl) was added to
200 µl of substrate which consisted of cAMP (106, Sigma)
and 3H-cAMP (2 × 105 cpm, Amersham) in 40 mM Tris/
HCl buffer containing 4 mM 2-mercaptoethanol and 50 mM MgCl2, and incubated for 10 min at 30°C. The reaction was stopped by boiling for 45 s, followed by cooling on ice.
The samples were incubated with 5'-nucleotidase (0.1 mg/
ml, Sigma) for 10 min at 30°C. Unreacted substrate was removed by incubation (30 min, 4°C) with 1 ml AG1 X2
resin slurry (BioRad, Richmond, CA; 1 part resin to 4 parts 100% methanol, resin prepared by twice washing
in 1 M NaOH, followed by 5 washes in water). The samples were centrifuged (300 × g, 10 min) and the radioactivity of the supernatant was measured.
Expression of Results
Cellular purity was expressed as mean ± SD for n preparations. In order to compare the levels of mediators produced by AM and monocytes, the data were normalized using measurements of monocyte/macrophage purity and expressed as ng or U/106 monocytes/macrophages. The mean value for duplicate or triplicate cultures of each test variable for individual donors was used to determine an overall mean ± SD for n donors. Because the amount of cytokine produced by different individuals varied, some results are expressed as changes relative to LPS-induced levels, normalized to 100%. Similarly, PDE activity was expressed as either pmol cAMP hydrolysed/min/106 monocytes/macrophages or as a percentage of control levels, mean ± SD.
Statistical Analysis
The effect of the reagents on the mean level or percentage change from the LPS-induced mediator levels or PDE activity from n donors was compared using a one-way analysis of variance (Fisher PLSD) or Student's t test, where appropriate (Stat View II; Abacus Concepts Inc., Berkeley, CA). Differences were considered significant if P < 0.05.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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TNF- Production by AM and Monocytes
AM and PBMC were incubated with LPS (500 ng/ml) for
22 h and secreted or cell-associated TNF- levels assayed.
The monocyte and macrophage purity for each preparation of cells was used to calculate cytokine levels as ng
(from immunoassay) or U (from bioassay) per 106 monocytes/macrophages. This was validated by demonstrating a positive linear relationship between mediator production
and monocyte content in PBMC preparations, suggesting
that monocytes/macrophages were quantitatively the major producers of TNF- (r2 = 0.89 and 0.95 for 2 donors,
P < 0.05). AM secreted significantly higher levels of TNF-
than did monocytes. However, in both cell types, significantly more TNF- was secreted than remained cell-associated (Table 1).
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Effect of Histamine on TNF- Production by
AM and Monocytes
Histamine (105 M) had no significant effect on TNF-
production by AM or monocytes cultured in the absence
of LPS (data not shown). In contrast, histamine significantly suppressed bioactive and immunoreactive TNF- in
supernatants harvested from LPS-stimulated monocytes
cultured for 22 h (Figure 1). Similarly, cell-associated TNF- was suppressed (Figure 1). In contrast, histamine
had no effect on bioactive TNF- detected in the supernatants of LPS-stimulated AM (Figure 1). Consistent with
this, histamine had no effect on secreted or cell-associated
immunoreactive TNF- production by LPS-stimulated AM
(Figure 1). This result was confirmed in dose-response studies for both LPS (5 to 500 ng/ml) and histamine (104 M to
107 M; data not shown).
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Because PBMC, but not AM, have a high percentage of
contaminating lymphocytes, the role of lymphocytes in the
response of PBMC to histamine needed investigation. Elutriated monocytes and lymphocytes were cultured together in varying proportions (total cell concentration; 5 × 105 cells/500 µl) and the effect of histamine on TNF- was
assayed. In all cell preparations (5% monocytes:95% lymphocytes to 95% monocytes:5% lymphocytes), histamine
suppressed LPS-stimulated TNF- production by approximately 80% (P < 0.05).
In contrast to its effect on monocyte TNF- production,
histamine had no significant effect on IL-1, IL-1
, and
IL-6 production by monocytes and AM (data not shown).
Effect of Histamine on TNF- Secretion by Mononuclear
Cells Isolated from Various Sources
To determine whether the lack of responsiveness of AM
to histamine was a function of their site-specific differentiation status, we examined the effect of histamine on
TNF- production (immunoassay or bioassay) by monocytes/macrophages isolated from various sites. After 22 h
of exposure, histamine (105 M) suppressed LPS-stimulated TNF- secretion by mononuclear cells isolated from
CAPD fluid and synovial fluid. Histamine (105 M) also
suppressed LPS-stimulated TNF- secretion by breast
milk mononuclear cells and in vitro monocyte-derived macrophages (M-CSF-differentiated and GM-CSF-differentiated) (Table 2).
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In these cell preparations, the major contaminating cell
was the lymphocyte. PBMC and synovial fluid-derived
mononuclear cells were adhered for 1 h and nonadherent
lymphocytes removed by washing. Removal of nonadherent cells had no effect on either the induction of TNF- by
LPS or the suppression of TNF- production by histamine as assessed by immunoassay (data not shown).
Effect of PGE2 and IL-10 on TNF- Secretion
by AM and Monocytes
Because histamine failed to suppress TNF- production
by AM, the effects of other mediators, previously shown
to regulate monocyte and macrophage TNF- production,
were examined. AM and PBMC were cultured for 22 h
with LPS (500 ng/ml) in the presence or absence of PGE2
(100 ng/ml) or IL-10 (100 U/ml). Culture supernatants were harvested and immunoreactive TNF- levels assayed. Both PGE2 (Figure 2A) and IL-10 (Figure 2B) significantly suppressed LPS-stimulated TNF- secretion by
AM and monocytes.
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3H-Histamine Uptake by AM
To determine whether AM can take up histamine, AM were incubated for 2 h at 37°C in the presence of 3H-histamine (0.5 µM) and unlabelled histamine (0 to 500 µM) and cell-associated radioactivity determined. In the presence of unlabelled histamine (500 µM) (n = 4), the uptake of 3H-histamine by AM was inhibited by 87% (Figure 3).
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Effects of Histamine and PGE2 on cAMP Levels in AM and Monocytes
Our studies showed that although histamine was taken up
by AM (Figure 3), no effect on TNF- production was observed (Figure 1). As histamine reduces LPS-stimulated
TNF- production by monocytes via increased cAMP, we
next determined whether histamine increased cAMP levels in AM. AM and PBMC were incubated for 1 min (8) with LPS (500 ng/ml) in the presence of histamine (105 M)
or PGE2 (100 ng/ml). PGE2 served as a control because it
suppressed LPS-stimulated TNF- secretion by AM (Figure 2A) by increasing cAMP (17).
Both histamine and PGE2 significantly increased cAMP levels in LPS-stimulated monocytes (Figure 4A). However, whereas PGE2 significantly increased cAMP levels in LPS-stimulated AM, histamine was without effect (Figure 4A). The basal cAMP levels in LPS-stimulated monocytes and AM were not statistically different (24 ± 5 pmol/ml, n = 3, monocytes; 23 ± 8 pmol/ml, n = 4, AM).
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In order to investigate further whether the different responses of AM and PBMC were due to the high percentage of lymphocytes in PBMC, cAMP levels were assayed in elutriated monocytes, lymphocytes, PBMC, and AM cultured under identical conditions. Both histamine and PGE2 increased levels in monocytes and PBMC, while only PGE2 increased levels in AM (data not shown). Furthermore, neither histamine nor PGE2 had a significant effect on cAMP levels of elutriated lymphocytes (data not shown).
Because PDE is involved in the breakdown of cAMP
within cells, AM and PBMC were cultured for 15 min (15)
with the nonspecific PDE inhibitor IBMX (400 µM) plus
LPS (500 ng/ml) in the presence or absence of histamine
(105 M). In the presence of IBMX, histamine increased
cAMP levels in both monocytes and AM (Figure 4B). In
addition, IBMX alone increased cAMP levels in PBMC
and AM (Figure 4B) and significantly suppressed LPS-stimulated TNF- secretion (data not shown).
Because histamine increased cAMP levels in AM only when incubated in the presence of IBMX (Figure 4B), it was necessary to determine whether IBMX influenced histamine uptake. AM were incubated for 1 or 15 min at 37°C with 3H-histamine (0.5 µM) either alone or together with IBMX (400 µM), and cell-associated radioactivity was measured. IBMX had no effect on 3H-histamine uptake by AM cultured for 1 or 15 min (Figure 5).
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PDE Activity
Because histamine increased AM intracellular cAMP levels in the presence of the PDE inhibitor IBMX (400 µM),
we hypothesized that AM had elevated PDE activity when
compared with monocytes. AM and PBMC were isolated
and lysates prepared immediately by freezing and thawing.
In addition, AM or monocytes were cultured for 1 or 15 min at 37°C with LPS (500 ng/ml) in the presence or absence of histamine (105 M). There was no significant difference in basal PDE activity between AM (n = 7) and
monocytes (n = 10; Figure 6A). In contrast, histamine significantly stimulated PDE activity in AM (n = 5; Figure
6B) but not in monocytes (n = 3; Figure 6B) after culturing for 15 min. The effect of histamine on AM PDE activity was not seen at 1 min (Figure 6C). The stimulatory effect was inhibited by the addition of the H2 receptor
antagonist cimetidine (Figure 6D).
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), or with LPS in the presence or absence of histamine (Hist) and cimetidine (Cim), and PDE activity was assayed. For triplicate cultures, data were expressed as mean ± SD. An asterisk
indicates a significant increase in PDE activity compared with control cultures (P < 0.05).
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We found that whereas histamine acts on H2 receptors
to increase cAMP levels and decrease LPS-stimulated
TNF- production in monocytes (7, 8), it had no such effect on AM (Figure 1). This difference in response did not
reflect the high percentage of lymphocytes in the PBMC
preparations because histamine suppressed TNF- production by both elutriated monocytes (95 ± 3% monocytes) and PBMC (35 ± 5% monocytes). Furthermore, elutriated monocytes and lymphocytes cultured together in
increasing proportions (5% monocytes:95% lymphocytes
to 95% monocytes:5% lymphocytes) responded similarly
to histamine with approximately 80% suppression of LPS-stimulated TNF- production (data not shown). Lymphocytes did not respond to LPS or histamine with either cAMP
accumulation or TNF- production (data not shown). Moreover, adherent cells from mononuclear cell preparations
from blood and synovial fluid responded in a manner very
similar to that of their unseparated counterparts to LPS in
terms of TNF- induction and to histamine in terms of the
percentage decrease in TNF- production (data not shown).
In contrast, monocyte and AM IL-1, IL-1, and IL-6 levels were unchanged by histamine (data not shown).
AM stimulated with LPS produced significantly higher
levels of TNF- than did monocytes (Table 1). These results are consistent with those of Becker and colleagues
(18) who observed that LPS-stimulated human AM produce 5.7 times the level of TNF- made by monocytes after a 20-h incubation period. In addition, it has been reported that AM stimulated with LPS express higher levels
of cell-associated TNF- than do blood monocytes (19).
The possibility that the AM in this study were producing
levels of TNF- too great to be regulated was unlikely because we found that histamine was ineffective in suppressing TNF- production by AM stimulated with lower LPS doses (5 ng/ml; data not shown). Moreover, in contrast to
histamine, other regulators (PGE2 and IL-10) significantly
suppressed AM TNF- production (Figure 2).
Because histamine suppressed LPS-stimulated TNF-
production by monocytes but not by AM, we hypothesized
that the lack of responsiveness of AM reflected their site-specific differentiation status (7, 20, 21). In contrast to
AM, mononuclear cells isolated from sites of acute (CAPD
fluid-derived mononuclear cells) and chronic (synovial
fluid-derived mononuclear cells) inflammation, as well as
those isolated from noninflamed tissue (breast milk-derived mononuclear cells) and in vitro monocyte-derived macrophages (M-CSF- and GM-CSF-incubated), responded like
monocytes, with histamine significantly suppressing LPS-stimulated TNF- secretion (Table 2). The monocyte/macrophage purity of these cell preparations ranged from 49 ± 6% (breast milk-derived mononuclear cells) to 68 ± 8%
(synovial fluid-derived mononuclear cells). As for PBMC, the major contaminating cell was the lymphocyte.
The inability of AM to respond to histamine in terms of
increased cAMP and decreased LPS-stimulated TNF-
does not appear to be due to a lack of expression of H2 receptors, which have been shown to be expressed by both
human (15) and guinea-pig AM (22, 23). Furthermore, in
the presence of the nonspecific PDE inhibitor IBMX, histamine increased AM cAMP levels (Figure 4B). When this
is considered with 3H-histamine experiments (Figure 3), it
strongly suggests that AM can both take up histamine and
respond to it.
Because there was no significant difference in basal cAMP levels between LPS-stimulated AM (23 ± 8 pmol/ ml, n = 4) and PBMC (24 ± 5 pmol/ml, n = 3), and because PGE2 could stimulate AM cAMP generation to an extent similar to that seen in PBMC (Figure 4A), there was no evidence of alteration in the ability of the enzyme adenylate cyclase to generate cAMP in AM (23).
In this study, we hypothesized that AM have elevated
levels of PDE activity, making them unresponsive to histamine. However, we found that basal PDE activity in AM
and monocytes did not differ (Figure 6A). At 15 min, a
time chosen to correlate with the increased cAMP levels in
AM stimulated with histamine in the presence of IBMX
(Figure 6B), histamine significantly increased PDE activity in AM but not in monocytes (Figure 6B). This response
was inhibited by cimetidine (Figure 6D), providing further
evidence for functional H2 receptors on human AM (15).
Under different culturing conditions, histamine has been
shown to increase PDE activity in monocytes: pre-incubation of monocytes with histamine (106 M) increased PDE
activity and rendered them less responsive to subsequent
histamine-stimulated cAMP production (24). Because monocytes and AM express a different profile of PDE isoenzyme activities (monocytes express predominantly PDE IV,
whereas AM express PDE I, PDE III, PDE IV, and PDE V)
(25), it is unclear which PDE isoenzyme is increased in AM
stimulated with histamine (Figure 6B). Further studies involving specific PDE inhibitors are currently underway.
In the present study, exposure to histamine for 15 min resulted in accumulation of cAMP in AM, but only in the presence of IBMX (Figure 4B). We suggest that the increased PDE activity in AM cultured for 15 min with histamine (Figure 6B) may be insufficient to account for the complete unresponsiveness of AM to histamine in the absence of IBMX. Methylxanthines, such as IBMX, are nonspecific PDE inhibitors which also block both inhibitory A1 adenosine receptors and inhibitory guanine nucleotide regulatory proteins (26). Possibly, the increased cAMP levels in AM exposed to histamine in the presence of IBMX are due to the inhibitory actions of IBMX on the inhibitory guanine nucleotide regulatory proteins associated with transmembrane signalling from the histamine receptor, leading to an increase in adenylate cyclase activity (23, 27).
We have shown that AM, isolated from either healthy
volunteers or an unaffected lobe of patients undergoing
investigative bronchoscopy, do not respond to histamine
with cAMP accumulation and suppressed TNF- production. The precise mechanism for this remains to be determined. The lung represents a large interface with the
body, and the AM plays an important role in the first line
of defense against inhaled aerosols in the lower respiratory tract. Upon stimulation in vivo with respiratory syncytial virus or P. aeruginosa, AM from mice produce increased
levels of TNF- (2), the potent pro-inflammatory cytokine important in the regulatory cascade of IL-1 and IL-6
production (28). In addition, TNF- has been implicated
in inhibiting the in vitro replication of respiratory syncytial
virus in human epithelial cells (5) and in priming AM to
inhibit the growth of Mycobacterium avium (29). Because
of the beneficial role of TNF- in host defense, a role
which might be more critical in the lungs, we suggest that it
would be counterproductive for histamine, produced by
mast cells degranulating in response to microbial stimuli,
to suppress TNF- production by AM.
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Footnotes |
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Address correspondence to: Julie Rowe, Department of Microbiology and Infectious Diseases, Flinders University of South Australia, GPO Box 2100, Adelaide, Australia, 5001. E-mail: pmnjur{at}pippin.cc.flinders.edu.au
(Received in original form July 29, 1996 and in revised form January 15, 1997).
Acknowledgments: The authors are very grateful to Dr. J. Alpers and Dr. H. Whitford (Respiratory Medicine Unit, Flinders Medical Centre, Adelaide, Australia) for supplying patient BAL fluid, and to Dr. I. Doyle (Human Physiology, School of Medicine, Flinders University of South Australia) for helpful discussions. Financial support for this study was provided by Flinders 2000 and the National Health and Medical Research Council of Australia.
Abbreviations
AM, alveolar macrophages;
BAL, bronchoalveolar lavage;
CAPD, continuous ambulatory peritoneal dialysis;
GM-CSF, granulocyte-macrophage colony stimulating factor;
H2, histamine type 2;
IBMX, 3-isobutyl-1-methylxanthine;
LPS, lipopolysaccharide;
M-CSF, macrophage-colony
stimulating factor;
PBMC, peripheral blood mononuclear cells;
PDE, phosphodiesterase;
TNF-, tumor necrosis factor alpha.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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