1 Intestinal Diseases Research Program and 3 Gastrointestinal Division and Department of Medicine, Health Sciences Center, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and 2 Department of Surgical and Gastroenterological Science, Section of Gastroenterology, University of Padua, 35131 Padua, Italy
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ABSTRACT |
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Intestinal
inflammation due to nematode infection impairs enteric cholinergic
nerve function and induces hypercontractility of intestinal muscle.
Macrophages have been implicated in the neural changes, but the
subpopulation and mechanism involved are unknown. We examined whether
macrophages alter nerves by virtue of their ability to activate
lymphocytes via major histocompatibility complex (MHC) II-restricted
antigen presentation. We also attempted to evaluate the role of
macrophage subsets using op/op mice deficient in macrophage
colony-stimulating factor (M-CSF). ACh release from the myenteric
plexus was measured in MHC II- and M-CSF-deficient (op/op)
mice infected with Trichinella spiralis. F4/80-positive macrophages and interleukin-1 were constitutively present in op/op and op/? mice but increased only in
op/? mice postinfection. After infection, a marked
suppression of ACh release occurred only in infected MHC II-deficient
and op/? mice. Muscle hypercontractility remained evident in
infected op/? mice. Treatment with M-CSF restored macrophage
number, and this was accompanied by suppression of cholinergic nerve
function during infection. Thus M-CSF plays a critical role in this
model by recruiting a subset of macrophages that selectively suppresses
enteric neural function.
macrophage colony-stimulating factor; op/op mice; nematode; enteric nerves; inflammatory bowel disease
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INTRODUCTION |
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THE INTERACTION BETWEEN IMMUNE cells and nerves during inflammatory processes is known to cause functional and structural damage of neural tissue. This is evident in animal models of intestinal inflammation, in which functional impairment of the enteric nerves, characterized by a deficit in the release of neurotransmitters (5, 6) and changes in neurotransmitter content (21), has been described. These alterations may account for the motility disorders observed during intestinal inflammation as well as for the persistence of altered motility after acute gastrointestinal infections.
In a model of intestinal inflammation induced by nematode infection in rodents, there is suppressed enteric cholinergic nerve function together with hypercontractility of longitudinal muscle (6, 22). In this model, muscle hypercontractility is T cell dependent (23). We (8) have shown that macrophages infiltrate the myenteric plexus and that depletion of macrophages abrogates the suppression of ACh release seen in this model (7). However, we do not know whether the role of macrophages involves T cells via antigen presentation or whether it represents a T cell-independent action. Furthermore, the macrophage subset involved has not yet been identified.
Macrophage colony-stimulating factor (M-CSF, CSF-1) is a critical cytokine in the development, differentiation, and recruitment of macrophages (20). Specifically, the M-CSF-dependent macrophage subset is involved in the inflammatory response and production of proinflammatory cytokines (25), whereas a different population of macrophages, likely granulocyte M-CSF (GM-CSF) dependent, is involved in the antigen presentation and immunoregulatory functions (17).
In this study, we examined the roles of major histocompatibility complex (MHC) II-restricted antigen presentation and M-CSF-dependent macrophages in the development of functional changes in cholinergic enteric nerves during intestinal inflammation. We conducted our investigation using nematode infection in mice lacking MHC class II expression (9) and mice (op/op) lacking M-CSF-dependent macrophages because of a spontaneous mutation in the M-CSF-encoding gene (27).
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MATERIALS AND METHODS |
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Animals
Mice lacking MHC-II (class IIM-CSF-deficient mice (op/op) as well as heterozygote op/+ breeding pairs were purchased from Jackson (Bar Harbor, ME) and kept under SPF conditions. A colony was obtained by breeding op/+ mice. Nonmutant mice, which are op/+ or +/+, are indistinguishable from each other (therefore named op/?) and do not exhibit either osteopetrosis or macrophage deficiency (16). Mutant op/op littermates were identified by a lack of incisors at day 10 after birth and, after weaning, were fed powdered Purina chow. Op/? mice received conventional Purina chow. The experiments were approved by the McMaster Animal Care Committee and the Canadian Council on the use of Laboratory Animals.
Study Design
Mice underwent infection with 375 Trichinella spiralis larvae by gavage. In MHC II-deficient and C57Bl/6 mice, [3H]ACh release from jejunal longitudinal muscle-myenteric plexus (LMMP) preparations was evaluated at days 0, 2, and 6 postinfection.In op/op as well as op/? mice, [3H]ACh release from LMMP preparations and jejunal longitudinal muscle contractility were evaluated at day 6 postinfection. Tissues from jejunum were taken to evaluate the presence of macrophages by immunohistochemistry.
Immunohistochemistry for Macrophages
Immunohistochemistry for macrophages was performed on cryopreserved sections of mouse jejunum using a monoclonal antibody recognizing F4/80 antigen, a glycoprotein expressed by mature murine macrophages. Uninfected mice were used as controls.Tissues were washed in cold PBS, embedded in OCT compound, frozen using
isopentane and liquid nitrogen, and stored at 70°C. Cross sections
4 µm thick were cut and fixed in ice-cold acetone. A rat-anti-mouse
monoclonal antibody against F4/80 antigen was used at 1:50 dilution
(clone Cl:A3-1, Serotec, Oxford, UK). Biotinylated polyclonal goat
anti-rat antibody (Cedarlane Laboratories, Hornby, ON, Canada) was used
in conjunction with Cl:A3-1 at 1:200 dilution. All antibodies were
diluted in 1% BSA solution in Tris-buffered saline.
Immunohistochemistry was performed by using the
streptavidin-biotin-peroxidase technique, as previously described
(7).
[3H]ACh Release
[3H]ACh release was measured on LMMP preparations obtained from mouse jejunum as previously described (7). Segments 2 cm long were tied at each end in the longitudinal axis and incubated in Krebs buffer containing 0.5 mM [3H]choline (sp act 81 Ci/mmol; Dupont) for 45 min. Tissues were then suspended in a water-jacketed bathing chamber maintained at 37°C inside an inner chamber lined with electrode rings and superfused with Krebs buffer containing 10 mM hemicholinium-3 (Sigma, St. Louis, MO) for 80 min. Superfusate was collected every 2 min using a fraction collector (Ultrorac 7000). Tissues were allowed to equilibrate for 40 min and then stimulated with 50 mM KCl Krebs solution. At the end of the experiments, LMMP preparations were weighed and solubilized with 1 ml of NCS tissue solubilizer (Amersham, Oakville, ON, Canada). Aqueous counting scintillant (4 ml; Amersham) was then added to each sample, and tritium content was analyzed with a Beckman liquid scintillation counter (model LS 5801). Tritium release was calculated as previously described (7). A baseline of spontaneous outflow of tritium was obtained by fitting a linear regression line of log dpm of the samples before the stimulation and the final five samples of the experiment. The evoked release of tritium was calculated from the difference between the total tritium released during stimulation and the baseline value. This was determined as a percentage of the total tritium taken up by the tissue and expressed as the "fractional release" (7).Longitudinal Muscle Contractility
Longitudinal muscle contractility was measured on strips from the jejunum. Strips were isolated and mounted into a muscle bath as previously described (22) and attached to force transducers (FT03C, Grass, Quincy, MA). Responses were recorded on a Grass 7E polygraph. After 30 min of equilibration in oxygenated Krebs buffer, experiments were performed to evaluate the contraction after electrical field stimulation (EFS) and pharmacological stimulation.EFS. EFS was induced by using a Grass S88 stimulator with two platinum ring electrodes placed 1 cm apart around each end of the tissue. Experiments were performed by using the parameters previously assessed (2): voltage of 60 V, pulse width of 0.5 ms, stimulus duration of 20 s, and frequency of 10 Hz.
Carbachol and KCl-induced contraction.
Gut segments were exposed to 106 M carbachol and 50 mM
KCl by adding microliter aliquots to the 20-ml baths. After each
stimulation, tissues were rinsed twice and allowed to recover for 15 min before the next stimulation.
Semiquantitative Analysis of Cytokine mRNA Expression
RNA extraction and cDNA preparation.
From full-thickness jejunal specimens, the mucosa was scraped off and
the remaining muscularis externa was then placed in guanidine
thiocyanate buffer and homogenized. Total RNA was isolated from tissue
specimens by acid guanidium thiocyanate-phenol-chloroform extraction as
described previously (4). RNA samples were stored at
70°C in 1% diethylpyrocarbonate-treated water, and quantity and
quality were determined by the ratio of absorbency at 260 nm to that at
280 nm. cDNA was synthesized from 2.5 mg of total RNA, using 400 U of
Moloney murine leukemia virus-derived RT (GIBCO-BRL, Gaithersburg, MD)
and 0.5 mg of oligo(dT)12-18 in a final reaction
volume of 40 µl in the presence of 40 U of RNase inhibitor (RNase
OUTTM; GIBCO BRL) and 10 mM dNTP (GIBCO BRL). The reaction was
incubated at 37°C for 60 min followed by heating for 7 min at 95°C
to inactivate the enzyme. The samples were stored at
20°C until use.
PCR and semiquantification of mRNA levels.
PCR reactions were performed in a total volume of 50 µl in presence
of 1 U of Taq DNA polymerase (GIBCO BRL), 10 mM dNTP (GIBCO BRL) and 15 pg of 5' and 3' primers. Amplification was performed by
25-40 cycles consisting of denaturation at 94°C for 45 s,
primer annealing at 56-57°C for 45 s, and primer extension
at 72°C for 60 s, using a Perkin Elmer thermal cycler 480 (Branchburg, NJ). The following primers specific for housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cytokines were
used: GAPDH, upstream 5'-CCATGGAGAAGGCTGGGG-3', downstream
5'-CAAAGTTGTCATGGATGACC-3'; interleukin-1 (IL-1
),
upstream 5'-GACAGTGATGAGAATGACCTG-3', downstream
5'-CCTGTAGTGCAGCTGTCTAAT-3'; and IL-4, upstream 5'-GAATGT ACCAGGAGCCATATC-3', downstream 5'-CTCAGTACTACGAGTAATCCA-3'. To exclude
the amplification of genomic DNA contaminating the samples, experiments
were also performed using RNA as substrate for PCR. After amplification
15 µl of PCR products were separated electrophoretically in 2%
agarose gel, visualized by ethidium bromide staining, and photographed
using Polaroid land film type 55 (Kodak, Rochester, NY). The negatives
were used for densitometric quantitation of band intensity using Kodak
Digital Science 1D 2.0 image analysis software. The results were
normalized to the housekeeping gene and expressed as the ratio of
cytokines to GAPDH expression.
Human Recombinant M-CSF Reconstitution
In parallel experiments, M-CSF-deficient op/op mice were reconstituted with recombinant human M-CSF (rhM-CSF; kindly donated by Chiron, Emeryville, CA). Mice received 50 µg of rhM-CSF intraperitoneally twice per day, starting 2 days before infection and daily until day 5 postinfection. At day 6 postinfection, mice were euthanized and ACh release and presence of macrophages by immunohistochemistry were evaluated.Data Analysis
Immunohistochemistry was performed on tissues from four animals per group; a blinded observer examined four sections for each animal. Mucosal macrophages are expressed as the number of positive cells per villus-crypt unit (vcu), whereas F4/80-positive cells in the neuromuscular layers are expressed as the number of positive cells per section. Longitudinal muscle contractility and [3H]ACh release studies involved six mice per group, and experiments with rhM-CSF involved four mice per group. RT-PCR was performed on tissues from four animals per group. Three independent PCR were performed for each sample, and data are expressed as densitometry units.Values are expressed as means ± SE. Statistical analysis was performed by using Kruskal-Wallis, ANOVA, and Bonferroni tests. Statistical significance was inferred for P < 0.05.
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RESULTS |
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[3H]ACh Release from LMMP
We first evaluated the putative involvement of macrophages by virtue of their ability to activate T cells via MHC II-restricted antigen presentation. We studied ACh release in C57Bl/6 and MHC II-deficient mice on a C57Bl/6 background.In uninfected C57Bl/6 mice KCl-stimulated fractional release of ACh from LMMP was 1.8 ± 0.05%. A transient 32% increase was observed by day 2 postinfection (2.64 ± 0.28%), whereas at day 6 the fractional release of ACh was significantly suppressed (0.91 ± 0.13%; P < 0.05). After infection with T. spiralis, MHC II-deficient mice showed suppression in ACh release similar to that observed in C57Bl/6, as the fractional release of ACh dropped from 1.7 ± 0.3% in uninfected animals to 0.51 ± 0.05% (P < 0.05). These results thus indicate that the development of altered cholinergic nerve function does not require MHC II antigen presentation and effectively exclude a contribution of antigen-presenting macrophages in the development of nerve dysfunction. Therefore, we next evaluated the role of M-CSF-dependent macrophages using op/op mice.
Under normal conditions, the KCl-evoked fractional release of ACh from
LMMP was similar in op/? and M-CSF-deficient mice, being
respectively 3.6 ± 0.4% and 3.4 ± 0.6%. Infection with
T. spiralis was associated with a 72% suppression in the
fractional release of ACh from LMMP in op/? mice. As shown
in Fig. 1, infected op/? mice
showed a significant decrease in the fractional release of ACh compared
with uninfected controls (0.8 ± 0.3% and 3.6 ± 0.4%,
respectively; P < 0.05). In contrast, in
M-CSF-deficient mice, ACh release was far less affected by the
infection, as in op/op-infected mice ACh release was similar
to that seen in uninfected controls (2.3 + 0.6% and 3.4 + 0.5%, respectively; not significant). This showed that in the absence
of M-CSF-dependent macrophages cholinergic enteric nerve function is
not impaired during nematode infection (Fig. 1).
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Presence of Macrophages in Jejunum During T. spiralis Infection in M-CSF-Deficient Mice
We next evaluated the presence of macrophages in M-CSF-deficient mice by immunohistochemistry. In op/? uninfected animals, F4/80-positive cells were present in the mucosa (6.5 ± 0.8 cells/vcu) but absent in the muscle layers. A similar distribution was found in uninfected M-CSF-deficient mice in which a similar number of positive cells (7 ± 1.8 cells/vcu) were observed in the mucosa (Fig. 2). At day 6 postinfection with T. spiralis, an increased number of F4/80-positive cells was found in the mucosa (14.9 ± 1.5 cells/vcu) (Figs. 2 and 3A), and numerous positive cells were evident in the serosa and the muscle layers, particularly in the area of the myenteric plexus. In contrast, in M-CSF-deficient mice, infection with T. spiralis was not accompanied by an increase of F4/80-positive cells in the mucosa, because the number of macrophages was similar to that observed in uninfected op/op mice (6.4 ± 0.4 cells/vcu) (Figs. 2 and 3B). Furthermore, no F4/80-positive cells were found in the serosa, muscle layers, or in the area of the myenteric plexus in infected op/op mice.
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IL-1 and IL-4 mRNA Expression
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Longitudinal Muscle Contractility in M-CSF-Deficient Mice
In uninfected op/? mice, the tension developed by longitudinal muscle strips was 655 ± 60.6 mg/mm2 after 10A similar hypercontractile response was observed in M-CSF-deficient (op/op) mice postinfection. In uninfected op/op mice, tension developed by longitudinal muscle strips after stimulation with carbachol and KCl was 765 ± 54.0 and 934.8 ± 78.1 mg/mm2, respectively. At day 6 postinfection, longitudinal muscle from op/op mice developed significant greater tension after either carbachol (1,883.8 ± 578.9 mg/mm2; P < 0.05 compared with uninfected op/op mice) or KCl (2,353.7 ± 398.2 mg/mm2; P < 0.05 compared with uninfected op/op mice), indicating that M-CSF-dependent macrophages are not necessary for the development of jejunal longitudinal muscle hypercontractility during infection with T. spiralis.
To evaluate the impact of impaired ACh release on muscle contraction, we examined jejunal longitudinal muscle contraction after EFS at parameters that stimulate cholinergic nerves (2). In op/? mice, T. spiralis infection resulted in a reduction of the EFS-evoked contraction; tension generated after EFS, expressed as a percentage of the KCl response, dropped from 70.8 ± 6.2% to 41.3 ± 7.2% at day 6 postinfection (P < 0.05), reflecting a substantial functional impairment of cholinergic nerves. In contrast, there was no difference in the magnitude of the EFS-mediated contractile response between infected and control op/op mice (44.2 ± 4.4% and 52.4 ± 5.6% of KCl evoked contraction, respectively, P > 0.05).
rhM-CSF Reconstitution
To verify the specific role of M-CSF-dependent macrophages, we reconstituted op/op mice with rhM-CSF in parallel experiments. Treatment with rhM-CSF restored the macrophage population in the inflamed jejunum in op/op mice as the number of F4/80-positive cells rose from 6.4 ± 0.4 cells/vcu in infected mice to 12.6 ± 2.7 cells/vcu in infected mice treated with M-CSF, a value similar to that seen in op/? infected mice (14.9 ± 1.5 cells/vcu) (Figs. 2 and 3C). Reconstitution with M-CSF also restored the macrophage infiltrate in the muscularis externa in infected op/op mice, as the number of positive cells per section rose from 0.15 ± 0.09 to 11.6 ± 3.1 in op/op mice reconstituted with M-CSF, reaching values comparable to those seen in op/? infected mice (18.7 ± 8.5).As described above, infection with T. spiralis led to a 72% suppression of the fractional release of ACh from LMMP in op/? mice, whereas in op/op mice the suppression was only 32%. However, after reconstitution of op/op mice with rhM-CSF op/op the suppression of the fractional release of ACh was 68%, similar to that observed in op/? mice (Fig. 1).
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DISCUSSION |
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This study builds on our previous work (7, 18) identifying the role of macrophages in the suppression of cholinergic nerve function in the model of nematode-induced intestinal inflammation. In this study, we show that macrophage involvement is not via MHC II-restricted antigen presentation but involves M-CSF-dependent macrophages.
Enteric neuromuscular changes have been described in a variety of inflammatory models. Particular attention has been focused on models of nematode infection, because much is known about the underlying immunologic response. It has been clearly shown (22) in T. spiralis-infected mice that changes in muscle function are T cell dependent. In addition, we (7) have previously demonstrated that the impairment of enteric nerve function is abrogated by macrophage depletion. However, it remains unclear whether macrophages contribute to nerve alterations via MHC class II-restricted antigen presentation or production of proinflammatory mediators. Furthermore, the subclass of macrophage involved has not been identified.
MHC II-expressing macrophages have been described in the muscularis externa in T. spiralis-infected mice, suggesting a potential role of these cells in the development of enteric nerve changes in this model (24). In the present study, we evaluated enteric cholinergic nerve function in MHC II-deficient mice and found, during nematode-induced intestinal inflammation, suppression in ACh release similar to that observed in MHC II-expressing mice. These findings indicate that MHC II-restricted antigen presentation is not required for the development of altered nerve function in this model. We thus investigated the role of a different subpopulation of macrophages, using op/op mice that carry a spontaneous inactivating mutation in the M-CSF-encoding gene (27). M-CSF, produced predominantly by endothelial cells, fibroblasts, and stromal cells of the bone marrow, regulates the survival, proliferation, and differentiation of the mononuclear-phagocyte lineage (20). Other target cells for this growth factor include osteoclasts and decidual and trophoblastic cells (20). The characteristic phenotype of M-CSF-deficient mice includes osteopetrosis and poor fertility. However, their best-described feature is the absence of peripheral monocytes and a substantial reduction of macrophages in several tissues (16), making op/op mice a unique tool for studying the role of macrophages in vivo. Nevertheless, to date few studies have utilized op/op mice to examine the role of macrophages during intestinal inflammation and none has addressed the impact of this cell type on changes in intestinal physiology in inflammatory conditions. Different functional subpopulations of macrophages have been identified in vivo using op/op mice. In particular, M-CSF-deficient mice exhibit normal phagocytic function in vivo, whereas the production of proinflammatory cytokines is almost abolished (25). This suggests that the M-CSF-dependent subpopulation of macrophages is principally involved in cytokine secretion, In contrast, M-CSF-independent macrophages, which probably require GM-CSF as growth factor, appear to be responsible for immunoregulatory functions, including phagocytosis and antigen presentation (16, 25).
We have shown by immunohistochemistry that under normal conditions, the number of lamina propria macrophages was similar in mutants and op/? mice, whereas in op/op mice macrophages were absent from the muscularis externa and serosa. These data are consistent with previous findings (26), showing that in op/op mice the surface marker of mature macrophage F4/80 is normally expressed in the intestinal lamina propria but is absent from the peritoneal cavity.
During T. spiralis infection of op/? mice, we observed a >200% increase in the number of lamina propria macrophages, and macrophages were also evident in the muscle layers and myenteric plexus. In contrast, in infected op/op mice, the number of lamina propria macrophages was similar to that found in uninfected mice and no macrophages were evident in the muscle and myenteric plexus. These results indicate that M-CSF is necessary for the development and recruitment of monocytes macrophages during intestinal inflammation.
In op/? mice, infection was accompanied by a 72% suppression in the fractional release of ACh from LMMP, and this is in keeping with previous data in both rats and mice (6, 7). In contrast, in the absence of M-CSF-dependent macrophages, T. spiralis infection did not cause a significant suppression in neurotransmitter release, because op/op mice showed no significant change in ACh release postinfection, identifying a critical role for macrophages in inducing functional neural damage. This is consistent with our (7) previous findings showing normal ACh release in nematode-infected mice after liposome-mediated macrophage depletion. However, we now have identified the specific subpopulation of macrophages involved in neural impairment. Indeed, we have provided evidence of a specific role of M-CSF-dependent macrophages by showing that treatment of op/op mice with M-CSF restored the macrophage infiltrate in the jejunum after infection and this was accompanied by a suppression of ACh release similar to that seen in M-CSF-expressing mice. Thus our findings indicate that M-CSF plays a crucial role in recruiting macrophages into the intestine during inflammation, and the M-CSF-dependent macrophage is the cell type responsible for the enteric cholinergic nerve changes in this model.
Previous studies (11, 14, 15, 18, 19) have identified
proinflammatory cytokines as putative mediators of altered enteric
neural function. In noninflamed tissue, incubation with IL-1
and tumor necrosis factor-
(TNF-
) suppressed neurotransmitter release similar to during T. spiralis infection (11,
15, 19). In addition, expression of these cytokines has been
shown (13) in the muscularis externa and myenteric plexus
during T. spiralis infection. A role for IL-1
in
mediating the observed changes in cholinergic nerve function is further
supported by our demonstration that the increased expression of this
cytokine was markedly attenuated in infected op/op mice.
Although there was some expression of IL-1
in noninfected
op/op mice, presumably from other cell sources, the lack of
any increase in this cytokine, coupled with the absence of
F4/80-positive macrophages, suggest that IL-1
produced by M-CSF-dependent macrophages mediates the suppression of cholinergic nerve function in this model.
In the rat, the expression of IL-1 precedes the changes in
cholinergic nerves, peaking within 24 h postinfection
(13). This delay suggests that IL-1
is the initiating
factor in a cascade of events that culminates in functional neural
changes, and other substances produced during inflammation may be the
final mediators. It has already been demonstrated (18)
that the ability of IL-1
and TNF-
to suppress neurotransmitter
release from myenteric plexus requires protein synthesis and the
production of prostaglandins. Therefore, taken in conjunction with this
previous work (13, 18), the results of the present study
support the hypothesis that IL-1
, expressed locally during the early
stages of the infection by infiltrating M-CSF-dependent macrophages,
initiates a cascade of events involving prostaglandins and possibly
other mediators, leading to cholinergic nerve dysfunction.
Cholinergic nerves are important modulators of enteric smooth muscle contraction. As ACh release is not totally suppressed during infection, we determined whether the degree of suppression observed in the model was sufficient to influence neurally mediated muscle contraction. To address this, we examined the tissue exposed to EFS at parameters known to activate cholinergic nerves (2). As expected, the atropine-sensitive EFS-stimulated contraction was decreased during the infection in op/? mice, reflecting the impaired release of ACh from enteric nerves. In contrast, tissues from infected op/op mice showed no impairment in the EFS-stimulated contractile response. These findings indicate that the magnitude of M-CSF macrophage-mediated cholinergic dysfunction is sufficient to alter the electrical stimulation of muscle.
Our results suggest that M-CSF-dependent macrophages influence the cholinergic neural regulation of muscle contraction also under normal conditions. In uninfected op/op mice, there was a 38% reduction in EFS-evoked muscle contraction compared with the response in noninfected wild-type mice. This implies a constitutive role for M-CSF-derived macrophages in the cholinergic regulation of muscle contraction. However, the contractility of muscle observed after direct stimulation by carbachol was similar in uninfected op/op and op/? mice, indicating that the changes in smooth muscle cell function are not subject to regulation by M-CSF-dependent macrophages.
The deficiency in M-CSF-dependent macrophages did not prevent the development of smooth muscle hypercontractility during T. spiralis infection. This is in keeping with our (24) previous observation that muscle changes in this model are T cell dependent, as they are abrogated in infected athymic mice as well as in MHC-II-deficient mice. We also observed that op/op mice are capable of mounting an immune response leading to increased expression of IL-4, a cytokine that plays a key role in the T helper 2 (Th2) cell dominant immune response to this infection and is a putative mediator of the muscle changes in this model. In a recent study, we have shown that in mice deficient in signal transduction and activator of transcription factor 6 (Stat6), which is critical for the actions of the Th2 cytokines IL-4 and IL-13, the hypercontractility associated with T. spiralis infection was markedly attenuated (14). These findings are consistent with previous studies (26) in op/op mice demonstrating appropriate expression of MHC II and a normal presence of dendritic cells, which are GM-CSF rather than M-CSF dependent (17). The preservation of muscle hypercontractility in the absence of suppressed ACh release in infected op/op mice indicates that the changes in muscle responsiveness are not secondary to a denervation phenomenon resulting from the reduction on cholinergic nerve function.
In conclusion, we have provided direct evidence that M-CSF-dependent
macrophages mediate the alterations in enteric cholinergic nerve
function during intestinal inflammation. A similar functional impairment in enteric nerves has also been observed during
hapten-induced experimental colitis in rats (12),
indicating a common response of the enteric nervous system to
inflammatory stimuli in different regions of the intestine. Functional
alterations have also been shown in the brain of transgenic mice
overexpressing TNF-, as they present with a loss of choline
acetyltransferase immunoreactivity (1, 3). Thus
proinflammatory cytokines appear to share the ability to alter nerve
structure and function, and this is demonstrable outside and within the
central nervous systems. Therefore, the identification of the
M-CSF-dependent macrophage as the cell responsible for functional
neural changes provides an important step in the understanding of the
pathophysiological mechanism of inflammation-induced changes in
peripheral and central cholinergic nerves and has broad implications
for the development of new therapeutic strategies aimed at preserving
neural function during inflammation.
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ACKNOWLEDGEMENTS |
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This study was funded by The Canadian Institutes for Health Research.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Stephen M. Collins, Rm. 4W8, McMaster Univ. Medical Center, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: scollins{at}mcmaster.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 November 2000; accepted in final form 9 February 2001.
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