Neural change in Trichinella-infected mice is MHC II independent and involves M-CSF-derived macrophages

Francesca Galeazzi1,2, Paola Lovato1, Patricia A. Blennerhassett1, Eric M. Haapala1, Bruce A. Vallance1, and Stephen M. Collins1,3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Mice lacking MHC-II (class II -/-), originally obtained by targeted gene mutation (9), were purchased from Taconic (Germantown, NY). These mice were backcrossed onto the C57Bl/6 background five times; the colony has been bred continuously under specific pathogen-free (SPF) conditions in McMaster University's central animal facilities. C57Bl/6 mice, also purchased from Taconic, were used as control.

M-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 10-6 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.

Contractile activity was analyzed visually and expressed as milligrams of tension per cross-sectional area (expressed in mm2) of tissue, as described previously (22). In experiments involving EFS, the response was standardized by the contractile response obtained with 50 mM KCl and expressed as a percentage of KCl response.

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-1beta (IL-1beta ), 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

[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).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   [3H]ACh release from longitudinal muscle-myenteric plexus preparation in normal op/? and op/op mice, 6 days after Trichinella spiralis infection and in infected op/op mice treated with recombinant human macrophage colony-stimulating factor (rhM-CSF). op/? Cont, uninfected op/? mice; op/? day 6, 6 days after infection with T. spiralis; op/op Cont, uninfected op/op mice; op/op day 6, op/op mice 6 days after infection with T. spiralis; op/op day 6 + M-CSF, op/op mice treated with rhM-CSF 6 days after infection with T. spiralis. * Statistical significance vs. uninfected controls and infected op/op mice not treated with M-CSF.

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.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Presence of F4/80-positive cells in the mucosa of op/? and op/op mice under normal conditions, 6 days after T. spiralis infection, and after treatment with rhM-CSF. Values are means ± SE given in number of positive cells/villus-crypt units (vcu). * Statistical significance vs. uninfected controls and infected op/op mice not treated with M-CSF.



View larger version (110K):
[in this window]
[in a new window]
 
Fig. 3.   Macrophage infiltration in the jejunum 6 days after T. spiralis infection. Immunohistochemistry for F4/80. A: cryopreserved cross section of jejunum from infected op/? mouse. Note the presence of a large number of F4/80-positive cells in the mucosa (arrow) and in the muscularis externa. B: cryopreserved cross section of jejunum from infected M-CSF-deficient op/op mouse. Note that only a few positive cells are evident in the mucosa and none in the muscle layers. C: cryopreserved cross section of jejunum from infected M-CSF-deficient op/op mouse treated with rhM-CSF. Note the presence of numerous positive cells in mucosa, serosa, and muscle layers. A-C: magnification, ×200.

IL-1beta and IL-4 mRNA Expression

We next examined the presence of macrophage- and T cell-derived cytokines in the muscularis externa using semiquantitative PCR. The transcripts for both IL-1beta and IL-4 were consistently detected in all samples. Quantitative IL-1beta expression by densitometric analysis is presented in Fig. 4. IL-1beta was constitutively expressed in both op/? and op/op mice. Compared with controls, mRNA levels were significantly increased during the early phase (24-48 h) of the infection in op/? mice and were restored to basal levels at day 6 postinfection (controls, 0.3 ± 0.1; day 1 postinfection, 0.7 ± 0.04; day 6 postinfection, 0.27 ± 0.08). In contrast, in M-CSF-deficient mice no changes in IL-1beta mRNA levels were detected at any time point after the infection (Fig. 4), suggesting that M-CSF-dependent macrophages are involved in IL-1beta mRNA expression in the muscularis externa during nematode infection. In contrast, infection with T. spiralis led to increased mRNA expression of the lymphokine IL-4 both in op/? and op/op mice, which reached a peak at day 6 after infection. Because suppression of cholinergic nerve function was not evident in op/op mice, the increased expression of IL-4 in these mice provides further support for a T cell-independent mechanism.


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 4.   mRNA expression for interleukin-1beta (IL-1beta ) in the muscularis externa in op/? and op/op mice. A: ethidium bromide-stained agarose gel indicating the level of IL-1beta mRNA in muscularis externa in op/? and op/op mice after T. spiralis infection. Lanes 1 and 4, uninfected mice; lanes 2 and 5, day 1 postinfection with T. spiralis; lanes 3 and 6, day 6 postinfection. Arrows indicate the position of IL-1beta and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR products after 33 and 25 amplification cycles, respectively. B: semiquantitative analysis of IL-1beta mRNA expression expressed as densitometric units. Open bars, uninfected controls; hatched bars, 1 day after T. spiralis infection; solid bars: 6 days after T. spiralis infection. *Statistical significance vs. uninfected op/? and op/op at all time points.

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 10-6 M carbachol and 827.5 ± 117.1 mg/mm2 after stimulation with 50 mM KCl. A significant increase in the tension generated by longitudinal muscle with both the stimuli was seen 6 days after infection with T. spiralis. After stimulation with carbachol, muscle strips from infected animals generated a tension of 1,669.5 ± 331.1 mg/mm2 (P < 0.05 compared with uninfected op/? mice), and a similar pattern was observed after KCl stimulation, as the tension generated was 2,234.2 ± 326.6 mg/mm2 (P < 0.05 compared with uninfected op/?).

A 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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta and tumor necrosis factor-alpha (TNF-alpha ) 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-1beta 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-1beta 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-1beta produced by M-CSF-dependent macrophages mediates the suppression of cholinergic nerve function in this model.

In the rat, the expression of IL-1beta precedes the changes in cholinergic nerves, peaking within 24 h postinfection (13). This delay suggests that IL-1beta 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-1beta and TNF-alpha 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-1beta , 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-alpha , 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.


    ACKNOWLEDGEMENTS

This study was funded by The Canadian Institutes for Health Research.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aloe, L, Fiore M, Probert L, Turrini P, and Tirassa P. Overexpression of tumor necrosis factor alpha in the brain of transgenic mice differentially alters nerve growth factor levels and choline acetyltransferase activity. Cytokine 11: 45-54, 1999[ISI][Medline].

2.   Barbara, G, Vallance BA, and Collins SM. Persistent intestinal neuromuscular dysfunction after acute nematode infection in mice. Gastroenterology 113: 1224-1232, 1997[ISI][Medline].

3.   Campbell, IL. Structural and functional impact of the transgenic expression of cytokines in the CNS. Ann NY Acad Sci 840: 83-96, 1998[Abstract/Free Full Text].

4.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

5.   Collins, SM. The immunomodulation of enteric neuromuscular function; implications for motility and inflammatory disorders. Gastroenterology 111: 1683-1689, 1996[ISI][Medline].

6.   Collins, SM, Blennerhassett PA, Blennerhassett MG, and Vermillion DL. Impaired acetylcholine release from the myenteric plexus of Trichinella-infected rats. Am J Physiol Gastrointest Liver Physiol 257: G898-G903, 1989[Abstract/Free Full Text].

7.   Galeazzi, F, Haapala EM, van Rooijen N, Vallance BA, and Collins SM. Inflammation-induced impairment of enteric nerve function in nematode-infected mice is macrophage dependent. Am J Physiol Gastrointest Liver Physiol 278: G259-G265, 2000[Abstract/Free Full Text].

8.   Galeazzi, F, Vallance BA, and Collins SM. Macrophages infiltrate intestinal muscle layers and myenteric plexus during T. spiralis infection in mice (Abstract). Gastroenterology 114: A981, 1998.

9.   Grusby, MJ, Johnson RS, Papaioannou VE, and Glimcher LH. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice. Science 253: 1417-1420, 1991[ISI][Medline].

10.   Haapala, EM, Blennerhassett PA, Vallance BA, and Collins SM. Inflammation-induced impairment of cholinergic nerve function is CD4 independent in nematode-infected mice (Abstract). Gastroenterology 14: A1148, 1998.

11.   Hurst, S, and Collins SM. Mechanism underlying tumor necrosis factor-alpha suppression of norepinephrine release from rat myenteric plexus. Am J Physiol Gastrointest Liver Physiol 266: G1123-G1129, 1994[Abstract/Free Full Text].

12.   Jacobson, K, McHugh K, and Collins SM. Experimental colitis alters myenteric nerve function at inflamed and noninflamed sites in the rat. Gastroenterology 109: 718-722, 1995[ISI][Medline].

13.   Khan, I, and Collins SM. Expression of cytokines in the longitudinal muscle-myenteric plexus of the inflamed intestine of rat. Gastroenterology 107: 691-700, 1994[ISI][Medline].

14.   Khan, WI, Vallance BA, Blennerhassett PA, Deng Y, Verdu EF, Matthaei KI, and Collins SM. Critical role for signal transducer and activator of transcription factor 6 in mediating intestinal muscle hypercontractility and worm expulsion in Trichinella spiralis-infected mice. Infect Immun 69: 838-844, 2001[Abstract/Free Full Text].

15.   Main, C, Blennerhassett PA, and Collins SM. Human recombinant interleukin 1beta suppresses acetylcholine release from rat myenteric plexus. Gastroenterology 104: 1648-1654, 1993[ISI][Medline].

16.   Marks, SC, and Lane PW. Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse. J Hered 67: 11-18, 1976[ISI][Medline].

17.   Palucka, KA, Taquet N, Sanchez-Chapuis F, and Gluckman JC. Dendritic cells as the terminal stage of monocyte differentiation. J Immunol 160: 4587-4595, 1998[Abstract/Free Full Text].

18.   Ruhl, A, Berezin I, and Collins SM. Involvement of eicosanoids and macrophage-like cells in cytokine-mediated changes in rat myenteric nerves. Gastroenterology 109: 1852-1862, 1995[ISI][Medline].

19.   Ruhl, A, Hurst S, and Collins SM. Synergism between interleukins 1beta and 6 on noradrenergic nerves in rat myenteric plexus. Gastroenterology 107: 993-1001, 1994[ISI][Medline].

20.   Stanley, ER, Berg KL, Einstein DB, Lee PS, and Yeung YG. The biology and action of colony-stimulating factor-1. Stem Cells (Dayt) 12 Suppl 11: 15-24, 1994[ISI][Medline].

21.   Swain, MG, Agro A, Blennerhassett PA, Stanisz A, and Collins SM. Increased levels of substance P in the myenteric plexus of Trichinella-infected rats. Gastroenterology 102: 1913-1919, 1992[ISI][Medline].

22.   Vallance, BA, Blennerhassett PA, and Collins SM. Increased intestinal muscle contractility and worm expulsion in nematode-infected mice. Am J Physiol Gastrointest Liver Physiol 272: G321-G327, 1997[Abstract/Free Full Text].

23.   Vallance, BA, Croitoru K, and Collins SM. T lymphocyte-dependent and -independent intestinal smooth muscle dysfunction in the T. spiralis-infected mouse. Am J Physiol Gastrointest Liver Physiol 275: G1157-G1165, 1998[Abstract/Free Full Text].

24.   Vallance, BA, Galeazzi F, Collins SM, and Snider DP. CD4 T cells and major histocompatibility complex class II expression influence worm expulsion and increased intestinal muscle contraction during Trichinella spiralis infection. Infect Immun 67: 6090-6097, 1999[Abstract/Free Full Text].

25.   Wiktor-Jedrejczak, W, Ansari AA, Szperl M, and Urbanoska E. Distinct in vivo functions of two macrophage subpopulations as evidenced by studies using macrophage-deficient op/op mouse. Eur J Immunol 22: 1951-1954, 1992[ISI][Medline].

26.   Witmer-Pack, MD, Hughes DA, Schuler G, Lawson L, McWilliam A, Inaba K, Steinman RM, and Gordon S. Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse. J Cell Sci 104: 1021-1029, 1993[Abstract/Free Full Text].

27.   Yoshida, H, Hayashi SI, Kunisada T, Ogawa M, Nishikawa S, Okamura H, Sudo T, Shultz LD, and Nishikawa S. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345: 442-443, 1990[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 281(1):G151-G158
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society