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The Journal of Immunology, 2003, 170: 2316-2322.
Copyright © 2003 by The American Association of Immunologists

Induction of Low Dose Oral Tolerance in Monocyte Chemoattractant Protein-1- and CCR2-Deficient Mice1

Patricia A. Gonnella2, Dhatri Kodali and Howard L. Weiner

Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The chemokine monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR2 have been shown to play an important role in the migration and trafficking of macrophages and Th1 effector cells in experimental autoimmune encephalomyelitis. Also, MCP-1 has been reported to regulate oral tolerance induction by inhibition of Th1 cell-related cytokines and by the ability of Abs to MCP-1 to inhibit oral tolerance. This study demonstrates that neither MCP-1 nor its receptor CCR2 is required for the induction of oral tolerance. Mice deletional for either MCP-1 or CCR2 had suppressed cell-proliferative and Th1 responses following oral administration and immunization with myelin oligodendrocyte glycoprotein (MOG35–55). TGF-{beta} was up-regulated in fed and immunized deletional mice, while IL-4 was absent from deletional mice, but up-regulated in controls. Decreased experimental autoimmune encephalomyelitis severity was found in MOG35–55-fed MCP-1 deletional mice, indicating induction of oral tolerance. These results demonstrate that MCP-1 is not required for induction of oral tolerance and that MCP-1 and CCR2 are essential for up-regulation of IL-4 in tolerized mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The interactions of chemokines and their receptors mediate the recruitment and trafficking of specific subpopulations of lymphocytes. Chemokine family members are subdivided into four groups (cxc, cc, c, cx3c) based upon the positioning of conserved cysteine residues. In addition to molecular structure, the cell types for which they are chemotactic may distinguish chemokines. The cc chemokines have adjacent cysteine residues and attract monocytes, basophils, eosinophils, and selective populations of lymphocytes. Monocyte chemoattractant protein-1 (MCP-1)3 is a prototype cc chemokine that has been shown to play a critical role in innate immunity by directing the migration of monocytes to sites of inflammation (1). In experimental autoimmune encephalomyelitis (EAE), inflammation in the CNS is characterized by the infiltration of macrophages and Ag-specific and nonspecific CD4+ and CD8+ lymphocytes. MCP-1 has been implicated in EAE pathogenesis, as evidenced by markedly elevated levels in the CNS during disease (2) and the blockage of relapse of EAE by MCP-1 Abs (3). In addition, absence of MCP-1 in deletional mice results in decreased macrophage recruitment and Ag-specific Th1 immune response in EAE (4). Furthermore, resistance to EAE has been reported in mice lacking CCR2, the receptor for MCP-1 (5). CCR2 expression is found on monocytes and activated T cells (6), and CCR2 signaling has been reported to promote Th1 immune response in vivo (7, 8, 9). Thus, the data indicate that MCP-1 and CCR2 play an important role in the pathogenesis of EAE.

In addition, MCP-1 has been reported to regulate immune responses that restrain CNS inflammation. Oral administration of Ag is often characterized by marked suppression of cell-mediated immune response to immunization with the same Ag and is known as oral tolerance. Feeding of myelin Ags has been shown to ameliorate disease severity in EAE (10). MCP-1 expression was elevated in the intestinal mucosa, Peyer’s patch, and mesenteric lymph node of orally tolerized mice and was correlated with down-regulation of mucosal IL-12 and an increase in IL-4 production (11). MCP-1 was reported to regulate oral tolerance because treatment with anti-MCP-1 in vivo resulted in abrogation of tolerance induction (11). MCP-1 was hypothesized to regulate tolerance through two possible mechanisms. Either MCP-1 up-regulates IL-4 expression, which potentiates the differentiation of Th2 cells (12), or MCP-1 may down-regulate IL-12 production, which could result in a block of Th1 differentiation (13) and/or enhanced production of TGF-{beta} (14). To determine whether MCP-1 is an essential regulatory chemokine for induction of oral tolerance or the up-regulation of IL-4, we examined oral tolerance induction in MCP-1-deficient mice. Also, we examined the effect of deletion of CCR2, the receptor for MCP-1. Our results show that neither MCP-1 nor its receptor CCR2 is required for the induction of oral tolerance, but both are essential for up-regulation of IL-4 in orally tolerized mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

MCP-1-deficient mice (MCP-1-/-) were obtained from B. Rollins (Dana-Farber Cancer Institute, Boston, MA). Disruption of the MCP-1 gene has been previously described (15). F10 mice on the C57BL/6 background were used. Wild-type C57BL/6 mice (MCP-1+/+) were obtained from The Jackson Laboratory (Bar Harbor, ME). CCR2-deficient mice (CCR2-/-) were obtained from I. Charo (Gladstone Institute of Cardiovascular Disease, San Francisco, CA). Methodology for the generation of CCR2-/- mice by homologous recombination has been previously described (7). CCR2-/- and CCR2+/+ (wild-type) controls were generated by mating between heterozygous (CCR2+/-) mice (50/50 sv129 x C57BL/6). For studies of MCP-1-/- and MCP-1+/+ mice, we used mice 8–14 wk of age. Female mice were used for two experiments, and male mice were used for one experiment. No difference in oral tolerance between male and female mice was detected. For experiments with CCR2-/- and CCR2+/+ mice, males 10–14 wk of age were used. All animals were raised in pathogen-free conditions.

Ags, Abs, and recombinant cytokines

Purified myelin oligodendrocyte glycoprotein (MOG35–55) was obtained from the Peptide Synthesis Core Facility (Massachusetts General Hospital Endocrine Unit, Boston, MA). Abs used for ELISA were purified rat anti-mouse IFN-{gamma} (clone R4-6A2), IL-2 (clone JES6-1A12), IL-4 (clone BVD4-1D11), IL-10 (clone JES5-2A5) mAb; biotinylated rat anti-mouse IFN-{gamma} (clone XMG1.2), IL-2 (clone JES6-5H4), IL-4 (clone BVD4-24G2), and IL-10 (clone SXC-1) mAb (BD PharMingen, San Diego, CA); polyclonal chicken anti-TGF-{beta} (R&D Systems, Minneapolis, MN); and monoclonal mouse anti-TGF-{beta} (clone 1D11.16; R&D Systems). Recombinant cytokines were mouse IL-2, IL-4, IL-10, IFN-{gamma} (BD PharMingen), and purified bovine TGF-{beta}1 (R&D Systems).

Oral administration of Ag

Deletional and wild-type mice were fed 250 µg MOG35–55 every other day for a total of five times. MOG35–55 was dissolved in PBS and fed by gastric intubation (0.5 cc) with an 18-gauge stainless steel feeding needle (Thomas Scientific, Swedesboro, NJ). Three mice per group were used, and each experiment was repeated three times. All fed mice were immunized and compared with unfed immunized mice.

Induction of EAE

Mice were immunized in the flank with an s.c. injection of 50 µg MOG35–55 in 0.1 ml PBS emulsified in an equal volume of CFA containing 4 mg/ml of Mycobacterium tuberculosis H37 RA (Difco, Detroit, MI). Two injections were given for a total of 100 µg MOG35–55 per mouse, followed by an i.v. injection in the tail vein of 150 ng pertussis toxin (List Biological Laboratories, Campbell, CA) in 0.2 ml of PBS. Animals received a second injection of pertussis toxin 48 h later. Severity of clinical disease was scored as follows: 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb plus forelimb paralysis; 5, moribund. One blinded and two unblinded experiments were conducted. No difference in tolerance induction in blinded and unblinded experiments was seen.

Cell culture and proliferation assay

Mice were immunized in the footpad with MOG35–55 in CFA, as detailed above, and popliteal nodes were harvested 8 days after immunization. Nodes from three animals per group were pooled. Cells were cultured at 0.5 x 106 cells/well in 0.2 ml of serum-free medium X Vivo 20 (BioWhittaker, Walkersville, MD) supplemented with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin, and containing 0, 1, 10, and 100 µg of MOG35–55. Culture supernatants were collected for cytokine assay for IL-2, IL-4, IL-10, and IFN-{gamma} at 48 h and for TGF-{beta} at 72 h. For proliferation assays, 1 µCi of [3H]thymidine (1 Ci = 376{beta}) was added to each culture at 72 h. Cells were harvested and radioactivity incorporated was counted 16 h later using a flatbed beta counter (Wallac, Gaithersburg, MD). Analysis was performed by Student’s t test.

ELISA for cytokines

Quantitative ELISAs for IL-2, IL-4, IL-10, and IFN-{gamma} were performed using paired mAbs specific for corresponding cytokines, per manufacturer’s recommendations (BD PharMingen). TGF-{beta} ELISA was performed, as previously described (16). Results are expressed as mean values from triplicate cultures. Analysis was performed by Student’s t test. Data presented are representative of three experiments.

Immunostaining of light-microscopic sections

Excised tissues from anesthetized animals were embedded in OCT Tissue-TeK (Miles, Elkhart, IL) and snap frozen in liquid nitrogen-cooled isopentane. Acetone-fixed cryosections were stored at -20°C. For immunohistochemistry, frozen sections were thawed and fixed in acetone (2 min), and endogenous peroxidase activity was quenched by incubation in 0.005 M periodic acid (10 min) and 0.003 M sodium borohydride (30 min) (17). Subsequently, tissues were washed in PBS and incubated in diluted normal blocking serum (20 min), which was prepared from the species from which the secondary Ab was made. Endogenous biotin was blocked by use of the avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA), per manufacturer’s instructions. PBS-washed sections were incubated overnight in primary Ab diluted in blocking serum. Sections were rinsed in PBS and incubated in diluted biotinylated secondary Ab (1 h). PBS-washed sections were incubated in Vectastain Elite ABC reagent, washed again, and incubated in peroxidase substrate solutions containing 3,3'-diaminobenzidine (DAB substrate kit; Vector Laboratories) for 5–7 min. Sections were washed in tap water (5 min) counterstained in hematoxylin, cleared, and mounted. Up-regulation of cytokine staining was determined by comparing fed immunized with unfed immunized mice. Quantification of data was performed by computerized image analysis (IP Labs Systems, Scanalytics, Fairfax, VA). Three mice per group were studied, and experiments were repeated three times. Nine sections (three per animal) were randomly selected and quantitated for each group. The region of interest (ROI) represents a defined area (116 µm x 44 µm) at x100 magnification. The percentage of staining within the ROI was measured in 20 randomly selected regions. This analysis was conducted for the dome, corona, germinal center, and interfollicular region of the Peyer’s patch and for the lamina propria of the villi. Data are expressed as the mean percentage of staining per ROI ± SD and were analyzed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oral tolerization in MCP-1-deficient mice

To determine whether oral tolerance could be induced in MCP-1-/- mice, we examined MCP-1+/+ and MCP-1-/- mice after feeding 250 µg MOG35–55 five times, followed by immunization in the footpad. Popliteal node cells were harvested 8 days after immunization. Upon exposure to Ag in vitro, oral tolerance was found to be induced in MCP-1+/+ and in MCP-1-/- mice, as evidenced by suppression of cellular proliferation (Fig. 1) and decreased production of IFN-{gamma} (Fig. 2A) and IL-2 (Fig. 2B). IL-4 was found to be up-regulated in fed MCP-1+/+ mice, but not in MCP-1-/- mice (Fig. 2C). Small increases in TGF-{beta} were found in fed wild-type and MCP-1-/- mice (Fig. 2D). No IL-10 was detected in either group (not shown).



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  		FIGURE 1. Proliferation of cells from the popliteal node of wild-type and MCP-1-/- C57BL/6 mice. Suppression of proliferation was seen in wild-type and MCP-1-/- mice fed MOG35–55. Results are compared with unfed immunized mice. SDs not visible are within the symbol. Significant differences were found with cells cultured with 10 and 100 µg MOG35–55 (p = 0.001).

 


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  		FIGURE 2. Cytokine production in the popliteal node of wild-type and MCP-1-/- C57BL/6 mice following oral administration of MOG35–55. Suppression of IFN-{gamma} (A) and IL-2 (B) production in wild-type and MCP-1-/- mice fed MOG35–55. C, Up-regulation of IL-4 with feeding in wild-type, but not in MCP-1-/- mice. D, Up-regulation of TGF-{beta} with feeding in both wild-type and MCP-1-/- mice. *, p = 0.001.

 
Oral tolerization in CCR2-deficient mice

CCR2+/+ and CCR2-/- mice were examined for induction of oral tolerance following feeding with 250 µg MOG35–55 five times and immunization in the footpad. Popliteal node cells were harvested 8 days after immunization and incubated in varying concentrations of MOG35–55 in vitro. Oral tolerization was found in both CCR2+/+ and CCR2-/- mice, demonstrated by suppression of cellular proliferation to Ag (Fig. 3) and decreased production of IFN-{gamma} (Fig. 4A) and IL-2 (Fig. 4B). Up-regulation of IL-4 was found in fed CCR2+/+ mice, but not in CCR2-/- mice (Fig. 4C). Increased production of TGF-{beta} in response to feeding was found in both CCR2+/+ and CCR2-/- mice (Fig. 4D). No up-regulation of IL-10 was detected in either group (not shown).



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  		FIGURE 3. Proliferation of cells from the popliteal node of wild-type and CCR2-/- (129 x C57BL/6) mice. Suppression of proliferation was seen in wild-type and CCR2-/- mice fed MOG35–55. SDs not visible are within the symbol (p = 0.001).

 


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  		FIGURE 4. Cytokine production in the popliteal node of wild-type and CCR2-/- (129 x C57BL/6) mice. Suppression of IFN-{gamma} (A) and IL-2 (B) production in wild-type and CCR2-/- mice fed MOG35–55. C, Up-regulation of IL-4 production with feeding in wild-type, but not in CCR2-/- mice. D, Up-regulation of TGF-{beta} with feeding in both wild-type and CCR2-/- mice. *, p = 0.001.

 
Induction of oral tolerance in MCP-1-deficient EAE mice

MCP-1+/+ (C57BL/6) mice fed MOG35–55 had less disease severity compared with unfed mice, indicating induction of oral tolerance (Fig. 5). Animals were examined from 10 to 25 days postimmunization. MCP-1-/- unfed immunized mice developed only mild disease, with a clinical score of one from 10 to 15 days. Oral tolerance was induced in MOG35–55-fed MCP-1-/- mice, as evidenced by complete absence of disease. In previous studies, no disease was detected in CCR2-/- unfed immunized mice (5, 18). Similarly, we detected no disease in unfed CCR2-/- mice immunized with MOG35–55 and given pertussis toxin.



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  		FIGURE 5. MOG35–55 induced EAE in MCP-1-/- and wild-type C57BL/6 mice fed 250 µg MOG35–55 five times. Decreased disease severity was seen in MCP-1-/- mice compared with wild type. Suppression of disease with feeding was seen in both wild-type and MCP-1-/- mice. For wild-type mice, p <= 0.05 for days 12, 13, 14, 18, 19, and 20–25. For MCP-1-/- mice, p = 0.02 for days 10–15. Analysis by Student’s t test.

 
Immunohistochemistry of the gut-associated lymphoid tissue (GALT) of wild-type and deletional mice

Due to the results in the popliteal node in vitro, we examined the in situ immune response to oral Ag in the GALT at 8 days after immunization. Results of immunohistochemical staining show the up-regulation of TGF-{beta} in the lamina propria of fed and immunized MCP-1-/- and CCR2-/- mice (Fig. 6). Results were compared with unfed immunized deletional mice. Quantification of the data shows that similar to the results found in the popliteal node, suppression of IFN-{gamma} production and up-regulation of TGF-{beta} were found in lamina propria of fed and immunized MCP-1+/+ and MCP-1-/- mice (Fig. 7, A and B), indicating oral tolerization in these mice. These changes were also seen in CCR2+/+ and CCR2-/- mice (Fig. 7, C and D). Although a large SD was detected in the lamina propria, significant up-regulation of TGF-{beta} was assessed by Student’s t test (p = 0.001). No significant changes were seen in the Peyer’s patch. In addition, no up-regulation of IL-4 or IL-10 was seen in the GALT of deletional mice (not shown).



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  		FIGURE 6. Immunohistochemical staining of the lamina propria for TGF-{beta}. Tissues were excised 8 days after immunization. TGF-{beta} is up-regulated with MOG35–55 feeding and immunization. A, Unfed immunized MCP-1-/- mice. B, MOG35–55-fed and immunized mice. TGF-{beta}-stained cell (arrow). C, A few TGF-{beta}-positive cells are found in unfed immunized CCR2-/- mice (arrow). D, Increase in TGF-{beta} staining (arrow) in MOG35–55-fed and immunized CCR2-/- mice (x200).

 


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  		FIGURE 7. Immunohistochemical quantification of cytokines in the lamina propria of wild-type and MCP-1-/- mice. Suppression of IFN-{gamma} (A) and up-regulation of TGF-{beta} (B) in wild-type and MCP-1-/- mice. *, p = 0.001. Similar suppression of IFN-{gamma} (C) and up-regulation of TGF-{beta} (D) are found in wild-type and CCR2-/- mice. *, p = 0.001. Results are compared with unfed immunized mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MCP-1 has been reported to regulate oral tolerance induction because Abs to MCP-1 were found to inhibit the protective effect of oral PLP139–151 on disease severity (19). Also, the down-regulation of IFN-{gamma} production was inhibited. In contrast, the present study shows that oral tolerance can be induced in MCP-1-/- mice, as evidenced by reduction in disease severity, and suppression of cellular proliferation and IFN-{gamma} production with concurrent up-regulation of TGF-{beta} in the popliteal node and GALT. Similar results were found in mice deficient for the MCP-1 receptor CCR2. The reason for the discrepancy between our results and those of the previous study (19) is not clear, although there are significant differences between the experimental systems. In the previous study, a relatively high dose of PLP139–151 (2 mg) to SJL mice was used, whereas we fed a low dose (250 µg) of MOG35–55 to C57BL/6 animals. They did not observe the induction of TGF-{beta}, whereas we did. Low doses of Ag are associated with the active suppression mechanism of tolerance and the up-regulation of Th2 cytokines and TGF-{beta}-producing regulatory cells. In contrast, high doses of Ag are associated with the induction of anergy. Thus, it may be that different mechanisms related to dose, animal strain, and Ag used account for the differences between our results. Alternatively, the differences may be due to methodology. In the previous study, Abs were used to block MCP-1, whereas we used MCP-1-deficient mice. Thus, the inability to obtain tolerance may be due to deleterious effects of exogenous protein on diverse cell types. What is clear, however, from our studies is that, although MCP-1 may be important for oral tolerance under some conditions (perhaps related to induction of anergy), MCP-1 is not a requirement for low dose oral tolerance induction, which is characterized by the induction of Th2- and/or TGF-{beta}-secreting regulatory cells along with up-regulation of these cytokines in the GALT.

Previous studies have shown that absence of MCP-1 (4) or CCR2 (5, 18) results in little or no induction of EAE. Either of these genetic deficiencies was shown to result in impaired recruitment of macrophages to the CNS; however, the generation of T cells that transferred severe disease to wild-type recipients was not impaired. Our results demonstrate that despite decreased disease severity in MCP-1-/- mice, oral tolerance can be induced, as evidenced by the complete absence of disease following MOG35–55 feeding and immunization. Thus, MCP-1 is not essential for the generation or trafficking of cells necessary for the induction of oral tolerance. The reason for the increase in MCP-1 in the Peyer’s patch and mesenteric lymph nodes in oral tolerance is not known, but may be related to the up-regulation of IL-4. MCP-1 has been shown to be a critical factor for T cell commitment to the Th2 phenotype (20). We observed that IL-4 was up regulated in popliteal node cell supernatants from orally tolerized MCP-1+/+ mice, but was absent in MCP-1-/- mice. Similarly, IL-4 was increased in fed and immunized CCR2+/+ controls, but was not detected in supernatants from CCR2-/- mice. Although we observed some endogenous IL-4 in the gut of MCP-1-/- and CCR2-/- mice, no up-regulation with feeding was seen (not shown). Thus, the results indicate that the expression of MCP-1 on mucosal immune cells potentiates the production of IL-4. Up-regulation of IL-4 could skew the mucosal cytokine microenvironment in wild-type mice, augmenting Th2 differentiation. We observed that oral tolerance could be induced in deletional mice in the absence of up-regulation of IL-4 or IL-10. Previous studies demonstrating the induction of oral tolerance in IL-4-deficient mice support this finding (21). Our results showing absence of disease in CCR2-/- mice are in agreement with previous studies implicating CCR2 as an important receptor mediating monocyte recruitment in vivo (5, 18). We detected mild disease in MCP-1-/- mice and no disease in CCR2-/- mice; thus, the data suggest that other CCR2 agonists as well as MCP-1 may be important for the recruitment of peripheral blood monocytes in EAE.

Another postulate of MCP-1 regulation of tolerance suggests that MCP-1 may down-regulate IL-12 production, which could result in a partial or complete block of peripheral Th1 differentiation (13) and/or enhancement of TGF-{beta} expression (14). Our results demonstrate that down-regulation of Th1 differentiation evidenced by suppression of IFN-{gamma} production can occur in the absence of MCP-1. In addition, TGF-{beta} was up-regulated in both the popliteal node and the GALT. Similar results were found in the absence of the MCP-1 receptor CCR2. The reason for the virtual abrogation of IFN-{gamma} secretion in fed MCP-1-/- mice is not known, but may be related to the role of MCP-1 in the trafficking of pathogenic T cells. The migration of Th1 cells would be impaired in deletional mice, while the trafficking of TGF-{beta}-producing regulatory cells proceeds, resulting in efficient down-regulation of Th1 responses. Whether MCP-1 exerts a partial effect on Th1 differentiation or the up-regulation of TGF-{beta} is not known, but appears unlikely because no significant difference compared with wild type was seen in the GALT. Also, no difference compared with wild type was seen in CCR2-deficient mice.

It was previously thought that MCP-1 up-regulation in the mucosa promoted the infiltration of T cells that may be activated in a Th2 environment to become Th2 or TGF-{beta} regulatory cells (12, 19). These T cells could then migrate to the CNS (19) and suppress pathogenic Th1 cells in EAE (22). In addition, MCP-1 was hypothesized to induce migration of macrophages to the GALT, the site of Ag absorption (19). These cells may present Ag in the gut mucosa and/or migrate to the CNS, inducing the production of regulatory T cells (19). Our findings of oral tolerance induction in MCP-1-deficient mice suggest that T cell and/or macrophage trafficking associated with TGF-{beta} production proceeds unimpaired. These findings suggest that other chemokines and receptors may play a role in the trafficking of T cells and/or macrophages in the absence of MCP-1. Chemokines are known to be redundant in their action on target cells and promiscuous in receptor usage (23). For example, monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 interact with CCR2, and MCP-2 and MCP-3 have been shown to interact with other receptors (CCR1 and CCR3) (24, 25). Also, mononuclear phagocytes have been reported to respond to the widest range of chemokines (23).

Our study indicates that MCP-1 is not essential for the trafficking of TGF-{beta}-producing regulatory cells in oral tolerance. MCP-1 and CCR2 appear to have a more pronounced effect on macrophage trafficking to the CNS responsible for Th1 cell migration and resultant disease severity in EAE. Because decreased disease severity is found in MCP-1-/- mice, the effect of the absence of IL-4-producing Th2 cells on the strength of tolerance induction compared with MCP-1+/+ mice cannot be evaluated. Nevertheless, similar suppression of cellular proliferation and IFN-{gamma} production was seen in both groups. Future studies are indicated to identify chemokines affecting the migration and trafficking of TGF-{beta}-producing regulatory cells in oral tolerance.


   Acknowledgments
 
We thank Dr. Barrett Rollins and Dr. Israel Charo for provision of deletional mice.


   Footnotes
 
1 This work was supported by a grant from the National Multiple Sclerosis Society (to P.A.G.) and a grant from the National Institutes of Health (to H.L.W.). Back

2 Address correspondence and reprint requests to Dr. Patricia A. Gonnella at the current address: The Children’s Hospital, Enders Research Building Room 1355, Boston, MA 02115. E-mail address: patricia.gonnella{at}tch.harvard.edu Back

3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; EAE, experimental autoimmune encephalomyelitis; GALT, gut-associated lymphoid tissue; MOG, myelin oligodendrocyte glycoprotein; ROI, region of interest. Back

Received for publication June 13, 2002. Accepted for publication December 20, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Oppenheim, J. J., C. O. Zachariae, N. Mukaida, K. Matsushima. 1991. Properties of the novel proinflammatory supergene "intercrine" cytokine family. Annu. Rev. Immunol. 9:617.[Medline]
  2. Juedes, A. E., P. Hjelmstrom, C. M. Bergman, A. L. Neild, N. H. Ruddle. 2000. Kinetic and cellular origin of cytokines in the central nervous system: insight into mechanisms of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. J. Immunol. 164:419.[Abstract/Free Full Text]
  3. Kennedy, K. J., R. M. Strieter, S. L. Kunkel, N. W. Lukacs, W. J. Karpus. 1998. Acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the CC chemokines macrophage inflammatory protein-1a and monocyte chemotactic protein-1. J. Neuroimmunol. 92:98.[Medline]
  4. Huang, D., J. Wang, P. Kivisakk, B. J. Rollins, R. M. Ransohoff. 2001. Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis. J. Exp. Med. 193:713.[Abstract/Free Full Text]
  5. Izikson, L., R. S. Klein, I. F. Charo, H. L. Weiner, A. D. Luster. 2000. Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2. J. Exp. Med. 192:1075.[Abstract/Free Full Text]
  6. Frade, J. M., M. Mellado, G. del Real, J. C. Gutierrez-Ramos, P. Lind, A. C. Martinez. 1997. Characterization of the CCR2 chemokine receptor; functional CCR2 receptor expression in B cells. J. Immunol. 159:5576.[Abstract]
  7. Boring, L., J. Gosling, S. Chensue, S. Kunkel, R. J. Farese, H. Broxmeyer, I. Charo. 1997. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice. J. Clin. Invest. 100:2552.[Medline]
  8. Warmington, K. S., L. Boring, J. H. Ruth, J. Sonstein, C. M. Hogaboam, J. L. Curtis, S. L. Kunkel, I. R. Charo, S. W. Chensue. 1999. Effect of C-C chemokine receptor 2 (CCR2) knockout on type-2 (schistosomal antigen-elicited) pulmonary granuloma formation: analysis of cellular recruitment and cytokine responses. Am. J. Pathol. 154:1407.[Abstract/Free Full Text]
  9. Traynor, T. R., W. A. Kuziel, G. B. Toews, G. B. Huffnagle. 2000. CCR2 expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection. J. Immunol. 2000:2021.
  10. Faria, A. M. C., H. L. Weiner. 1999. Oral tolerance: mechanisms and therapeutic applications. Adv. Immunol. 73:153.[Medline]
  11. Karpus, W. J., R. M. Ransohoff. 1998. Chemokine regulation of experimental autoimmune encephalomyelitis: temporal and spatial expression patterns govern disease pathogenesis. J. Immunol. 161:2667.[Abstract/Free Full Text]
  12. Karpus, W. J., N. W. Lukacs, K. J. Kennedy, W. S. Smith, S. D. Hurst, T. A. Barrett. 1997. Differential CC chemokine-induced enhancement of T helper cell cytokine production. J. Immunol. 158:4129.[Abstract]
  13. Abbas, A. K., K. M. Murphy, A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[Medline]
  14. Marth, T., W. Strober, B. L. Kelsall. 1996. High dose oral tolerance in ovalbumin TCR-transgenic mice: systemic neutralization of IL-12 augments TGF-{beta} secretion and T-cell apoptosis. J. Immunol. 157:2348.[Abstract]
  15. Lu, B., B. Rutledge, L. Gu, J. Fiorillo, N. Lukacs, S. Kunkel, R. North, C. Gerard, B. J. Rollins. 1998. Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J. Exp. Med. 187:601.[Abstract/Free Full Text]
  16. Chen, Y., J. Inobe, H. L. Weiner. 1995. Induction of oral tolerance to myelin basic protein in CD8-depleted mice: both CD4+ and CD8+ cells mediate active suppression. J. Immunol. 155:910.[Abstract]
  17. Isobe, Y., S. Chen, P. Nakane, W. Brown. 1977. Studies of translocation of immunoglobulins across intestinal epithelium. I. Improvements in the peroxidase-labelled antibody method for application to study of human intestinal mucosa. Acta Histochem. Cytochem. 10:161.
  18. Fife, B. T., G. B. Huffnagle, W. A. Kuziel, W. J. Karpus. 2000. CC chemokine receptor 2 is critical for induction of experimental autoimmune encephalomyelitis. J. Exp. Med. 192:899.[Abstract/Free Full Text]
  19. Karpus, W. J., K. J. Kennedy, S. L. Kunkel, N. W. Lukacs. 1998. Monocyte chemotactic protein 1 regulates oral tolerance induction by inhibition of T helper cell 1-related cytokines. J. Exp. Med. 187:733.[Abstract/Free Full Text]
  20. Gu, L., S. Tseng, R. Hormer, C. Tam, M. Loda, B. Rollins. 2000. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404:407.[Medline]
  21. Garside, P., M. Steel, E. A. Worthey, A. Satoskar, J. Alexander, H. Bluethmann, F. Y. Liew, A. M. Mowat. 1995. T helper 2 cells are subject to high dose oral tolerance and are not essential for its induction. J. Immunol. 154:5649.[Abstract]
  22. Khoury, S. J., W. W. Hancock, H. L. Weiner. 1992. Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis as associated with down-regulation of inflammatory cytokines and differential up-regulation of transforming growth factor {beta}, interleukin 4, and prostaglandin E expression in the brain. J. Exp. Med. 176:1355.[Abstract/Free Full Text]
  23. Mantovani, A.. 1999. The chemokine system: redundancy for robust outputs. Immunol. Today 20:254.[Medline]
  24. Combadiere, C., S. K. Ahuja, J. Van Damme, J. L. Tiffany, J. L. Gao, P. M. Murphy. 1995. Monocyte chemoattractant protein-3 is a functional ligand for CC chemokine receptors 1 and 2B. J. Biol. Chem. 270:29671.[Abstract/Free Full Text]
  25. Franci, C., L. M. Wong, D. J. Van, P. Proost, I. F. Charo. 1995. Monocyte chemoattractant protein-3, but not monocyte chemoattractant protein-2, is a functional ligand of the human monocyte chemoattractant protein-1 receptor. J. Immunol. 154:6511.[Abstract]



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