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ILAR Journal V40(4) 1999
Animal Models of Inflammation
William J. Karpus, Kevin J. Kennedy, Brian T. Fife, and Lisa M. Hoffman
| All authors are from the Department of Pathology, Northwestem University Medical School, Chicago, Illinois, where William J. Karpus, Ph.D., is Assistant Professor, Kevin J. Kennedy, M.S., is Research Technician II, Brian T. Fife, B.S., is Graduate Assistant, and Lisa M. Hoffman, Ph.D., is Research Associate. |
Introduction
Experimental autoimmune encephalomyelitis (EAE1) is a CD4+ T-helper (Th1)1-mediated, inflammatory demyelinating disease of the central nervous system (CNS1) that serves as a model for multiple sclerosis (MSl) (Amason 1983; Wekerle 1991). EAE can be induced in genetically susceptible animals by immunization with neuro-antigens such as proteolipid protein, myelin basic protein, myelin oligodendrocyte glycoprotein, and the immuno-dominant, encephalitogenic peptide sequences of these molecules emulsified in complete Freund's adjuvant (CFA1) or by the adoptive transfer of antigen-activated T lymphocytes (Amor and others 1994; Falk and others 1968; Pettinelli and others 1982; Satoh and others 1988; Tuohy and others 1988, 1992). The disease is characterized either by a progressive ascending clinical paralysis followed by periods of remission and subsequent relapses (McRae and others 1992) or as monophasic, acute, and nonrelapsing (Miller and Karpus 1994). Analysis of mononuclear cell infiltration in the CNS has revealed that antigen-specific and -nonspecific CD4+ and CD8+ T cells as well as macrophages constitute the CNS-migrating cell populations (Cross and others 1990; Hickey and others 1983, 1991). The mechanism by which these cells traffic to the CNS and accumulate before and during clinical disease is not well understood. One important mechanism involved in the trafficking of T cells to the CNS is the interaction of integrins expressed on the surface of T cells with the appropriate counter ligands expressed on cere-brovascular endothelium. Specifically, expression of the (z4[31 integrin (very late antigen-4) on the surface of activated T cells has been shown to be required for entry of those cells into the CNS (Yednock and others 1992). Although integrin expression is important for T cell migration to target tissues, there also appears to be a role for chemotactic factors (Springer 1994).
Chemokines such as macrophage inflammatory protein (MIP1)-1a, monocyte chemotactic protein (MCP1)-1, interleukin (IL1)-8, and regulated upon activation normal T cell expressed and secreted (RANTES1) are chemotactic molecules that induce leukocyte accumulation in tissue sites of inflammation (Oppenheim and others 1991). Chemokines are potent chemoattractants that can be divided into four highly conserved but distinct subfamilies--CXC, CC, C, and CX3C--based on the position of the first two cysteines in the amino terminus as well as the remaining cysteines in the carboxy portion of the molecule (Luster 1998). CC chemokine family members have been implicated as candidates in the immunopathology of EAE, with T cell production of MIP-1a and T cell activation-3 shown to be required for induction ofEAE (Kuchroo and others 1993) and MCP-1 and gamma interferon inducible protein- 10 (IP- 101) expression by parenchymal astrocytes (Glabinski and others 1997). Additionally, MIP-1a and MCP-1 production in the CNS has been correlated with the development of acute clinical disease symptoms in both rat (Hulkower and others 1993) and mouse EAE models (Hayashi and others 1995; Karpus and others 1995; Ransohoff and others 1993). These examples raise the possibility that chemokine production in the CNS of MS patients functions to drive pathogenesis of disease through the recruitment of leukocytes into the brain.
The role of chemokines in the pathogenesis of MS has not been well established. An early study demonstrated elevated MIP-1a expression in the cerebrospinal fluid (CSF1) of MS patients compared with control patients with other neurological diseases, and the increased levels correlated with increased CSF leukocyte counts (Miyagishi and others 1995). Findings in MS studies support the relevance of chemokine tissue distribution, as demonstrated in EAE (Simpson and others 1998). CNS expression of RANTES in MS brain was shown to predominate at the edge of active plaques in T cell-rich areas of the lesion (Hvas and others 1997). More recently, the expression of chemokines in the CSF of MS patients was examined and revealed the presence of IP- 10 in new onset MS and clinically definite MS patients compared with control neurological patients (Sorensen and others 1999). IP-10 and RANTES CSF levels were elevated in MS patients compared with controls, and the levels of IP-10 correlated with increased CSF leukocyte counts. Moreover, the CSF-derived T cells expressed the CXCR3 receptor for IP-10. Because both IP-10 and RANTES are potent T cell chemoattractants, it is reasonable to postulate that the elevated levels of these chemokines during active episodes of MS induce accumulation of T cells into the CNS through chemokine receptor expression on T cells and monocytes.
We have begun to address the role of CC chemokines in EAE as well as the effector function of T cells by studying the production and biological function of chemokines in both acute and relapsing EAE. Here we review the kinetics of CC chemokine production in the CNS during the progression of acute and relapsing EAE and the biological roles of MIP-1a, MCP-1, RANTES, and macrophage inflammatory protein (MIP1)- 1b in the pathogenesis of relapsing EAE.
Methods for Disease Induction and Chemokine Detection
Animals
Female SJL mice (H-2s) were purchased from Harlan Sprague Dawley (Indianapolis, Indiana). Mice were 6 to 7 wk old at the initiation of the experiment. Animal care was provided in accordance with Northwestern University and National Institutes of Health guidelines.
Antigens and Antibodies
PLP 139-151 peptide (HSLGKWLGHPDKF) was purchased from Peptides International (Louisville, Kentucky). The amino acid composition was verified by mass spectrometry, and purity (> 98%) was assessed by high-performance liquid chromatography. Rabbit antimurine MIP-1a, MCP-1, and RANTES antibodies were prepared by multiple site immunization of New Zealand white rabbits with recombinant proteins (R & D Systems, Minneapolis, Minnesota) emulsified in CFA. Polyclonal antibodies (anti-MIP-1a, anti-MCP-1, and anti-RANTES) were titered by direct enzyme-linked immunosorbent assay (ELISA1), and specificity was verified by the failure to cross-react with any other cytokine or chemokine tested (such as mIL-1a, mIL-2, IL-6, hlL-8, TNF, hRANTES, hMIP-1, mMCP-1/JE, mMIP-2, MIP-1b, or ENA-78) as measured by either direct ELISA or the ability to neutralize in vitro chemokine-induced mononuclear cell migration (Lukacs and others 1994a; Strieter and others 1992). At the time of the experiments, the titer of the antichemokine antibodies was >106.
Priming of Donor Lymphocytes, Cell Culture, and Transfer of EAE
Donor lymphocytes were primed by subcutaneous immunization of normal SJL/J mice with 25 mg of PLP139-151 in CFA containing 4 mg/mL of Mycobacterium tuberculosis (Difco, Detroit, Michigan). Seven days later, draining lymph node cells were pooled and cultured in vitro in complete Dulbecco's minimum essential medium (Biowhitaker, Bethesda, Maryland) containing 5 x 10-5 M 2-ME (GIBCO), 2 mM L-glutamine (GIBCO), 100 U/mL of penicillin (GIBCO), 100 mg/mL of streptomycin (GIBCO), 0.1 mM nonessential amino acids (GIBCO), and 5% fetal calf serum (Hy-Clone) (Karpus and others 1994) at a concentration of 6 x 106 cells/mL in the presence of 50 mg/mL of PLP 139-151 for 72 hr. The cells were harvested and washed, and 3 x 106 viable T cell blasts were transferred intraperitoneally to normal SJL/J recipients. After cell transfer, mice were evaluated for the development of EAE.
Chemokine ELISA
Assessment of MIP-1a, MCP-1, and MIP-2 was quantitated from tissue samples and culture supernatants using previously described ELISA (Lukacs and others 1993, 1994b). Spinal cord samples were homogenized in 1 mL of phosphate-buffered saline and clarified by centrifugation (400 x g) for 10 min. Flat-bottomed microtiter plates (Nunc, Naperville, Illinois) were coated with capture antibody diluted to 3.2 mg/ mL in borate-buffered saline coating buffer and incubated ovemight. Nonspecific binding sites were blocked with 2% bovine serum albumin in phosphate-buffered saline for 1 hr at 37°C, and samples were subsequently added in triplicate for 2 hr at 37ºC. Biotinylated goat anti-rabbit detection antibody was added, and the plates were incubated for an additional 1 hr at 37ºC. The wells were developed using strepavidin-peroxidase and O-phenylenediaminedihydrochloride substrate, and absorbance was read at 490 nm. Standard curves for the individual chemokines were generated using a series of dilutions of purified recombinant protein (R & D Systems). Chemokine levels in spinal cord were quantitated by comparison with the standard curves and expressed as ng/mL. The detection limit of these ELISA is at least 0.05 ng/mL. The individual chemokine antibodies used in the ELISA are specific and do not cross-react with any other chemokine or cytokine as described above.
Clinical Evaluation
Adoptive R-EAE was induced by the transfer of 3 x 106 in vitro-stimulated PLP139-151-specific T cell blasts from PLP 139-151 peptide-primed mice. Individual animals were observed daily and graded according to their clinical severity as follows: 0 = no abnormality; 1 = limp tail; 2 = limp tail and partial hind limb weakness (waddling gait); 3 = complete hind limb paralysis; and 4 = death. In most experiments, there was a range of maximum severity between grades 1 and 3. Mice rarely died from EAE; however, the score of 4 was used when no other cause could be attributed to a death. We did not induce a severe form of EAE or use death as an endpoint. A relapse was scored when a mouse developed additional neurological deficits (an increase of at least one clinical grade) after a period of stabilization or improvement.
Differential Chemokine Production during Acute and Chronic Relapsing EAE
We previously demonstrated that MIP-1a production in the CNS correlated with the development of acute EAE (Karpus and others 1995) whereas MCP-1 expression in the CNS correlated with increasing relapsing disease severity (Kennedy and others 1998). EAE was induced by intravenous adoptive transfer of PLP 139-151-activated T cells as a means to model the effector arm of the demyelinating disease course and ask questions about the role of chemokines in CNS leukocyte infiltration. After EAE induction, the mice were monitored for the development of clinical disease. Spinal cord tissue from naYve and preclinical mice was analyzed for chemokine expression. Chemokine-specific ELISA was used to detect the presence of the CC chemokines MIP-1a, MIP- 1b, RANTES, and MCP- 1 and the CXC chemokine MIP-2. As summarized in Table 1, we round that there was no detectable chemokine expression in the CNS of naive mice. In addition, there was very little detectable chemokine protein in the CNS of preclinical mice. However, during acute EAE, both MIP-1a and RANTES were produced in the CNS in relatively substantial quantities. The difference between the two chemokines was that increasing MIP-1a production showed a correlation with increasing acute EAE severity (Karpus and others 1995) whereas increasing RANTES production did not correlate as well with clinical disease development. MIP-1b expression was detectable, albeit at much lower levels than either MIP-1a or RANTES. We could never detect the presence of the CXC chemokine MIP-2 in preclinical, acute, remission, or relapsing EAE. Both MIP-1a and RANTES production remained elevated in the CNS of mice that had gone into clinical remission, and the expression of MCP-1 was just beginning to become detectable (Kennedy and others 1998).
Because the SJL mouse EAE model displays a relapsing-remitting clinical presentation, we also examined CNS chemokine expression during the relapsing phase of disease. A notable difference between acute and relapsing EAE was that CNS MCP-1 production, which was not detectable during acute disease, correlated with increasing relapsing EAE severity (Table I). In comparison, MIP-1a and RANTES levels remained elevated during relapses but did not directly correlate with increasing relapse severity. These data suggest that there was differential chemokine production during the course of EAE and that it was specific to the affected CNS inflammatory site.
Biological Role of Chemokines during EAE
Because MIP-1a production in the CNS correlated with acute clinical disease severity and MCP-1 production in the CNS correlated with relapsing disease severity, we wanted to test the biological significance of these observations in vivo. The approach we used was similar to that of Lukacs and others (1993, 1995), wherein antibodies directed against different chemokines were administered at varying time points during EAE. The summary of our results is shown in Table 2. Anti-MIP-1a administration before acute disease development inhibited both acute and relapsing EAE (Karpus and others 1995). Additionally, we observed that anti-MIP-1a treatment resulted in the reduction of mononuclear infiltrating cells in the CNS, including both antigen-specific T cells and antigen-nonspecific bystander T cells and macrophages. However, when administered during remission, before relapsing disease development, anti-MIP-1a had no effect on clinical relapses (Kennedy and others 1998) or the T cell composition of the mononuclear cell infiltrate (unpublished observations).
Because RANTES expression was detected in the CNS during the course of both acute and relapsing EAE, we investigated whether anti-RANTES treatment had an effect on the clinical disease course. Anti-RANTES treatment at the time of disease induction, during acute disease presentation, and during relapsing disease did not have an effect on the clinical progression of EAE (Kennedy and others 1998). Therefore, we speculate that RANTES expression in the CNS may be a result of the inflammation rather than the cause of disease induction and/or progression. Likewise, anti-MIP-2 administration at any time during the course of EAE had no effect on modulating the clinical disease course. As opposed to the anti-RANTES experiments, anti-MIP-2 administration did not reduce clinical severity because MIP-2 is not significantly expressed during the course of EAE. We have not assessed the role of anti-MIP-1b during the course of EAE because the chemokine content in the CNS has never been correlated with any phase of the disease course.
We tested the biological role of MCP-1 expression using a similar strategy. The surprising observation was that administration of anti-MCP- 1 before the onset of acute clinical EAE had no effect on decreasing acute disease or relapses (Karpus and others 1995). This regimen also did not affect the degree of CNS mononuclear cell infiltration. The explanation for these findings is most likely related to the lack of MCP-1 expression during the induction and acute phase of EAE (Table 1). However, when anti-MCP-1 was administered during the remission phase of disease, before the appearance of relapsing symptoms, the severity of clinical relapsing EAE was significantly reduced (Kennedy and others 1998). Additionally, anti-MCP-1 administered after acute EAE but before the relapsing phase of disease resulted in a decreased percentage of macrophages infiltrating the CNS (Kennedy and others 1998). The decrease in infiltration of the cell type responsible for demyelination may explain the accompanying reduction in clinical severity mechanistically. Collectively, these data suggest that the different phases of EAE, acute and relapsing, are regulated by the local differential production of chemokines.
Discussion
Chemokines were originally described as molecules that induce directional migration of leukocytes (Oppenheim and others 1991), increase affinity of integrin receptor ligand binding (Lloyd and others 1996), increase integrin expression (Vaddi and Newton 1994), increase macrophage function (Fahey and others 1992), and regulate hematopoeisis (Broxmeyer and others 1993). Recently, a role for chemokine-induced costimulation of T cell activation has been demonstrated that has significantly altered the thinking about the biological activities of this family of molecules (Taub and others 1996). Our recent observations that chemokines can differentially influence antigen-specific T cell effector mechanisms (Karpus and others 1997) also suggest a more broad role for chemokines in the regulation of the immune response.
In the present report, we have summarized our recent results showing that MIP-1a regulates the immunopathogenesis of EAE during acute disease and MCP-1 regulates the relapsing phase of disease (Tables 1 and 2). This differential regulation appears to result from MIP-1a being produced primarily by infiltrating macrophages and T cells whereas MCP-1 is produced primarily by CNS resident astrocytes (Glabinski and others 1997; Ransohoff and others 1993). The reason for this differential chemokine production is unknown, but it is possibly related to the nature of the CNS mononuclear cell infiltrate at a particular time during the disease course. For example, during the acute phase of disease, the CNS mononuclear cell infiltrate is rich with T cells whereas during the relapsing phase of disease, mononuclear cells with the macrophage/monocyte immunophenotype are more prevalent (Kennedy and others 1998). Kuchroo and others (1993) have reported that EAE-inducing T cells express MIP-1a Subsequent to the initial T cell infiltration is a macrophage infiltration that could be responsible in part for additional MIP-1a production (Glabinski and others 1997). Therefore, it is likely that both infiltrating T cells and macrophages are responsible for the initial production of MIP-1a in the CNS. As disease progresses, there is a CNS infiltration by bystander T cells without self-antigen specificity (Cross and others 1990). Demyelination is a hallmark of EAE and results from activated macrophages in the CNS phagocytosing myelin (Cammer and others 1978). In spite of this severe structural damage as a result of a massive mononuclear cell infiltration, clinical disease spontaneously remits. The mechanism of this remission is not well understood, but it has been suggested that either antiinflammatory cytokine production in the CNS (Kennedy and others 1992) or antigen-specific T cell apoptosis (Pender and others 1991) may be responsible.
After remission, a second phase of clinical disease ensues. The induction of this relapsing clinical disease has been hypothesized to involve in situ recognition of newly released self-antigens by naive T cells (McRae and others 1995). Whether these new antigen-specific cells are recruited from outside the CNS or are in the target organ at the time of clinical relapse is not well understood. Chemokines could play a functional role in the secondary recruitment of these new T cells responsible for relapsing disease.
How chemokines exert these differential effects on T cells is not known, however, one possibility is through integration of signaling pathways through the differential expression of chemokine receptors. MIP-1a is a ligand for multiple murine CC chemokine receptors (Boring and others 1996; Gao and Murphy 1995; Hoogewerf and others 1996), and MCP- 1 is a ligand for murine CCR2 (Boring and others 1996). It is possible that T cells express multiple CC chemokine receptors differentially, and binding MIP-1a leads to a different signaling outcome than binding MCP-1. The regulation of chemokine receptor expression is not well understood. Chemokine receptors signal through the mitogen activated protein-kinase pathway whose downstream events can lead to gene transcription (Jones and others 1995). Therefore, it is possible that the MIP-1a-induced Th1 differentiation results from signals generated by a distinct pathway compared with the Th2 differentiation driven by MCP- 1. Indeed, differential signaling has been reported for the IL-8A and IL-8B receptors (Damaj and others 1996) when IL-8 and GRO-a were the respective ligands. We are currently testing these hypotheses in an effort to find the direct mechanism of chemokine-induced T cell differentiation and regulation of EAE pathogenesis.
These observations emphasize the multifactorial role of chemokines during inflammatory immune responses. Originally shown to be chemotactic regulators, chemokines can now be thought of as molecules that regulate the developing as well as the ongoing immune response. All of the chemokines' precise roles during inflammatory responses are not known; however, they present as regulatory targets for manipulation during developing and progressing immune responses, including autoimmune disease and allergic reactions. The EAE model has been used to study the role of chemokine-mediated mononuclear cell migration. In Table 3, the variety of observations on chemokine expression and regulation of a number of different clinical forms of EAE are summarized. What is easily discernible is that chemokine expression patterns are not identical for the different clinical forms of EAE, which may reflect the complexity of chemokine expression patterns in MS patients as well. Our results, as well as those of others, demonstrate that EAE is an appropriate animal system to study chemokine-regulated mononuclear cell trafficking as models for human inflammatory diseases.
1 Abbreviations used in this article: CFA, complete Freund's adjuvant; CNS, central nervous system; CSF, cerebrospinal fluid; EAE, experimental auto-immune encephalomyelitis; ELISA, enzyme linked immunosorbant assay; IL, interleukin; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; MS, multiple schlerosis; RANTES, regulated upon activation normal T cell expressed and secreted; Th, T-helper; VLA-4, very late antigen-4;
Acknowledgments
The authors thank Drs. Richard Ransohoff, Nicholas Lukacs, Robert Strieter, and Steven Kunkel for their support and helpful discussions. This research was supported by National Institutes of Health grants NS34510 and AI35934.
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Table 1 CNS chemokine expression
| Clinical presentation | MIP-1ab | MIP-1bb | RANTESb | MCP-1b | MIP-2b |
| Naive | <0.05 | <0.05 | <0.05 | <0.05 | <0.05 |
| Preclinical | <0.05 | <0.05 | 0.8 | <0.05 | <0.05 |
| Acute | 5.2 | 0.1 | 1.6 | 0.6 | <0.05 |
| Remission | 5.2 | 0.1 | 1.8 | 1.0 | <0.05 |
| Relapse | 5.6 | 0.3 | 2.4 | 5.4 | <0.05 |
a Chemokine levels were determined by enzyme-linked immunosorbent assay as described in the text (see Methods).
b MIP-1a, macrophage inflammatory protein-1a; MIP-1b, macrophage inflammatory protein-1b; RANTES, regulated upon activation normal T cell expressed and secreted; MCP-1, monocyte chemotactic protein-I; MIP-2, macrophage inflammatory protein-2.
Table 2 Effects of antichemokine treatment on clinical disease presentation
| Time of treatmentb | MIP-1ac | MIP-1bc | RANTESc | MCP-1c | MIP-2c | IP-10c |
| Preclinical | ¯d | NEc | NE | NE | NE | ¯ |
| Acute disease | ¯ | NDc | NE | NE | ND | ND |
| Remission | NE | ND | NE | ¯ | ND | ND |
| Relapsing disease | NE | ND | NE | NE | ND | ND |
b Antibodies were administered to mice induced to develop experimental autoimmune encephalomyelitis by the adoptive transfer of encephali-togenic T lymphocytes. The treatment regimens were either before disease development (preclinical), at the peak of initial disease (acute disease), when the mice had entered a time period of remission from disease signs (remission), or during the peak of relapsing disease (relapsing disease).
c MIP-1a macrophage inflammatory protein-1a; MIP-1b, macrophage inflammatory protein-1b; RANTES, regulated upon activation normal T cell expressed and secreted; MCP-1, monocyte chemotactic protein-1; MIP-2, macrophage inflammatory protein-2; IP-10, gamma interferon inducible protein-10; NE, no effect on clinical disease progression; ND, not done.
d Down arrow indicates that a particular treatment decreased clinical disease severity.
Table 3 Summary of chemokine expressiona in various models for experimental autoimmune encephalomyelitis
| Disease induction | MCP-1b | RANTESb | MIP-1ab | IP-10b | MCP-1b | RANTES | MIP-1a | IP-10 | MCP-1 | RANTES | MIP-1a | IP-10 | |
| Strainc | Antigend | Initial attack | Remission | Relapse | |||||||||
| SJL | PLP139-151/CFA | ++ | + | +++ | +++ | + | + | + | + | ++ | ++ | +++ | +++ |
| PLP139-151 T cells | ± | + | +++ | NDb | ± | + | +++ | ND | +++ | + | +++ | ND | |
| SWxJ | PLP139-151/CFA | +++ | ++ | +++ | +++ | _ | _ | _ | _ | ++ | ++ | ++ | ++ |
| BALB/ GKO PL/J | MBP/CFA | +++ | ND | ND | - | ND | ND | ND | ND | ND | ND | ND | ND |
| PLP43-64/CFA | +++ | ND | +++ | ND | ++ | ND | ++ | ND | Nonrelapsing disease model | ||||
| Lewis | MPB/CFA | +++ | ND | ND | ND | - | ND | ND | ND | Nonrelapsing disease model | |||
| MBP T cells | +++ | + | +++ | ++ | ND | ND | ND | ND | Nonrelapsing disease model | ||||
| S100b T cells | + | + | + | + | ND | ND | ND | ND | Nonrelapsing disease model | ||||
b MCP-1, monocyte chemotactic protein-1; RANTES, regulated upon activation normal T cell expressed and secreted; MIP-1a macrophage inflammatory protein-1a; IP-10, gamma interferon inducible protein-10; ND, not done.
c SWxJ, (SWR xSJL)F1; BALB/GKO, IFN-g/- mice on BALB/c background.
d PLP139-151/CFA, proteolipid protein 139-151 peptide in CFA; PLP43-64, proteolipid protein 43-64 peptide in CFA; MBP, myelin basic protein; S100b, astrocyte calcium binding protein
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