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(Investigative Ophthalmology and Visual Science. 2001;42:2894-2903.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Expression of CC Chemokines and Their Receptors in the Eye in Autoimmune Anterior Uveitis Associated with EAE

Grazyna Adamus1, Maria Manczak1 and Michal Machnicki1,2

1 From the Neurologic Sciences Institute, Oregon Health and Science University, Portland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the pattern of expression of CC chemokines and their receptors in the eyes of Lewis rats and to establish their role in autoimmune anterior uveitis (AU) associated with experimental autoimmune encephalomyelitis (EAE).

METHODS. EAE/AU was induced in Lewis rats with myelin basic protein in complete Freund’s adjuvant (CFA). The rats were scored for the development of clinical EAE and AU. The expression of CCL5/regulated on activation normal T-cell expressed and secreted (RANTES), CCL2/monocyte chemotactic protein (MCP)-1, CCL3/macrophage inflammatory protein (MIP)-1{alpha}, and CCL4/MIP-1ß and their receptors was examined at the preclinical stage, onset, peak, and recovery by RT-PCR and ELISA. EAE/AU rats were treated with neutralizing polyclonal antibodies against CCL3/MIP-1{alpha}, CCL4/MIP-1ß, CCL2/MCP-1, and CCL5/RANTES and tested for the suppression of onset of clinical AU and EAE. The control group received normal rabbit IgG at the same dose.

RESULTS. The gene expression of those chemokines was upregulated concurrently with symptom onset of EAE/AU and correlated with the intensity of inflammatory changes in the eye and central nervous system (CNS). The highest expression of CCL4/RANTES, CCL2/MCP-1, and CCL3/MIP-1{alpha} in the eye was detected at onset of clinical uveitis, whereas CCL4/MIP-1ß was elevated at the peak of AU. The expression of chemokine receptors associated with T-helper (Th)1-type response, CCR1 and CCR5, correlated with their appropriate ligands and was the highest at the peak of AU, whereas CCR2, the receptor for CCL2/MCP-1, was present before the onset of the disease. Treatment of anti-MIP-1ß and anti-MCP-1 significantly delayed the onset and shortened the duration of AU and EAE. Anti-MIP-1{alpha} treatment had no effect on clinical EAE but inhibited the clinical signs of AU. Although CCL5/RANTES expression was observed during the entire course of the disease, anti-RANTES treatment had no effect on clinical disease progression.

CONCLUSIONS. The data suggest that CCL2/MCP-1, CCL3/MIP-1{alpha}, and CCL4/MIP-ß contribute to the recruitment of inflammatory cells into the eye and CNS and to disease activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Uveitis is an inflammatory eye disease characterized by intraocular infiltration of various populations of leukocytes. In experimental autoimmune uveitis (AU) the migration of leukocytes plays an essential role in the initiation and development of disease. Migration of lymphocytes from the blood to the site of antigenic challenge, including the eye, is controlled by a series of steps involving adhesion molecules and chemoattractants. The role of adhesion molecules in eye inflammation has been investigated, whereas the involvement of chemokines and their receptors in eye inflammation has not been extensively studied.1 In the pathogenesis of AU, T-cell–mediated autoimmune responses are considered to play a crucial role in the initiation of disease. Early infiltrating T helper (Th)1 cells are key to later inflammatory events, and T cells themselves may play an active role in the recruitment of other leukocytes into inflamed tissues over the course of disease.

Chemokines are divided into two major subfamilies based on the spacing of the first pair of N-terminal cysteine residues. This molecular subdivision generally correlates with function. The CXC chemokines usually recruit neutrophils, whereas CC chemokines tend to attract monocytes. The CC chemokines are involved in the pathogenesis of immune-mediated inflammation through monocyte-macrophage activation and recruitment.2 3 The CC chemokines, such as CCL3/macrophage inflammatory protein (MIP)-1{alpha}, CCL4/MIP-1ß, and CCL5/regulated on activation normal T-cell expressed and secreted (RANTES), are efficient chemoattractants for Th1 but not for Th2.4 CCL5/RANTES, a chemoattractant for macrophages and mast cells, also attracts memory T cells and NK cells.5 Another CC chemokine, CCL2/monocyte chemotactic protein (MCP)-1, has an effect on both Th1 and Th2 cells. The receptors for chemokines also comprise two major groups: CC receptors (CCR), which bind CC chemokines, and CXC receptors (CXCR), which bind CXC chemokines.6

Studies of human uveitis have shown that CXCL8/IL-8, CXCL10/IP-10 (interferon {gamma}-inducible protein-10), CCL2/MCP-1, CCL5/RANTES, and CCL4/MIP-1ß are significantly increased during the active stages of AU and correlate with the clinical severity of disease. These chemokines probably play a critical role in leukocyte recruitment in acute AU7 ; however, the mechanism by which these cells traffic to the eye and accumulate before and during clinical disease is not well understood. It is a multistep process that includes entry of activated T cells from peripheral lymphoid organs into the eye, recognition of endogenous antigen in the eye, chemotactic-induced recruitment of lymphocytes into the tissue, and disease phase.8 9 10 11 T cell ability to secrete chemokines induces the accumulation of other mononuclear cells and nonspecific T cells in the perivascular space. The chemokines produced at the sites of inflammation are likely to play a major role in the recruitment of particular cell types that infiltrate and participate in the pathologic lesions. They are also important for the selective migration of particular T-cell subsets.12 Moreover, chemokines produced by other cells including tissue-resident cells can regulate the movement of T lymphocytes. Therefore, the relation between the development of disease and chemokine production is important in the pathogenic process.

To investigate the involvement of chemokines in eye inflammation, we used the rat experimental autoimmune encephalomyelitis (EAE) model. Rats injected with myelin basic protein (MBP) showed development of EAE and AU.13 14 The target autoantigens in EAE and possibly in AU include myelinated neurons found within the central nervous system (CNS) and the iris, respectively. CD4+ T cells of Th1 phenotype (IL-2 and IFN-{gamma}-producers) mediate EAE as well as AU in response to encephalitogenic-uveitogenic peptides. Those MBP-specific T cells are needed to initiate the induction of uveitis, followed by the recruitment of polymorphonuclear leukocytes and mononuclear leukocytes.13 15 T cells found in the iris-ciliary body during the acute phase share characteristics with the T cells from the spinal cord.13 16

Previously, we have shown that CCL2/MCP-1 contributes to the initial recruitment of inflammatory cells to the eye and CNS.16 The CCL2/MCP-1 expression was detected at the preclinical phase in the iris-ciliary body and lumbar spinal cord and increased during the course of EAE/AU. Mononuclear infiltrating cells, endothelial cells, and astrocytes of the CNS were identified as a source of CCL2/MCP-1 by in situ hybridization. Kinetics of expression of Th1-type cytokines, IL-2 and IFN-{gamma}, was in agreement with the expression of CCL2/MCP-1.16 In view of the fact that CC chemokines have a potential to influence the migration of T cells and monocytes across the blood–ocular barrier during inflammation and that they accumulate in the eye, we examined the role CCL5/RANTES, CCL2/MCP-1, CCL3/MIP-1{alpha}, and CCL4/MIP-1ß, members of the CC superfamily of chemokines, in the pathogenesis of MBP-induced AU in Lewis rats. In the present studies, we investigated the expression of those chemokines in the iris-ciliary body and spinal cord of the rat with actively induced EAE/AU. We also assessed the potential role of those chemokines in clinical disease development by application of anti-chemokine antibodies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Lewis rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN), 6 to 8 weeks old, were used in these studies. The rats were housed at the Oregon Health Sciences University Animal Care Facility according to institutional and federal guidelines. All animal experimentation procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the institutional animal committee.

Induction and Assessment of AU and EAE
EAE was induced by subcutaneous injection of 25 µg guinea pig myelin basic protein (MBP) in complete Freund’s adjuvant (CFA) supplemented with 150 µg Mycobacterium tuberculosis strain H37Ra (Difco, Detroit, MI). The rats were assessed daily for changes in clinical signs according to the following clinical rating scale: 0, no signs; 1, limp tail; 2, hind leg weakness and ataxia; 3, paraplegia; and 4, paraplegia with forelimb weakness and moribund condition. Rats were also scored for clinical signs of ocular inflammation by biomicroscopy according to the following scale: 0, normal; 1, slight iris vessel dilation and thickened iris stroma, a few scattered inflammatory cells, or both; 2, engorged blood vessels in the iris, abnormal pupil contraction, and occasional vitreous cells; 3, hazy anterior chamber, and decreased red reflex; and 4, marked vitreous cells.

Preparation of RNA and cDNA
The iris-ciliary tissue was dissected from diseased and normal eyes at various times during the course of EAE/AU, along with the lumbar section of the spinal cord. Tissues were stored at -80°C before RNA extraction. Total RNA was extracted from both tissues with an extraction reagent (TRIzol; Life Technologies, Gaithersburg, MD). All RNA preparations were treated with RNAase-free DNase. RNA concentration was determined by spectrophotometry. First-strand cDNA was prepared from 5 µg total RNA, each sample was annealed for 5 minutes at 65°C with 300 ng oligo(dT)12-18 and reverse transcribed to cDNA using 80 U Moloney murine leukemia virus reverse transcriptase (MMLV-RT) per 50 µl reaction for 1 hour at 37°C. The reaction was stopped by heating the sample for 5 minutes at 90°C.

Chemokine Expression by Touchdown RT-PCR
PCR was performed on the resultant cDNA from each sample with specific primers for rat cytokines, chemokines, and GAPDH (Table 1) . The amplification was performed with a thermocycler (Ericomp Inc., San Diego, CA). The 25-µl reaction mixture consisted of 2.5 µl cDNA, 0.3 µM of sense and antisense primers, 200 µM of each deoxynucleotide, 1.3 U Taq polymerase, 1.5 mM MgCl2, and 1x Taq polymerase buffer. Conditions for cytokine amplification were as follows: denaturation 1 minute at 94°C and elongation 3 minutes at 72°C. The annealing temperatures for cytokines and chemokines were as follows: from 62°C to 42°C was designed for MCP-1, RANTES, MIP-1ß IL-10, and IFN-{gamma}, declining at 1°C increments and followed by 20 cycles at 50°C. The annealing temperature for IL-2, IL-10, CCR2, and IL-4 was from 67°C to 50°C, declining at 1°C increments and followed by 21 cycles at 55°C. The annealing temperature for MIP-1{alpha}, CCR1, CCR2, CCR3, and CCR5 was from 67°C to 50°C, declining at 1°C increments followed by 21 cycles at 60°C. At the end of amplification, the reaction mixture was heated for 10 minutes at 72°C and then cooled to 4°C. A 10-µl sample of each PCR product was separated by gel electrophoresis on 2% agarose containing ethidium bromide and then analyzed under UV light against the DNA molecular markers. Ethidium bromide–stained bands were photographed by a digital camera for densitometry (Digital 1D Science; Eastman Kodak, Rochester, NY). The sum of intensity and band area was determined for each PCR product and the housekeeping gene GAPDH.


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  		Table 1. Sequences of Rat Cytokine, Chemokine, and GAPDH Primers Used in RT-PCR

 
Rat Chemokine ELISA
Chemokines MCP-1 and RANTES were determined in the target tissue extracts by specific sandwich ELISA using kits (BioSource International, Camarillo, CA). Spinal cords and eyes were removed from diseased rats. For each experiment, three to five MBP-injected rats were used. Freshly isolated whole spinal cords and whole eyes were homogenized in 1 ml sterile PBS by sonication. The extracts were then clarified by centrifugation at 400g for 10 minutes. Collected supernatants were immediately frozen at -80°C. A 50- to 100-µl sample of each supernatant was used for tests. Optical density was determined at A450 with a plate reader (Microplate Reader; BioRad, Richmond, CA). Chemokine concentration was determined from standard curves using recombinant standards supplied by the manufacturer. For determination of MIP-1{alpha}, the ELISA kit for mouse MIP-1{alpha} (R&D Systems, Minneapolis, MN) was used. The standard curve was prepared using a rat MIP-1{alpha} recombinant protein (PeproTech, Inc., Rocky Hill, NJ).17

Isolation of Iris Cells
Cells infiltrating the iris and ciliary body were prepared from eyes isolated from MBP-injected rats. The eyes were removed and the iris-ciliary tissue was microdissected and placed in RPMI medium containing 10% fetal bovine serum and 1 mg/ml collagenase for 2 hours. A single-cell suspension was prepared by pipetting and filtering through a cell strainer. Red blood cells were lysed, washed three times, counted, and frozen for RNA isolation. Spinal cord mononuclear cells were prepared and purified on a density gradient (Percoll; Pharmacia-Upjohn, Uppsala, Sweden), according to Cohen et al.18

Treatment of EAE/AU with Anti-chemokine Antibodies
Lewis rats were divided into five groups. Each group of four rats received five doses of 100 µg rabbit polyclonal neutralizing antibodies against the following chemokines: MCP-1, RANTES, MIP-1{alpha}, and MIP-1ß (Torrey Pines Biolabs, Inc., San Diego, CA). The antibodies were administered on days 0, 3, 6, 9, and 12 after injection with MBP and CFA. The control group was untreated or received normal rabbit IgG at the same dose and times. The experiment was repeated three times. On day 18, peak of AU, rats were killed, and the eyes and spinal cords were collected for chemokine assessment by RT-PCR.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pattern of Chemokine mRNA Expression in the Eye
MBP-induced AU is characterized by infiltration of CD4 and CD8 T cells, monocytes-macrophages, and granulocytes.15 In our study, we selected those chemokines that attract T cells to the inflammation site. The expression of four CC chemokines, CCL5/RANTES, CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL2/MCP-1, was examined. Our initial goal was to determine whether the eye inflammation correlates with the presence of chemokines in the eye. To determine the overall chemokine expression during the course of ocular disease, the iris-ciliary body tissues and spinal cord were collected at the preclinical stage (day 6), onset (day 11), AU peak (day 18), and full recovery (day 35). Clinical assessments of EAE and AU were performed over a 40-day period. On each day of the study three to five rats were used. The total level of chemokine expression was determined by RT-PCR, using iris-ciliary body or spinal cord RNA. For controls, the tissues from naïve Lewis rats were obtained to determine the background expression for each chemokine tested.

Chemokine mRNA expression was increased in rats with EAE/AU and correlated with clinical ocular and neurologic symptoms in comparison with untreated control animals, but each represented a unique pattern. Densitometry was used to measure the products of PCR from normal rat eyes relative to that of GAPDH (Fig. 1) . All chemokine transcripts were detected at the preclinical phase before the onset of clinical signs. Therefore, they seem to be important in the recruitment of cells to the eye. The expression of CCL2/MCP-1 and CCL5/RANTES was more abundant at the mRNA level than CCL3/MIP-1{alpha} and CCL4/MIP-1ß in both organs, although there were differences in the transcript expression for CCL3/MIP-1{alpha} and CCL4/MIP-1ß. CCL3/MIP-1{alpha} was expressed at the highest level before onset of clinical signs of AU, whereas CCL4/MIP-1ß transcript was the highest at the peak of AU. No chemokine expression was detected in tissues without inflammation, suggesting that the cells migrate to the eye and secrete chemokines, which subsequently amplifies the inflammatory process.



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  		Figure 1. Densitometric analysis of MIP-1{alpha}, MIP-1ß, MCP-1, and RANTES mRNA expression in the iris-ciliary body and spinal cord by RT-PCR. Tissues from MBP-immunized rats were dissected at the preclinical stage, onset, peak and recovery and pooled for RNA extraction and RT-PCR analysis. Data are presented as the mean ± SD in five rats. The top graph shows a typical time course of AU and EAE in Lewis rats injected with MBP.

 
To test the hypothesis that the inflammation in the CNS and in the eye is differentially regulated by CC chemokine expression, we examined the chemokine production in the spinal cord over the entire course of AU. All chemokines tested were detected in the spinal cord before clinical symptoms of EAE except CCL4/MIP-1ß (Fig. 1) . There was a trend for the highest expression to be at the onset and peak of EAE, and then the mRNA levels were reduced with the resolution of clinical symptoms.

CCL5/RANTES, CCL2/MIP-1{alpha}, and CCL2/MCP-1 Protein Levels in the Eye
To determine whether mRNA expression correlates with the production of chemokine proteins we used ELISA to assay CCL5/RANTES, CCL3/MIP-1{alpha}, and CCL2/MCP-1 during the course of the ocular disease. Extracts from whole eyes and spinal cords were used. After the immunization, typical signs of EAE/AU developed in the rats (Fig. 2) . The increase in CCL5/RANTES and CCL2/MCP-1 was observed before onset of clinical signs in both organs. The levels of CCL2/MCP-1 and CCL5/RANTES remained elevated through recovery in the eye. Moreover, the protein level in the eye correlated with the expression of mRNA (Fig. 1) . In contrast, the level of CCL5/RANTES and CCL2/MCP-1 in the spinal cord decreased significantly to the background level after the clinical EAE subsided. In the case of CCL3/MIP-1{alpha}, we measured only small amounts, 20 pg per organ, at the onset of EAE and AU. This low measured level of MIP-1{alpha} was most likely due to the low sensitivity of the assay, rather than to the amounts of MIP-1{alpha} produced.



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  		Figure 2. Levels of MCP-1 and RANTES in eye and spinal cord during the course of AU and EAE determined by ELISA. Chemokine concentrations were determined from standard curves, by using recombinant MCP-1 or RANTES.

 
Expression of CC Chemokine Receptor Genes
Chemokines act through specific membrane receptors. We examined the expression of five CC chemokine receptors in the iris-ciliary body and spinal cord of Lewis rats during EAE/AU, including Th1-associated CCR5, and Th2-associated CCR3 and CCR4. Densitometry was used to measure the products of PCR from rat eyes relative to that of the housekeeping gene GAPDH (Fig. 3) . We detected that CCR3 was constitutively expressed in the normal iris tissues and CCR1, -2, -4, and -5 were expressed in normal spinal cords collected before the immunization with MBP. All CC chemokine receptor transcripts were detected as early as the onset of EAE/AU, although the pattern of gene expression was different (Fig. 3) . CCR3 and -5, the receptors for CCL5/RANTES, CCL3/MIP-1{alpha}, and CCL4/MIP-1ß, were significantly upregulated and maximized at the peak of AU, suggesting the amplification of the Th1 response. CCR2, the major receptor for CCL2/MCP-1, was detected at onset. In the spinal cord, all receptors showed a trend to peak at the height of EAE and were downregulated with recovery. In general, the expression of receptors correlated with the appropriate chemokines; however, the expression of CCR1 and -5 was far more abundant than CCR2 and -4 at the mRNA level in the iris-ciliary body.



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  		Figure 3. Comparison by RT-PCR of CC chemokine receptor expression in the iris-ciliary body and spinal cord during the course of EAE and AU. Tissues from MBP-immunized rats (five rats per experimental group) were dissected and pooled for RNA extraction and RT-PCR analysis. Data represent densitometric analyses of PCR products in comparison with the housekeeping gene GAPDH.

 
Expression of Chemokines and Chemokine Receptors in the Eye-Infiltrating Cells
To determine the chemokine profile of cells infiltrating the inflammation site, RNA was prepared from cells isolated from the iris-ciliary body after onset of clinical signs of MBP-induced AU. The cell population isolated from the iris and spinal cord included CD4+, CD8+ T cells and monocytes. The pattern of upregulated cytokines and chemokines observed in those cells was in agreement with a Th1 response. A significant production of mRNA for IL-2 and IFN-{gamma}, as well as CCL5/RANTES, CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL2/MCP-1, was detected (Fig. 4) . No IL-4 was detected in contrast to the cells from the spinal cord, where the message for IL-4 was detected. All receptors were expressed on the infiltrating cells from the eye and CNS (Fig. 4) . The transcript CCL2/MCP-1 receptor, CCR2, was present in the eye and spinal cord over the course of EAE/AU.



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  		Figure 4. Profile of cytokine, chemokine, and chemokine receptor expression in cells isolated from the eye and spinal cord after onset of clinical signs. mRNA expression was assessed by RT-PCR analysis. PCR products were separated on 2% agarose gels.

 
Effect of Anti-chemokine Antibodies on the Clinical Course of EAE/AU
To address the role of chemokines in the development of initial signs of acute EAE/AU, rats were treated with neutralizing polyclonal antibodies against CCL3/MIP-1{alpha}, CCL4/MIP-1ß, CCL2/MCP-1, and CCL5/RANTES on days 0, 3, 6, 9, and 12. The control group received normal rabbit IgG at the same dose. A positive control group remained untreated. The rats were scored for the development of EAE and AU for 18 days after MBP immunization. The summary of results is presented in Table 2 . MBP-immunized animals, which were treated with control antibodies or were not treated in any way, had a high cumulative score, indicating that they developed AU and EAE. Anti-RANTES had no significant effect on amelioration of EAE and AU symptoms. Anti-MIP-1{alpha} treatment had no effect on clinical EAE but inhibited the clinical signs of AU. Rats developed AU, but the mean disease severity was always lower than in control animals at the same time point. Anti-MCP-1 antibody treatment delayed the onset by 2 days with lower severity (average AU score, ~1.1) untreated control animals, suggesting its role in the recruitment of cells to the eye. Similarly, anti-MCP-1 influenced the onset and severity of EAE. We used antibodies from two different commercial sources with the same effect. The most striking effect on EAE and AU was the treatment with anti-MIP-1ß antibody. The protection from AU and EAE lasted as long as antibodies were administered. Two to 3 days after the last injection, EAE/AU developed. Nonetheless, treatment of anti-MIP-1ß delayed onset and shortened the duration of disease to 7 days (compared with 15 days in untreated rats), but it did not dramatically suppress clinical signs of AU. This treatment also produced a significant change in the course of EAE.


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  		Table 2. Treatment of MPB-Injected Rats with Anti-Chemokine Antibodies

 
To confirm that antibody treatment had an effect on levels of chemokines in the eye, we examined mRNA expression of inflammatory cytokines and chemokines neutralized in the iris-ciliary body and in the periphery. In the case of the most effective treatment with anti-MIP-1ß antibody we observed a marked reduction in mRNA expression for cytokines (IL-2, IFN-{gamma}, IL-10, and TNF-{alpha}) in the iris-ciliary body (Fig. 5A) and MIP-1ß in both target organs (Fig. 5B) . Treatment with anti-MIP-1{alpha} and anti-MCP-1 antibodies resulted in decreased levels of mRNA for chemokine and cytokine to half the level in control eyes (Figs. 5A 5B) . The level of mRNA for RANTES remained unchanged in the eye and periphery but decreased in the spinal cord in anti-RANTES–treated animals. In the spleen and lymph nodes we did not detect any marked reduction in the mRNA chemokine expression compared with control untreated animals (Fig. 5B) .



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  		Figure 5. Effect of antichemokine treatment on the expression of Th1 and -2 cytokines and chemokines and T-cell–specific responses. (A) Profile of cytokine expression in the iris-ciliary body of control and anti-chemokine antibody–treated animals. (B) Profile of chemokine expression in the lymphatic and target organs of control and anti-chemokine antibody–treated animals. (A, B) Tissues from MBP-immunized (A, control) and antibody-treated rats (B, four rats per experimental group) were dissected and pooled for RNA extraction and RT-PCR analysis. Data represent densitometric analysis of PCR products in comparison with the housekeeping gene GAPDH. (C) MBP proliferative responses of T cells from lymph nodes and spleens of control and anti-chemokine–treated rats. Mean values ± SD of four rats per experiment are shown.

 
In the spinal cords, the message for CCL4/MIP-1ß was not detected in antibody-treated animals (Fig. 5B) . No decrease in CCL5/RANTES, CCL2/MCP-1, and CCL3/MIP-1{alpha} transcripts was observed in the spinal cords of rats treated with anti-MCP-1 or MIP-1{alpha} antibodies, respectively.

To test the possibility that the administration of antibody may affect the activation of MBP-specific T cells we measured the proliferative activities against MBP of T cells obtained from draining lymph nodes or spleens. Spleen T cells showed proliferation in response to MBP to be half that in untreated rats; however, proliferative responses of lymph node cells were not altered (Fig. 5C) .


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In EAE/AU, inflammation is associated with infiltration by T cells and monocytes and is restricted to two organs, the eye and spinal cord. In these studies we have shown for the first time the expression of chemokines in the eye and spinal cord over the entire course of disease. We showed that all four chemokines—CCL2/MCP-1, CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES—that act toward T cells and monocytes were upregulated simultaneously before onset of AU, and their expression correlated with the intensity of inflammation in the eye and CNS. The early expression of those chemokines in both organs provides a mechanism for the later influx of macrophages and T cells. The expression of CC chemokines involved in Th1 cell type attraction correlated with the expression of their receptors (CCR5 and -1) in the eyes during the course of AU, which suggests a role for these chemokines in the pathogenicity of uveitis. In particular, CCL4/MIP-1ß and CCL2/MCP-1 seem to play an important role in the recruitment of inflammatory cells into the eye and CNS. Administration of antibodies delayed onset and suppressed the development of disease. Anti-MCP-1, but not anti-MIP-1{alpha} treatment decreased clinical severity of EAE. However, that the anti-chemokine treatment did not completely suppress clinical disease, but delayed onset or reduced severity, suggests that not one, but many chemokines together promote cell infiltration of the inflammatory site. We also do not exclude the possibility that higher doses of antibodies may be required to completely block those chemokines.

We do not directly know where the neutralization of endogenous chemokines, such as CCL2/MCP-1 or CCL4/MIP-1ß, occurred, in the inflamed tissue (eye or CNS) or in the periphery. Our data suggest that the inhibition of their activity starts in the periphery and occurrs in the target organs. The treatment probably affected the priming step during antigenic stimulation, which in consequence influenced the levels of Th1-type cytokine expression and MBP-specific responses. Thus, it is possible that the subsequent accumulation of leukocytes in the eye was reduced. Some chemokines trigger intravascular adhesion.19 CCL3/MIP-1{alpha} and CCL4/MIP-1ß have been shown to be potent chemoattractants for macrophages and T lymphocytes, but their effect on lymphocyte differs. CCL4/MIP-1ß selectively attracts and promotes adhesion of CD4+ T cells, whereas CCL3/MIP-1{alpha} is more effective in the attraction and adhesion of CD8+ T cells.20 21 Because CD4+ T cells mediate EAE/AU, the CCCL4/MIP-1ß antibody neutralization was more effective in suppression of clinical disease than anti-MIP-1{alpha} treatment. CCL2/MCP-1 has been reported as a major attractant for CD4+ T cells of the activated-memory phenotype.22 It attracts both Th1 and Th2, whereas CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES attract only Th1.4 CCR5 is a major receptor for CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES and therefore is considered to be a Th1-associated marker.23 24 25 26 CCR2 is a major receptor for CCL2/MCP-1.27 28 CCR2 and -5 were present in high levels in infiltrating lymphocytes, which is in agreement with the presence of their ligands.

The relationship between the production of chemokines in the CNS and development of EAE has been demonstrated by several investigators29 30 31 32 33 34 ; however, there is limited information on the role of chemokines in autoimmune eye inflammation. Our analysis of chemokine expression revealed differences in the level of expression between the target organs in EAE/AU in Lewis rats, although cells from both organs expressed a similar chemokine profile, representing CCL2/MCP-1, CCL3/MIP-1{alpha}, CCL4/MIP-1ß, and CCL5/RANTES. These chemokines can be produced by a variety of different cells, but T cells are a rich source of chemokines during a Th1 response, and those T-cell–secreted chemokines seem to play an important role in the regulation of the immune response. This suggests that the Th1 response is a principal effector mechanism in EAE/AU. Kuchroo et al.29 reported that encephalitogenic T cells were the source of CCL5/RANTES, CCL3/MIP-1{alpha}, and CCL4/MIP-1ß.T cells also can be the initial source of CCL2/MCP-1 in the EAE rat.35 We showed that the cells found in the eye during inflammation also expressed all those chemokines. In a recent study, CCL3/MIP-1{alpha} and CCL2/MCP-1 have been shown to play a role in the immunopathogenesis of experimental autoimmune neuritis in Lewis rats, in which the inflammatory infiltrates of the sciatic nerve consisted of T lymphocytes and macrophages.36

Our results are also in agreement with findings in patients with AU, which show that IL-8, IP-10, MCP-1, RANTES, and MIP-1ß were significantly increased in the aqueous humor and correlated with the clinical severity of the disease.7 37 38 The investigators suggested that these chemoattractant cytokines play a critical role in leukocyte recruitment in acute AU.7 For example, RANTES has the potential to influence the migration of memory T cells and monocytes across the blood–aqueous barrier during inflammatory eye disease.39 40 41 42 43 RANTES also appears to play a role in the pathogenesis of relapsing–remitting multiple sclerosis (RR-MS), enhancing the inflammatory response within the nervous system. RANTES production is increased in relapse and remission compared with levels in control subjects.44 In our studies, the RANTES level has been elevated but anti-RANTES treatment was not effective in suppression of acute AU. Nevertheless, its role in experimental uveitis should be investigated further. In contrast, anti-RANTES antibody treatment of MBP-immunized rats with recurrent AU had an effect on clinical disease severity, suggesting that RANTES plays a modulatory role in Th1-type selective migration during recurrent disease (Adamus et al. manuscript submitted).

The expression of chemokine receptors in the human eye has not been determined. Moreover, there is limited information about the receptors in experimental models of uveitis. Recently, the roles of a murine IL-8 receptor homologue (mIL-8Rh, neutrophil chemokine CXCR2) and CCL3/MIP-1{alpha} have been examined in the eyes of experimental animals in two eye inflammation models: endotoxin-induced uveitis (EIU) and immune complex-induced uveitis (reverse passive Arthus reaction [RPAR] uveitis). Chemokines acting through mIL-8Rh significantly influence the induction of neutrophil infiltration during EIU, but not during RPAR uveitis. However, CCL3/MIP-1{alpha} is not critical for either EIU or RPAR-induced uveitis. The differential dependence on IL-8-like chemokines is in agreement with the two forms of uveitides having different origins but mostly mediated by neutrophils.45 CXCL8/IL-8 mediates neutrophil infiltration, whereas CCL2/MCP-1 mediates mononuclear cell infiltration and protein leakage in lipopolysaccharide (LPS)-induced uveitis in rabbits.46 In another study, the investigators showed that a CXC chemokine, GRO, and IL-8 act in concert to mediate neutrophil infiltration in the same LPS-induced uveitis model.47 Our studies showed for the first time the expression of CC chemokine receptors in the iris, which correlated with the expression of their ligands during the course of AU.

Chemokine expression in AU provides important information regarding the pathogenesis of uveitis. The further evaluation of their roles in the initiation and regulation of disease activity may lead to the development of new therapies in human uveitis.


   Footnotes
 
2 Present affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta. Back

Supported by Grant EY12477 from the National Institutes of Health (GA).

Submitted for publication February 13, 2001; revised May 29 and July 13, 2001; accepted July 20, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Grazyna Adamus, Neurologic Sciences Institute, Oregon Health & Science University, West Campus, Building 1, 505 NW 185th Avenue, Beaverton, OR 97006. adamusg{at}ohsu.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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