January 2004
Volume 45, Issue 1
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Immunology and Microbiology  |   January 2004
Chemokines in Autoimmune Lacrimal Gland Disease in MRL/MpJ Mice
Author Affiliations
  • Esen Karamursel Akpek
    From the Departments of Ophthalmology and
  • Douglas A. Jabs
    From the Departments of Ophthalmology and
    Medicine, The Johns Hopkins University School of Medicine, Baltimore Maryland;
    The Department of Epidemiology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland; and the
  • Hervé C. Gérard
    Departments of Immunology and Microbiology
  • Robert A. Prendergast
    From the Departments of Ophthalmology and
  • Alan P. Hudson
    Departments of Immunology and Microbiology
  • Bella Lee
    From the Departments of Ophthalmology and
  • Judith A. Whittum-Hudson
    Departments of Immunology and Microbiology
    Medicine, and
    Ophthalmology, Wayne State University, Detroit, Michigan.
Investigative Ophthalmology & Visual Science January 2004, Vol.45, 185-190. doi:https://doi.org/10.1167/iovs.03-0812
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      Esen Karamursel Akpek, Douglas A. Jabs, Hervé C. Gérard, Robert A. Prendergast, Alan P. Hudson, Bella Lee, Judith A. Whittum-Hudson; Chemokines in Autoimmune Lacrimal Gland Disease in MRL/MpJ Mice. Invest. Ophthalmol. Vis. Sci. 2004;45(1):185-190. doi: https://doi.org/10.1167/iovs.03-0812.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. MRL/MpJ-fas +/fas + (MRL/+) and MRL/MpJ-fas lpr /fas lpr (MRL/lpr) mice undergo spontaneous development of inflammation of the lacrimal and salivary glands, similar to that in the human disorder Sjögren’s syndrome. Previous work has shown that these lesions appear to be largely T helper (Th)-2–driven, as evidenced by the substantially greater expression of IL-4 than interferon-γ. The relative contributions of selected chemokines associated with Th1 and Th2 immune responses were assessed.

methods. Lacrimal glands from MRL/+ and MRL/lpr mice, at ages 1.5 through 9 months were evaluated by immunohistochemistry for the chemokines monocyte chemoattractant protein (MCP)-1 (also known as chemokine ligand [CCL]-2), MCP-5 (CCL12), thymus activation regulated chemokine (TARC; or CCL17), and macrophage-derived chemokine (MDC; or CCL22). Additional lacrimal glands were tested by real-time RT-PCR for chemokines MCP-1 and -5, which are associated with Th2 and Th1 responses, respectively.

results. By immunohistochemistry a significantly greater proportion of mononuclear inflammatory cells in the lacrimal gland lesions stained for MCP-1 (29%–48% depending on age) compared with MCP-5 (1%–3% depending on age) both in MRL/+ (mean difference 34.2%, P < 0.001) and MRL/lpr (mean difference 33.6%, P < 0.001) substrains. Real-time RT-PCR studies showed higher transcript levels of MCP-1 compared with MCP-5, in both MRL/+ (median difference, 37.3; P < 0.0001) and MRL/lpr (median difference, 77.1; P < 0.0001) mice. Relative transcripts of MCP-1 increased with age in both MRL/+ mice (P = 0.02) and MRL/lpr mice (P = 0.03). Staining for TARC was present on lacrimal gland ductular cells but not on the infiltrating lymphocytes, and staining for MDC was present on scattered individual cells throughout the lacrimal gland, but not on infiltrating lymphocytes.

conclusions. The predominant expression of a Th2-associated chemokine in the lacrimal gland lesions in this murine model of Sjögren’s syndrome may contribute to the predominantly Th2-type lymphoid infiltrate in these tissues.

Sjögren’s syndrome is a chronic autoimmune disorder characterized by dry eyes and a dry mouth. There is a progressive loss of exocrine gland function due to glandular damage, which results from a mononuclear inflammatory cell infiltration into these target organs. Although the etiology and pathogenesis of Sjögren’s syndrome are incompletely understood, Sjögren’s syndrome appears to be an autoimmune process. 1 MRL/MpJ mice spontaneously develop lacrimal and salivary gland inflammation, providing a model human Sjögren’s syndrome. 2 3 4 MRL/MpJ-fas +/fas + (MRL/+) and MRL/MpJ-fas lpr /fas lpr (MRL/lpr) mice are congenic, spontaneously autoimmune mice, which differ only by a single autosomal recessive gene, the fas lpr mutation. 2 5 The fas lpr mutation results in defective Fas protein, defective peripheral lymphoid organ lymphocyte apoptosis, and accelerated autoimmune disease in MRL/lpr mice. 5  
Our work has demonstrated that the lacrimal gland disease in these mice is T-cell–driven with a predominance of CD4+ T cells at the site of inflammation. 3 4 On antigen stimulation, CD4+ T-helper (Th) cells may differentiate into one of two subpopulations, each producing its own set of cytokines and mediating separate effector functions. 6 7 8 Th1 responses generally initiate delayed-type hypersensitivity and are associated with the production of interleukin (IL)-2 and interferon (IFN)-γ. 9 Th2 responses stimulate antibody production and are characterized by the production of IL-4 and -5. There appears to be a balance between the two responses: IFN- γ inhibits Th2 responses, whereas IL-10 inhibits Th1 responses. 9 In experiments by our group evaluating the cytokine expression in the lacrimal glands of MRL/MpJ mice, the lacrimal gland lesions in both substrains appeared to be Th2-mediated, as evidenced by the greater expression of the cytokines IL-4 and -10 than of IFN-γ and IL-12, in both immunohistochemistry and RT-PCR. 10 11  
Chemokines, also known as chemotactic cytokines, are 8- to 10-kDa proteins that control the attraction of leukocytes to tissues and, thereby, the ensuing inflammatory process. Chemokines induce cell migration and activation by binding to specific cell surface receptors on target cells. Chemokine production is influenced by other cytokine production. 12 13 Hence, an inflammatory infiltrate that characterizes a specific disease appears to be controlled by the subgroup of chemokines expressed in the diseased tissue, and feedback loops augment the tissue response. The chemokine monocyte chemoattractant protein (MCP)-5, also known as chemokine ligand-12 (CCL12), is associated with the Th1 immune response, whereas the chemokine MCP-1, also known as CCL2, is associated with a Th2 immune response. 12 13 14 15 In this study, we sought to determine the expression profiles of the chemokines MCP-1 and -5, to assess the relative contributions of Th2- and Th1-associated chemokines in the lacrimal gland disease of MRL/MpJ mice of both substrains. We also evaluated the chemokines thymus activation regulated chemokine (TARC), also known as CCL17, and macrophage derived chemokine (MDC), also known as CCL22, both of which also are associated with Th2 processes. 16 17  
Materials and Methods
Animals
MRL/MpJ mice of MRL/+ and MRL/lpr substrains and control BALB/c mice were obtained from the Jackson Laboratories (Bar Harbor, ME) at age 1 month and kept under standard conditions. Groups of 15 mice of each strain were anesthetized and killed by exsanguination at ages 1.5, 3, and 5 months. A group of MRL/+ and control BALB/c mice also were killed at age 9 months, but not MRL/lpr mice, as they typically do not survive beyond 6 months. At the time of death, lacrimal glands were removed and processed for either immunohistochemistry or for real-time RT-PCR for selected chemokines. Lacrimal glands from 10 mice of each age and strain were processed for immunohistochemistry and from 5 mice of each age and strain for real-time RT-PCR. 
These experiments were approved by the Johns Hopkins Medical Institutions Animal Care and Use Committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Evaluation of the Lacrimal Gland Histology
Lacrimal gland sections were graded according to the previously published modified focus score scale. 2 3 With this scoring system, lacrimal gland histologic sections were graded from 0 to 4, based on the presence of inflammatory foci containing 50 or more mononuclear inflammatory cells: grade 0, no inflammatory cells; grade 1, inflammatory cell infiltration, without any foci; grade 2, the presence of at least one focus; grade 3, multiple foci; grade 4, multiple foci plus evidence of lacrimal gland destruction as evidenced by effacement of lobular architecture by mononuclear inflammatory cells, fibrosis or other evidence of damage. 2 18  
Immunohistochemistry
Harvested lacrimal gland tissues were embedded in optimal cutting temperature compound (OCT; Miles, Elkhart, IN), frozen in liquid nitrogen and sectioned at 8 μm on a cryostat. Staining of frozen sections was performed using our standard method with antibodies to selected chemokines and the avidin-biotin-peroxidase complex (ABC) technique. 3 4 Briefly, frozen sections were fixed in chilled (4°C) acetone, air dried, rehydrated in phosphate-buffered saline (PBS), and incubated with the appropriate blocking agent (Vector, Burlingame, CA) for 15 minutes. The primary antibody was applied, and the slides were incubated for 60 minutes. A second blocking step was then performed. The slides were washed in PBS, incubated with a biotinylated secondary antibody for 30 minutes, rinsed in PBS, incubated with the ABC reagent for 45 minutes, and washed again in PBS, and the reaction product was developed with 3% hydrogen peroxide and 3-amino-9-ethyl-carbazole containing acetate buffer, and counterstained with Harris’s hematoxylin (Sigma-Aldrich, St. Louis, MO). The proportion of cells staining positively was enumerated by using a 10 × 10-μm grid disk that covered a 0.16-mm2 area, using a ×25 objective and a ×10 ocular mounted on a standard binocular microscope (Carl Zeiss Meditec, Oberkochen, Germany). 3 4 Two separate foci of inflammation in one histologic section of each lacrimal gland were counted, except in tissue sections with only one focus, where the one focus was counted. 
The antibodies were affinity-purified polyclonal goat anti-mouse antibodies and were used at the following dilutions: anti-MCP-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:35, anti-MCP-5 (Santa Cruz Biotechnology, Inc.) at 1:30, anti-TARC (Santa Cruz Biotechnology, Inc.) at 1:25, and anti-MDC (Santa Cruz Biotechnology, Inc.) at 1:33. The secondary antibody was a biotinylated, rabbit anti-goat immunoglobulin (Ig; Vector, Burlingame, CA) used at a dilution of 1:100. For each staining run and each antibody, appropriate positive controls (peritoneal exudate cellular smears or lung sections) and negative controls (in which normal goat Ig was substituted for the primary antibody) were performed to ensure quality control. 
Real-Time RT-PCR
Total nucleic acids were isolated from tissues through homogenization in hot phenol 19 or with extraction reagent (TRIzol; Invitrogen-Gibco, Grand Island, NY). 20 Pure RNA was prepared by treatment of total nucleic acid preparations with DNase1 (RQ1; Promega Life Sciences, Madison, WI), followed by phenol-chloroform extraction and collection through ethanol precipitation. RNA preparations were assessed for residual DNA by standard PCR with primers targeting the 18S rRNA gene, in the absence of reverse transcription. cDNA was prepared for real-time RT-PCR analyses using the Moloney murine leukemia virus (MuLV) reverse transcriptase enzyme (Invitrogen, Carlsbad, CA) and random hexamers as primers, as described. 19 SYBR-green-based real-time PCR analysis (Molecular Probes, Eugene, OR) was used to assess relative transcript levels from host genes through real-time PCR. These analyses were performed as described by us and others, 20 21 22 23 the sequences targeted, and the primers used for these studies were generated from GenBank (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD) with commercial software (Hastings Software, Hastings, NY). Primers were confirmed to amplify the predicted products by testing under real-time RT-PCR conditions. Primer sequences were as follows: for MCP-1 5′-primer: 5′-TTAACGCCCCACTCACCTGCTG-3′; 3′-primer: 5′-GCTTCTTTGGGACACCTGCTGC-3′ to yield a 105-bp product; for MCP-5, 5′primer: 5′-TTGGCTGGACCAGATGCG-3′ and 3′-primer: 5′-GGGACACTGGCTGCTTGTGA-3′ to yield a 116-bp product; for TARC, 5′ primer: 5′-CAATGTAGGCCGAGAGTGCTG-3′ and 3′ primer: 5′-GCATCCCTGGAACACTCCACTG-3′ to yield a 102-bp product; and for 18S rRNA: 5′-primer: 5′-CGGCTACCACATCCAAGGAA-3′ and 3′-primer, 5′-GCTGGAATTACCGCGGCT-3′ to yield a 187-bp product. Each assay was repeated twice, with each tube run in duplicate each time; signals from the analyses were normalized to values obtained for the 18S rRNA gene run simultaneously with the experimental tubes. Analyses were performed in a sequence detector (model 7700; Applied Biosystems, Foster City, CA), and data were analyzed using the accompanying software (Sequence Detection Software ver.1.7; Applied Biosystems). Data from the two runs were averaged, and the results expressed as the relative level of chemokine transcript. 
Statistics
The comparison of the proportion of cells staining positively for MCP-1 and -5 and the comparison of the levels of mRNA transcripts between MCP-1 and -5 were performed using the sign test, a nonparametric paired analysis. The evaluation of trends over time within a substrain was performed using linear regression for chemokine immunohistology and a nonparametric test for trend for chemokine real-time RT-PCR. The comparison between substrains was performed using multiple linear regression. 24  
Results
Histopathological examinations of the lacrimal tissue sections demonstrated no inflammatory infiltrates in 6-week-old MRL/+ mice. However, all 6-week-old MRL/lpr mice had infiltrates, and the median grade of inflammation was grade 3 (Table 1) . At 3 months of age, both MRL/lpr and MRL/+ mice had a marked increase in the number of infiltrating mononuclear cells, although the grade of inflammation typically was greater in the MRL/lpr substrain than in the MRL/+ substrain at comparable ages. By 5 months, there was advanced lacrimal gland damage destruction as evidenced by loss of lobular architecture by infiltrating mononuclear inflammatory cells and fibrosis, more evident in MRL/lpr than in MRL/+ mice. Both the number and size of foci of inflammation increased with age in both MRL/+ and MRL/lpr mice. 
Results of immunohistochemistry are listed in Table 1 . MRL/+ mice at age 1.5 months are not included because there were no infiltrates present. There was a significantly greater proportion of mononuclear inflammatory cells staining for MCP-1 (29%–48% depending on age) than for MCP-5 (1%–3% depending on age) in both the MRL/+ (mean difference 34.2%, P < 0.001; Fig. 1 ) and the MRL/lpr (mean difference 33.6%, P < 0.001; Fig. 2 ) substrains. In both MRL/+ and MRL/lpr mice there was a decline in the proportion of cells staining positively for MCP-1 with increasing age. The slopes of the regression lines were −2.19 for the MRL/+ mice (P = 0.009) and −3.3 for the MRL/lpr mice (P = 0.009). However, because of the increase in lesion size, the absolute number of cells staining for MCP-1 increased with age. For MCP-5 there were no significant trends with age in MRL/+ mice (P = 0.19), but in MRL/lpr mice there was a suggestion of a decline with increasing age (slope = −0.39, P = 0.06). After adjustment for the later age of onset in MRL/+ mice, there were no differences between the two strains in the proportion of cells staining for either MCP-1 (P = 0.430) or MCP-5 (P = 0.364). Control BALB/c mice had only minimal inflammation, even at age 9 months and only a few cells staining with MCP-1 compared with the MRL/MpJ mice. 
Lacrimal gland ductular cells in both MRL/+ and MRL/lpr mice stained for TARC, but the infiltrating mononuclear inflammatory cells did not. Ductular cell staining for TARC was detected in mice at all ages, even MRL/+ mice at 1.5 months of age, which typically had no infiltrates. Similar, though less intense, staining for TARC was present on the ductular cells in BALB/c mice. 
Isolated single cells, presumably tissue macrophages (which produce MDC), scattered throughout the lacrimal glands of BALB/c mice stained for MDC 12 15 In both MRL/+ and MRL/lpr mice, a small number of MDC-positive staining cells were detected at the periphery of the lacrimal gland lesions, typically accounting for approximately 1% or less of the cells in the foci (Table 1) . However, staining for MDC was not detected on the lymphocytes within the lesions. 
Results of real-time PCR studies of lacrimal gland tissue, shown in Table 2 , are expressed as the median relative amount of transcripts normalized for 18S rRNA. The transcript levels of both MCP-1 and -5 were significantly higher in MRL/MpJ mice of both substrains compared with BALB/c mice. There was a greater transcription of MCP-1 than -5 mRNA in both MRL/+ mice (median difference 37.3, P < 0.0001) and MRL/lpr mice (median difference 77.1, P < 0.0001). Furthermore, the relatively greater amounts of MCP-1 mRNA than MCP-5 mRNA were detected as early as 1.5 months of age in MRL/+ mice, which preceded the histopathologically advanced lesions, suggesting that the chemokine MCP-1 may be important in the initiation of disease process. For MCP-1 there was an increase in relative transcript levels with increasing age in both MRL/+ (P = 0.02; Fig. 3 ) and MRL/lpr (P = 0.03; Fig. 4 ) mice. There also was an increase in MCP-5 transcript levels in MRL/+ mice (P = 0.01) and a suggestion of an increase in MRL/lpr mice (P = 0.06). In control BALB/c mice there was a modest excess of MCP-1 transcripts over MCP-5 (median difference 5.1, P < 0.001), but there were no increases in either chemokine, with increasing age (P = 0.36 for MCP-1; P = 0.10 for MCP-5). Comparison of real-time PCR results for TARC transcripts among the three strains showed no increase in TARC transcripts between MRL/+ mice (median 5.83) and control BALB/c mice (median 6.32) and between MRL/lpr mice (median 3.90) and control BALB/c mice. 
Discussion
Work from our group has suggested that the lacrimal gland lesions in both MRL/+ and MRL/lpr mice are predominantly Th2 in nature. 10 11 Competitive RT-PCR demonstrated that IL-4 transcripts were present in 100- to 1000-fold greater amounts than IFN-γ transcripts in both MRL/+ and MRL/lpr mice at all time points. In MRL/+ mice IL-4 transcripts increased approximately 10-fold with increasing age, whereas IFN-γ transcripts often were at the lower limit of detection. In MRL/lpr mice IL-4 transcripts increased approximately 15-fold with increasing age, whereas IFN-γ transcripts often were undetectable. In both MRL/+ and MRL/lpr mice, IL-10 transcripts increased with age, increasing approximately 15- and 100-fold, respectively, whereas IL-2 and -12 transcripts were below the limit of detection. 11 These results were supported by immunohistochemistry, which demonstrated a 10- to 20-fold greater proportion of cells staining for IL-4 than for IFN-γ in the lacrimal gland lesions of both substrains and that IFN-γ was detected on 5% or less of the inflammatory cells. B7-1 (CD80) and B7-2 (CD86) are costimulatory molecules, expressed on antigen-presenting cells, which appear to stimulate Th1 and Th2 responses, respectively. Immunohistochemistry of MRL/MpJ mice also revealed greater expression of B7-2 than B7-1. In both substrains, B7-1 was present on 10% or less of the inflammatory cells and the mean difference between the proportion of cells staining for B7-2 and B7-1 was 19% in MRL/+ mice (P = 0.006) and 15% in MRL/lpr mice (P = 0.0001). 10 Collectively, these data suggest that the lacrimal gland lesions in both MRL/+ and MRL/lpr were predominantly Th2 in nature. Although Th1 responses typically are associated with tissue damage, Th2 responses also can cause tissue inflammation 25 and can produce tissue damage, as demonstrated by the vascular and renal lesions in Palmerston-North mice, 26 and by experimental autoimmune uveitis in IFN-γ-deficient mice. 27 Data on lymphocyte apoptosis 28 suggest that cellular trafficking may be essential in the pathogenesis of lacrimal gland inflammation in the MRL/MpJ mice. Therefore, we sought to evaluate the chemokines present in the lacrimal glands. Because of our cytokine results, we hypothesized that those chemokines associated with a Th2 response were more likely to be upregulated than those associated with a Th1 response. 
Using both immunohistochemistry and RT-PCR, we observed a greater expression of MCP-1 than of MCP-5 in the lacrimal glands in both MRL/+ and MRL/lpr substrains. The results of these chemokine studies are consistent with the previous cytokine and accessory molecule data, which suggest that lacrimal gland lesions in MRL/+ and MRL/lpr mice are predominantly Th2 in nature. 10 11 The MCP-1 chemokine upregulation preceded the lymphocytic infiltration in MRL/+ mice and remained high during the destruction of the lacrimal glands in both substrains. This result suggests that MCP-1 may have an important role in regulating the recruitment of lymphocytes into the lacrimal gland and the predominantly Th2 nature of the autoimmune response. 14  
In both MRL/+ and MRL/lpr mice, the proportion of cells staining for MCP-1 did not increase with age, whereas the amount of MCP-1 transcripts increased substantially with increasing age. However, with increasing age there was an increase in the number of inflammatory cells infiltrating the lacrimal gland. Hence, even though there is a declining proportion of cells staining positively for a given chemokine, the absolute number of such cells increased, a result consistent with the increasing MCP-1 transcripts in both substrains. 
The greater expression of MCP-1 in MRL/lpr mice lacrimal glands is supported by the work of others 29 that has demonstrated a key role for this chemokine in the production of the systemic disease in this substrain. MRL/lpr mice in which the MCP-1 gene has been knocked out have less systemic disease than controls with intact MCP-1 production. 29 However, the presence of MCP-1 in the lacrimal glands of MRL/+ mice, which do not have the aggressive systemic disease of MRL/lpr mice, suggests that its role in the autoimmunity of MRL/MpJ mice may be intrinsic to the strain. Because MCP-1 is associated with Th2 responses, 12 30 31 it may initiate, or at least contribute, to the Th2-mediated process in the lacrimal gland disease in MRL/MpJ mice. 
We observed similar expression of TARC in the lacrimal glands of MRL/+, MRL/lpr, and BALB/c mice, where TARC was expressed primarily on ductules. In BALB/c mice lacrimal glands, MDC was expressed on scattered cells, presumably macrophages, and in MRL/+ and MRL/lpr mice, MDC was expressed on a small number of cells at the periphery of the infiltrates as well. In neither MRL/+ nor MRL/lpr mice was there either expression of TARC or much expression of MDC in the inflammatory infiltrates. TARC is constitutively expressed and inducible in both mucosal cells (such as nasal and bronchial epithelia) and dendritic cells, where it appears to direct the migration of lymphocytes, 32 33 34 and is associated with Th2 responses. 12 32 33 34 Its expression in normal BALB/c mice, as well as in diseased MRL/MpJ mice, suggests that it may play a role in attracting a Th2 lymphocytic response that contributes to the normal lacrimal gland immunoglobulin secretion in tears. Although there was a suggestion of more intense staining for TARC on the ductules of MRL/MpJ mice, there was no increase in TARC transcripts in MRL/MpJ mice when compared with control BALB/c mice. 
MDC is expressed by macrophages and dendritic cells and also is associated with Th2 responses. 17 35 Scattered MDC-positive cells were present normally in the lacrimal gland of control BALB/c mice and at the periphery of the pathologic lesions in MRL/MpJ mice. The small number of cells staining for MDC in the lacrimal gland lesions in MRL/MpJ mice suggests that, like TARC, MDC does not play a major role in lacrimal gland disease production. The lack of upregulation of TARC and MDC in the lacrimal glands of MRL/MpJ mice is consistent with these chemokines’ primarily homeostatic role, rather than with an inflammatory one. 36 Conversely, MCP-1 and -5 primarily have an inflammatory role, 36 and our results demonstrate upregulation of MCP-1, a Th2-associated inflammatory chemokine. 
As in MRL/MpJ mice, most lymphocytes in the glandular lesions in human Sjögren’s syndrome are CD4+ T cells. 37 38 Although evaluation of minor salivary gland biopsy specimens from patients with Sjögren’s syndrome has produced variable results for the cytokines detected, 39 40 41 Aziz et al. 41 reported that IL-4 mRNA was detected in a greater proportion of the infiltrating mononuclear inflammatory cells than was IFN-γ and that only IL-4 mRNA-positive cells were detected in a statistically significant excess over that in control biopsy specimens. They concluded that a Th2 process was present in the glandular inflammatory infiltrate in patients with Sjögren’s syndrome, 41 results similar to those in MRL/MpJ mice. Evaluation of the chemokines in minor salivary gland biopsy specimens has demonstrated the presence of several chemokines, including B-cell attracting chemokine (BCA-1), MDC, TARC, macrophage inhibiting protein (MIP)-1α (CCL3), and MIP-1β (CCL4), but these specimens have not been tested for either MCP-1 or -5. 42 43 44 45  
In conclusion, chemokine expression data, in which there is greater expression of MCP-1, a Th-2-associated chemokine, than of MCP-5, a Th-1–associated chemokine, in MRL/MpJ mice of both substrains are consistent with our previous cytokine data, 10 11 which suggested that the lacrimal gland inflammation in these mice is predominantly Th2-mediated. Because chemokines control cellular trafficking, these chemokine data may help explain why there is a predominant Th2 inflammatory infiltrate in the lacrimal glands of MRL/MpJ mice. Studies with selected chemokine knockout mice, such as MCP-1-knockout mice, may help clarify the contributions of these chemokines to the lacrimal gland disease process. 
 
Table 1.
 
Immunohistochemical Analysis of Th1 and Th2 Chemokine Expression in Lacrimal Gland Lesions in MRL/MpJ Mice
Table 1.
 
Immunohistochemical Analysis of Th1 and Th2 Chemokine Expression in Lacrimal Gland Lesions in MRL/MpJ Mice
Strain Age (mo) Grade* (Median) MCP-1, † (Mean) MCP-5, † (Mean) MDC, † (Mean)
MRL/+ 3 3 47.9 ± 10.1 1.9 ± 2.0 1.1 ± 1.4
5 3 29.1 ± 5.0 1.8 ± 2.9 0.4 ± 0.5
9 3 31.4 ± 9.1 0.9 ± 1.2 1.2 ± 0.8
MRL/lpr 1.5 3 40.2 ± 7.3 2.8 ± 2.3 1.1 ± 1.3
3 4 38.9 ± 16.1 0.4 ± 0.5 0.4 ± 0.6
5 4 27.1 ± 6.2 1.2 ± 1.2 1.0 ± 2.8
Figure 1.
 
Immunohistochemistry for (A) MCP-1 and (B) MCP-5 in the lacrimal glands of MRL/MpJ-fas + /fas + mice, showing staining for MCP-1 but not for MCP-5. Original magnification, ×200.
Figure 1.
 
Immunohistochemistry for (A) MCP-1 and (B) MCP-5 in the lacrimal glands of MRL/MpJ-fas + /fas + mice, showing staining for MCP-1 but not for MCP-5. Original magnification, ×200.
Figure 2.
 
Immunohistochemistry for (A) MCP-1 and (B) MCP-5 in the lacrimal gland of MRL/MpJ-fas lpr /fas lpr mice, showing staining for MCP-1 but not MCP-5. Original magnification, ×200.
Figure 2.
 
Immunohistochemistry for (A) MCP-1 and (B) MCP-5 in the lacrimal gland of MRL/MpJ-fas lpr /fas lpr mice, showing staining for MCP-1 but not MCP-5. Original magnification, ×200.
Table 2.
 
Real-Time RT-PCR Analysis of Relative Th1 and Th2 Chemokine mRNA Expression Levels in Lacrimal Gland Lesions in MRL/MpJ Mice
Table 2.
 
Real-Time RT-PCR Analysis of Relative Th1 and Th2 Chemokine mRNA Expression Levels in Lacrimal Gland Lesions in MRL/MpJ Mice
Strain Age (mo) MCP-1* MCP-5*
MRL/+ 1.5 15.2 5.4
3 148.7 44.5
5 1,768.7 83.0
9 18,318.3 1,131.0
MRL/lpr 1.5 39.3 19.7
3 184.6 136.3
5 1,093.6 43.0
BALB/c 1.5 5.7 4.4
3 24.7 3.6
5 6.3 2.5
9 25.0 15.0
Figure 3.
 
Median MCP-1 and -5 relative transcript levels in MRL/MpJ-fas + /fas + mouse lacrimal glands as determined by real-time RT-PCR, normalized to 18S rRNA.
Figure 3.
 
Median MCP-1 and -5 relative transcript levels in MRL/MpJ-fas + /fas + mouse lacrimal glands as determined by real-time RT-PCR, normalized to 18S rRNA.
Figure 4.
 
Median MCP-1 and -5 relative transcript levels in MRL/MpJ-fas lpr /fas lpr mouse lacrimal glands as determined by real-time RT-PCR, normalized to 18S rRNA.
Figure 4.
 
Median MCP-1 and -5 relative transcript levels in MRL/MpJ-fas lpr /fas lpr mouse lacrimal glands as determined by real-time RT-PCR, normalized to 18S rRNA.
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Figure 1.
 
Immunohistochemistry for (A) MCP-1 and (B) MCP-5 in the lacrimal glands of MRL/MpJ-fas + /fas + mice, showing staining for MCP-1 but not for MCP-5. Original magnification, ×200.
Figure 1.
 
Immunohistochemistry for (A) MCP-1 and (B) MCP-5 in the lacrimal glands of MRL/MpJ-fas + /fas + mice, showing staining for MCP-1 but not for MCP-5. Original magnification, ×200.
Figure 2.
 
Immunohistochemistry for (A) MCP-1 and (B) MCP-5 in the lacrimal gland of MRL/MpJ-fas lpr /fas lpr mice, showing staining for MCP-1 but not MCP-5. Original magnification, ×200.
Figure 2.
 
Immunohistochemistry for (A) MCP-1 and (B) MCP-5 in the lacrimal gland of MRL/MpJ-fas lpr /fas lpr mice, showing staining for MCP-1 but not MCP-5. Original magnification, ×200.
Figure 3.
 
Median MCP-1 and -5 relative transcript levels in MRL/MpJ-fas + /fas + mouse lacrimal glands as determined by real-time RT-PCR, normalized to 18S rRNA.
Figure 3.
 
Median MCP-1 and -5 relative transcript levels in MRL/MpJ-fas + /fas + mouse lacrimal glands as determined by real-time RT-PCR, normalized to 18S rRNA.
Figure 4.
 
Median MCP-1 and -5 relative transcript levels in MRL/MpJ-fas lpr /fas lpr mouse lacrimal glands as determined by real-time RT-PCR, normalized to 18S rRNA.
Figure 4.
 
Median MCP-1 and -5 relative transcript levels in MRL/MpJ-fas lpr /fas lpr mouse lacrimal glands as determined by real-time RT-PCR, normalized to 18S rRNA.
Table 1.
 
Immunohistochemical Analysis of Th1 and Th2 Chemokine Expression in Lacrimal Gland Lesions in MRL/MpJ Mice
Table 1.
 
Immunohistochemical Analysis of Th1 and Th2 Chemokine Expression in Lacrimal Gland Lesions in MRL/MpJ Mice
Strain Age (mo) Grade* (Median) MCP-1, † (Mean) MCP-5, † (Mean) MDC, † (Mean)
MRL/+ 3 3 47.9 ± 10.1 1.9 ± 2.0 1.1 ± 1.4
5 3 29.1 ± 5.0 1.8 ± 2.9 0.4 ± 0.5
9 3 31.4 ± 9.1 0.9 ± 1.2 1.2 ± 0.8
MRL/lpr 1.5 3 40.2 ± 7.3 2.8 ± 2.3 1.1 ± 1.3
3 4 38.9 ± 16.1 0.4 ± 0.5 0.4 ± 0.6
5 4 27.1 ± 6.2 1.2 ± 1.2 1.0 ± 2.8
Table 2.
 
Real-Time RT-PCR Analysis of Relative Th1 and Th2 Chemokine mRNA Expression Levels in Lacrimal Gland Lesions in MRL/MpJ Mice
Table 2.
 
Real-Time RT-PCR Analysis of Relative Th1 and Th2 Chemokine mRNA Expression Levels in Lacrimal Gland Lesions in MRL/MpJ Mice
Strain Age (mo) MCP-1* MCP-5*
MRL/+ 1.5 15.2 5.4
3 148.7 44.5
5 1,768.7 83.0
9 18,318.3 1,131.0
MRL/lpr 1.5 39.3 19.7
3 184.6 136.3
5 1,093.6 43.0
BALB/c 1.5 5.7 4.4
3 24.7 3.6
5 6.3 2.5
9 25.0 15.0
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