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The Journal of Immunology, 2007, 178: 2344-2351.
Copyright © 2007 by The American Association of Immunologists, Inc.

The Arthritis Severity Quantitative Trait Loci Cia4 and Cia6 Regulate Neutrophil Migration into Inflammatory Sites and Levels of TNF-{alpha} and Nitric Oxide1

Teresina Laragione*, Nuriza C. Yarlett*, Max Brenner*,{dagger}, Adriana Mello*, Barbara Sherry{ddagger}, Edmund J. Miller{ddagger}, Christine N. Metz§ and Pércio S. Gulko2,*,¶,||

* Laboratory of Experimental Rheumatology, Robert S. Boas Center for Genomics and Human Genetics, The Feinstein Institute for Medical Research, Manhasset, NY; {dagger} North Shore-LIJ Graduate School of Molecular Medicine, Manhasset, NY 11030; {ddagger} Center for Immunology and Inflammation, The Feinstein Institute for Medical Research, Manhasset, NY 11030; § Center for Patient-Oriented Research, The Feinstein Institute for Medical Research, Manhasset, NY 11030; Division of Rheumatology, Department of Medicine, North Shore University Hospital, Manhasset, NY; || Department of Medicine, New York University School of Medicine, New York, NY 11030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neutrophils are required for the development of arthritis, and their migration into the synovial tissue coincides with the onset of clinical disease. Synovial neutrophil numbers also correlate with rheumatoid arthritis disease activity and severity. We hypothesized that certain arthritis severity genes regulate disease via the regulation of neutrophil migration into the joint. This hypothesis was tested in the synovial-like air pouch model injected with carrageenan using arthritis-susceptible DA and arthritis-resistant F344 rats. DA had nearly 3-fold higher numbers of exudate neutrophils compared with F344 (p < 0.001). Five DA.F344(QTL) strains congenic for severity loci and protected from autoimmune arthritis were studied. Only DA.F344(Cia4) (chromosome 7) and DA.F344(Cia6) (chromosome 8) congenics had significantly lower exudate neutrophil counts compared with DA. TNF-{alpha} levels were 2.5-fold higher in DA exudates as compared with F344 exudates, and that difference was accounted for by the Cia4 locus. Exudate levels of NO, a known inhibitor of neutrophil chemotaxis, were higher in F344, compared with DA, and that difference was accounted for by Cia6. This is the first time that non-MHC autoimmune arthritis loci are found to regulate three central components of the innate immune response implicated in disease pathogenesis, namely neutrophil migration into an inflammatory site, as well as exudate levels of TNF-{alpha} and NO. These observations underscore the importance of identifying the Cia4 and Cia6 genes, and suggest that they should generate useful novel targets for development of new therapies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activated neutrophils and their products, including reactive oxygen species and myeloperoxidase, are present in increased concentrations in the synovial fluid of patients with active rheumatoid arthritis (RA),3 as well as in the pannus-cartilage junction, and have been implicated in disease pathogenesis and joint destruction (1). Although angiogenesis, synovial hyperplasia and lymphocytic infiltrates are present in the preclinical stages of arthritis in humans (2, 3) and rodents (4, 5), neutrophils are either absent or present in low numbers at this stage. However, synovial neutrophil numbers increase significantly around the onset of arthritis, and their numbers correlate with disease severity (5, 6, 7, 8, 9), suggesting that migration of neutrophils into the joint is a key step in the development of clinical disease. Neutrophils are also present in increased numbers in RA synovial fluid, and are the predominant cell type present during flare-up (10). Additionally, neutrophils are required for the development of experimental arthritis (6, 11, 12), and neutropenia in RA, as seen in Felty’s syndrome, is associated with reduced joint inflammation (13). Taken together, current evidence implicates neutrophils and their migration into the joint as central to the onset of arthritis and to the regulation of disease activity and severity.

Several neutrophil properties, including their numbers in peripheral blood (14, 15, 16), migration into inflammed tissues (14, 17) and some of its functional characteristics (14, 16, 18, 19), are genetically regulated. We considered that part of the difference in arthritis susceptibility and severity between DA (arthritis-susceptible) and F344 (arthritis-resistant) strains could be attributed at least in part to differences in the regulation of neutrophil migration into the inflammed synovial cavity. Furthermore, we hypothesized that there is a genetic component in the regulation of neutrophil migration into the synovial cavity, and neutrophil activation and function, and that this genetic contribution could be accounted for by arthritis severity quantitative trait loci (QTL). To test these hypotheses, the synovial-like air pouch model injected with carrageenan was used. Inbred DA, F344 rats, and five DAxF344 strains congenic for QTL known to account for the difference in arthritis susceptibility and severity in an intercross between the two parental strains, were studied (20, 21, 22, 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Rats

DA/BklArbNsi rats (DA) were originally purchased from Bantin & Kingman, maintained at Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, and then transferred to the Feinstein Institute for Medical Research (FIMR; formerly North Shore-LIJ Research Institute, NSI). F344/Hsd (F344) rats were purchased from Harlan.

DA.F344(Cia3), DA.F344(Cia4), DA.F344(Cia5a), DA.F344(Cia5d), and DA.F344(Cia6) congenics were generated as previously described (Fig. 1) (21, 22, 23). Briefly, each interval of interest was introgressed from arthritis-resistant F344 into the arthritis-susceptible DA genetic background through eight to ten genotype-guided backcrosses followed by at least five intercrosses. This strategy selects for the intervals of interest while at the same time excluding donor genome contamination at other loci known to regulate arthritis.


Figure 1
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  		FIGURE 1. DA.F344(QTL) congenic strains studied, and specific markers used in the breeding. Black bars, homozygous F344 alleles; white bars, homozygous DA alleles; striped bars, recombination interval (unknown genotype). RNO, rat (Rattus norvegicus) chromosome number.

 
All experiments involving animals were reviewed and approved by the FIMR Institutional animal care and use committee. Animals were housed in a pathogen free environment, under standard light and dark cycles, with free access to food and water.

Air pouch induction

The air-pouch model recreates a synovial-like cavity (24, 25), and is considered a standard model to evaluate acute inflammation following an injection with carrageenan (26, 27, 28). Air pouches were induced based on previously described methods (24, 29, 30, 31). Briefly, 8–14 wk-old females were anesthetized on day 0 and injected with 14 ml of air subcutaneously in the interscapular region to produce an air pouch. On day 3, rats were reinjected with 10 ml of air to boost the air pouch. On day 6, 1.0 ml of 1% carrageenan ({lambda}, type IV; Sigma-Aldrich) diluted in normal saline solution was injected into the air pouch. Six hours after the carrageenan injection rats were euthanized in a CO2 chamber and air pouches injected with 6 ml of sterile 2 mM EDTA in PBS (without calcium or magnesium), and exudate lavage fluids were collected for analyses.

Air pouch exudates and cell processing

Air pouch exudate fluids were centrifuged at 300 g for 7 min. Cell-free exudates were stored at –80°C until used. Cell pellets were resuspended in 1 ml of sterile PBS. Contaminating RBC were lysed with 4 ml Gey’s Solution for 2 min at 4°C, followed by the addition of 46 ml of PBS (32). After a brief centrifugation, cells were resuspended again in 1 ml PBS. Total cells were counted on a hemocytometer, using an Olympus CKX41 microscope.

Cytospins

Cytospin-3 slides (Shandon) were prepared with 30–100 µl of a suspension containing 2.5 x 106 cells/ml. Slides were stained with Diff-Quik (Allegiance Cardinal Health) and differential counts performed with an Olympus CKX41 microscope (40x magnification). Four random fields were counted (typically an average of 200 cells per field), and the mean used for analysis.

ELISAs

Five well-known and important neutrophil chemotaxis mediators (TNF-{alpha}, CINC1, fractalkine, leukotriene B4 (LTB4), and the LTB4 antagonist lipoxin A4 (LXA4)), were measured in the pouch exudates with Quantikine ELISA kits (R&D Systems), according to the manufacturer’s instructions. LXA4 was measured with an ELISA kit (Neogen), according to the manufacturer’s instructions with minor modifications. Briefly, 200 µl of the exudate was diluted with 200 µl of methanol and 1.5 ml of water for LXA4 extraction. Samples were acidified to pH 3.5 with 1 N HCl, and loaded onto C18 Sep-Pak light columns (Waters) preconditioned with 2 ml of methanol and 2 ml of water. Loaded columns were washed with 5 ml of water, followed by 5 ml of hexane. LXA4 was eluted with 2 ml of methylformate. Samples were reduced to residue under a stream of dry nitrogen gas and reconstituted with 200 µl of Neogen kit extraction buffer before use in ELISA.

Apoptosis assays

Neutrophil resistance to apoptosis was studied in freshly obtained pouch exudate cells. Cells were irradiated with either 2, 5, or 20 Gy, or cultured in the presence or absence of 5 µM camptothecin (Sigma-Aldrich). Treated cells were cultured in DMEM with 10% FBS plus antibiotics for 18 or 42 h, and then stained with annexin-V and 7-aminoactinomycin D and analyzed by flow cytometry. Single- or double-positive cells were considered apoptotic, and double-negative cells were considered to be viable.

Peripheral blood counts

Blood from arthritis-naive 8- to 12-wk-old DA and F344 female rats was collected by cardiac puncture or retroorbital puncture into EDTA-containing Microtainer tubes (BD Biosciences) for automated hematological analysis (AniLytics) of RBC indices and white blood cells total counts and differential.

Peripheral blood neutrophil isolation for functional and chemotaxis studies

Peripheral blood was obtained from 8- to 14-wk-old female rats into EDTA-containing tubes for in vitro functional assays, and into heparin containing tubes for chemotaxis. After density-gradient separation with Nycoprep (Accurate), neutrophils and RBC were sedimented with 5% dextran (Fisher) for 60 min, and erythrocytes lyzed with 0.2% NaCl. Resulting neutrophils were 93% pure, as determined by staining cytospins with Diff-Quik, and 98–99% viable, based on trypan blue exclusion. Cells were resuspended at 1 x 106/ml in HBSS for NO/superoxide dismutase (SOD) experiments, and in RPMI 1640 with 0.01% BSA for chemotaxis.

Chemotaxis Assay

Neutrophil migration was assessed in modified Boyden chambers as previously described (33). The two compartments of the 48-well Boyden microchemotaxis chamber (Neuro Probe) were separated by a polyvinylpyrrolidone-free polycarbonate filter with a 5-µm pore size (PFB5-50; Neuro Probe). The neutrophil chemotaxis mediators quantified in the pouch exudates were use in the in vitro chemotaxis assays. The lower chamber was loaded with 26 µl of control medium or chemoattractant. The upper chamber was loaded with 50 µl of a suspension containing 1.0 x 106 cells/ml in RPMI 1640 with 0.02% BSA. The chemotaxis chamber was incubated for 1 h at 37°C with 5% CO2. The filters were recovered, and the cells on the upper side of the filter removed by scraping. Cells migrating through the filter (bottom side) were fixed and stained with Diff-Quik. Filters were mounted on glass slides and counted using a grid eyepiece (Olympus). Number of cells appearing on the lower face of the filter was recorded and the mean of four high-power fields per well used as representative of that well. Each experimental condition was assayed in triplicate wells and the data expressed as the mean ± SD of triplicate wells.

In vitro neutrophil production of NO

Peripheral blood neutrophils (1.5 x 105) were suspended in 150 µl of HBSS and incubated with 100 µg/ml PMA per well (96-well plates) for 15 min at 37°C. Cells were removed by centrifugation (200 x g, 10 min) and the supernatant used to determine NOX levels (NO2, NO3). Nitrate in the medium was reduced to nitrite by Escherichia coli nitrate reductase (50 mU/100 µl) (34), and supernatants were diluted 1/1 with Griess reagent (Sigma-Aldrich) and incubated for 10 min (35). The absorbance was measured at 570 nm. A sodium nitrate solution reduced to nitrite by the same method was used as positive control and to generate a standard curve. All samples and standard controls were run in triplicate.

NO measurement in air pouch exudates

Air pouch exudates (100 µl) were treated with E. coli nitrate reductase (50 mU/100 µl) for 30 min at room temperature to reduce nitrate into nitrite. Samples were deproteinized with 900 µl of methanol/ether (3:1) mix for 1 h to avoid Griess reagent precipitation. The samples were next centrifuged at 16.1 x g for 20 min, the supernatant collected, and the total amount of nitrite assayed with Griess reagent (36). A standard curve was generated as described above.

SOD levels

Superoxide production was determined through the SOD-inhibitable reduction of cytochrome c in 96-well microtiter plates. Peripheral blood neutrophils (1.5 x 105 cells) were suspended in 150 µl of HBSS containing 50 µM cytochrome c and PMA (100 µg/ml) as activator. After 15 min at 37°C, absorbancies were measured at 570 nm. Samples were run in triplicate and compared with triplicate control wells containing 100 U/ml SOD (37).

Statistics

Means (normally distributed data) were analyzed with Student’s t test, and medians with the non-parametric Mann-Whitney U test using SigmaStat 3.0 (SPSS).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Increased in vivo neutrophil and mononuclear cell migration and accumulation in air pouches from DA compared with F344 rats

Exudates harvested from air pouches 6 h after the administration of 1 ml of 1% carrageenan contained 80–90% neutrophils. There were significantly higher numbers of total neutrophils and mononuclear cells in DA air pouches compared with F344 (Fig. 2) (mean ± SEM x 106 cells: neutrophils DA = 80.6 ± 3.7, F344 = 23.2 ± 2.4; p ≤ 0.001; mononuclear cells DA = 15.9 ± 1.4, F344 = 3.8 ± 0.4; p ≤ 0.001) suggesting a strong genetic component in the regulation of cell migration into the air pouch. These differences in pouch exudates cell counts reflected migration into the inflammatory site and not simply differences in peripheral blood cell counts as DA and F344 rats had similar mean peripheral neutrophil and mononuclear cell (monocytes and lymphocytes) blood counts (mean ± SD: peripheral blood leukocyte counts x 103 cells/µl: DA = 10.8 ± 2.7, F344 = 10.9 ± 4.3; neutrophil counts x 103 cells/µl: DA = 2.32 ± 1.3, F344 = 2.72 ± 1.0) (Table I).


Figure 2
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  		FIGURE 2. Cell counts in the synovial-like pouch exudates 6 h following an intrapouch injection of 1 ml of 1% carrageenan in different inbred strains and DA.F344(QTL) congenics. A, Neutrophil counts were significantly higher in DA compared with F344, a difference accounted for by the Cia4 and Cia6 loci; and B, mononuclear cells macrophage counts were significantly higher in DA compared with F344, but that difference was not explained by any arthritis QTL. Data are shown as mean ± SEM. *, p ≤ 0.05 compared with DA.

 

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  		Table I. Hematologic parameters in naïve DA and F344 rats

 
Cia4 and Cia6 account for most of the DA and F344 differences in neutrophil migration in vivo

To determine whether the arthritis QTLs known to regulate arthritis severity also account for the DA and F344 differences in neutrophil and mononuclear cell counts in the synovial-like inflammatory pouch exudates, DA.F344(Cia3), DA.F344(Cia4), DA.F344(Cia5a), DA.F344(Cia5d) and DA.F344(Cia6) congenic rats were studied (Fig. 1). Both DA.F344(Cia4) and DA.F344(Cia6) congenics had significantly lower pouch exudate neutrophil counts compared with DA (mean ± SEM neutrophil numbers: DA = 80.6 ± 3.7; DA.F344(Cia4) = 59.4 ± 5.3, p = 0.014; DA.F344(Cia6) = 65.8 ± 3.9, p = 0.038) (Fig. 2A). The number of neutrophils in the pouches of both DA.F344(Cia4) and DA.F344(Cia6) was still higher than that of F344, supporting the concept that neutrophil migration into inflammatory sites in the carrageenan-air pouch model, similar to arthritis, is under complex genetic regulation, and that the two loci operate in an additive or epistatic manner.

The number of neutrophils in the air pouches of the other congenic strains was not significantly different from DA, demonstrating that the other loci did not contain a gene involved in the regulation of neutrophil migration. These observations also support the specificity of the effect associated with Cia4 and Cia6.

Cia4 accounts for the difference in levels of TNF-{alpha} seen in DA and F344 rats

Pouch exudates were used to quantify neutrophil and macrophage chemotactic factors. Levels of TNF-{alpha} were 2.5-fold higher in DA exudates, compared with F344 exudates (mean ± SEM: DA = 2071 ± 271 pg/ml; F344 = 809 ± 143 pg/ml; p = 0.001) (Fig. 3A). We next determined whether rats congenic for the two loci involved in the regulation of neutrophil migration into the air pouch, Cia4 and Cia6, accounted for the TNF-{alpha} difference. Exudate levels of TNF-{alpha} in DA.F344(Cia4) were significantly lower than in DA (mean ± SEM: DA.F344(Cia4) = 899 ± 138 pg/ml, p ≤ 0.001 vs DA), and similar to F344 (p = 0.7). DA.F344(Cia6) TNF-{alpha} levels were similar to DA (mean ± SEM: DA.F344(Cia6) = 1570 ± 240 pg/ml, p = 0.2 and significantly higher than F344 (p = 0.019) (Fig. 3A). These results show that the non-MHC locus Cia4 regulates levels of TNF-{alpha} and accounts for nearly all the difference in TNF-{alpha} levels detected between DA and F344.


Figure 3
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  		FIGURE 3. Quantification of inflammatory mediators in the air pouch exudates 6 h following the injection of carrageenan. Data are shown as mean ± SEM, except for NO, which was not normally distributed and is shown as medians, with 25 and 75 percentile boxes, and 5 and 95 percentile bars. A, TNF-{alpha} (number of rats per strain: DA = 9, F344 = 9, DA.F344(Cia4) = 9, DA.F344(Cia6) = 11); B, NO (number of rats per strain: DA = 9, F344 = 10, DA.F344(Cia4) = 11, DA.F344(Cia6) = 14); C, LXA4 (number of rats per strain: DA = 9, F344 = 8, DA.F344(Cia4) = 8, DA.F344(Cia6) = 9); and D, LTB4 (nine rats per strain). *, p ≤ 0.05.

 
Cia6 accounts for the difference in levels of NO seen in DA and F344 rats

Levels of NO in the air pouch exudates were significantly higher in F344, compared with DA (median NO µmol/L: DA = 0.105, F344 = 0.115, p < 0.05; Fig. 3B). NO levels in DA.F344(Cia6) were similar to F344 (p = 0.5) and significantly higher than in DA (median DA.F344(Cia6) = 0.161 µmol/L, p < 0.05 vs DA), although DA.F344(Cia4) was similar to DA. These data show that the Cia6 locus regulates levels of NO and accounts for the differences in air pouch exudates NO levels seen between DA and F344.

Levels of CINC1, fractalkine, LTB4 and LXA4 in the air pouch were not regulated by Cia4 and Cia6

Levels of the chemokine CINC1 were not significantly different between DA and F344 (data not shown). Fractalkine levels were very low in DA, and could not be detected in F344 (data not shown).

Levels of LTB4 were significantly higher in F344 compared with DA (median pg/ml: DA = 250, F344 = 469, p = 0.001), but this difference was not accounted for by Cia4 or Cia6, as congenics for these loci had levels similar to DA (Fig. 3D). Levels of the LTB4 antagonist LXA4 also tended to be higher in F344 (median pg/ml: DA = 26 F344 = 41, p = 0.1; Fig. 3C), resulting in a similar LTB4 to LXA4 ratio in both DA and F344 (LTB4/LXA4 ratio: DA = 9.6, F344 = 11.4, p = 0.59). LXA4 levels in DA.F344(Cia4) and DA.F344(Cia6) congenics were similar to DA.

No evidence for in vitro neutrophil migration defects in F344

One potential explanation for the observed differences in neutrophil migration into the synovial-like inflammatory pouch in DA and F344 rats is that the neutrophils in these two strains are intrinsically different. Specifically, DA rats could have increased cell surface expression of chemokine receptors or LTB4 receptors, or more efficient signaling through them, while the opposite could be taking place in F344 rats. To address this possibility, peripheral blood neutrophils from naive DA, F344, DA.F344(Cia4) and DA.F344(Cia6) rats were isolated and studied in chemotaxis assays using Boyden chambers. The chemoattractants were titrated for maximum migration, and optimal concentrations used in the experiments shown in Fig. 4. Neutrophils from all four strains migrated efficiently in response to fMLP 5 µM, a widely used and well-established synthetic peptide that mimics the activity of bacterially derived peptides with formylated N-terminal methionine groups and is a potent neutrophil chemoattractant and activator, and 250 ng/ml CINC1 (Fig. 4). DA neutrophil chemotaxis in response to 100 ng/ml LTB4 and 250 ng/ml fractalkine was not significantly different from control medium, whereas F344 cell migration was nearly twice that of control, suggesting that F344 are more sensitive to these chemoattractants. These differences in responses to LTB4 and CINC1 do not explain the increased numbers of neutrophils detected in DA exudates.


Figure 4
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  		FIGURE 4. In vitro chemotaxis measured in Boyden chambers (mean ± SEM). Rats tested per strain: DA = 6, F344 = 12, DA.F344(Cia4) = 8, and DA.F344(Cia6) = 4.

 
Taken together, these chemotaxis data show that neutrophils derived from F344, DA.F344(Cia4) and DA.F344(Cia6) rats are responsive to several chemoattractants. In fact, for certain chemoattractants, F344 cells were even more responsive than DA cells. Therefore, differences in neutrophil numbers within the synovial-like pouch exudates are unlikely to be due to intrinsic defects in neutrophil responsiveness to chemoattractants.

No evidence for effector functional differences between DA and F344 neutrophils

It was also considered that functional/effector differences between neutrophils from DA and F344 reaching the inflammatory site could contribute to differences in disease (arthritis) outcome. The in vitro production of NO and SOD upon activation with PMA was analyzed using peripheral blood neutrophils. As shown in Fig. 5, neutrophils from DA, F344 and DA.F344(Cia6) rats produced similar levels of NO and SOD. These observations also suggest that the increased levels of NO detected in the air pouch exudates from F344 and DA.F344(Cia6), two strains with reduced numbers of exudate neutrophils, was not accounted for by neutrophils. Instead, it suggests that air pouch synovial-like cells, most likely macrophages, account for the difference. There were no significant interstrain differences in the number of apoptotic cells following irradiation or treatment with camptothecin (data not shown).


Figure 5
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  		FIGURE 5. In vitro neutrophil activity of SOD and synthesis of NO in response to PMA (mean ± SEM); n = 5 per strain.

 
DA and F344 differences in numbers of mononuclear cells in the synovial-like inflamed pouch were not explained by the five arthritis QTL

Numbers of mononuclear cells/macrophages in all five DA.F344(QTL) congenic strains studied were similar to DA, and therefore none of these loci accounted for the differences seen between DA and F344 6h after the injection of carrageenan (Fig. 3B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Neutrophils and their migration into the joint have been implicated in the onset of clinical arthritis and in the regulation of disease activity and severity (5, 6, 7, 8, 9, 10, 11, 12, 13). Therefore, understanding the regulation of neutrophil migration into the synovial tissue and fluid, and neutrophil functional regulation could be a critical step toward generating more effective therapies for arthritis. Several properties of neutrophils, including their numbers in peripheral blood (14, 15, 16), migration into inflamed tissues (14, 17, 18, 38), and certain functional characteristics such as spontaneous apoptosis (14), phagocytic capacity (16), and cell surface expression of CD11b (19) have been shown to be at least in part genetically regulated. In the present study we considered that part of the difference in arthritis susceptibility and severity between DA and F344 could be accounted for by intrinsic genetic differences in the regulation of neutrophil migration into the inflammatory synovial cavity. Furthermore, we hypothesized that arthritis severity QTL could account for part of the genetic regulation of neutrophil migration into the synovial cavity, and/or neutrophil activation and function.

To test the above hypotheses, we used the synovial-like pouch model injected with carrageenan, and studied inflammatory cell recruitment and activation in DA, F344 and five DA.F344(QTL) lines congenic for intervals known to regulate arthritis severity. A significant in vivo difference in neutrophil migration into the synovial-like air pouch was detected, with higher numbers in DA and lower numbers in F344 rats. This difference in pouch numbers of neutrophils was not explained by differences in peripheral blood neutrophil counts, which were similar in DA and F344. Therefore, the exudates’ difference in cell numbers is not merely a reflection of blood cell counts, but active migration of cells into an inflammatory site.

Neutrophils are known to contribute to joint pathology in RA and enhanced recruitment of neutrophils in arthritis-susceptible DA might underlie increased pathology in this strain. The introgression of F344 alleles at the arthritis severity loci Cia4 and Cia6 into DA background, as in DA.F344(Cia4) and DA.F344(Cia6) congenics, was enough to significantly reduce the number of neutrophils in the pouch. The magnitude of the reduction in neutrophil numbers was not identical with F344, consistent with a complex trait regulated by these two loci in either an additive or epistatic mode of action. Although it is possible that additional QTLs influence neutrophil migration, it is unlikely that such loci would have a significant role in the regulation of arthritis in DAxF344 crosses and congenics because these loci were not detected in the F2 intercross generated between these strains and studied for collagen-induced arthritis (20). To our knowledge, this is the first time that arthritis severity QTLs (Cia4 and Cia6) have been implicated in the regulation of neutrophil migration into an inflammatory synovial-like site.

Although our study is the first to implicate arthritis QTL in neutrophil migration, two previous studies analyzed the genetic regulation of the carrageenan-injected air pouch model. One study looked at exudate volume, not cellularity, 10 days after the air pouch injection of carrageenan in a genome-wide scan in a LEW x F344 F2 intercross (39), and identified loci in rat chromosome 2 and 10; the chromosome 10 locus partially overlaps the Cia5d locus studied herein. Both the phenotype (exudate volume) and time-point in the LEW x F344 study were different from the acute model reported herein, and possibly regulated through pathways different from those regulating neutrophil influx. In fact, Cia5d did not influence the number of infiltrating inflammatory cells, suggesting that the locus identified in the LEW x F344 study regulates a later, as opposed to an acute and early phenotype. It is also possible that the gene segregating in LEW x F344 does not segregate in DA x F344. The second genetic analysis of the carrageenan air pouch model was conducted in mice using an in silico strategy that did not involve any breeding, and identified eight female and eight male candidate QTL regulating numbers of cells in the exudates 4h after administration of carrageenan (40). However, none of these loci were syntenic to Cia4 or Cia6.

DA and F344 also differed in the number of mononuclear cells/macrophages present in the air pouch exudates; however, none of the DAxF344 QTLs studied accounted for that difference.

There were two potential overarching explanations for our results: a) an intrinsic difference in the neutrophil population of DA as compared with F344 neutrophils, or b) differences in the synthesis of chemotactic and activating factors by the joint or synovial-like cavity cells. The in vitro studies with peripheral blood neutrophils demonstrated that cells from both DA and F344 migrated efficiently in response to fMLP, CINC1, fractalkine and LTB4. Additionally, neutrophils from DA and F344 produced similar amounts of NO and SOD in response to PMA stimulation. These observations suggested that intrinsic neutrophil defective responses were unlikely to account for the in vivo differences in cell migration into inflammatory sites, and levels of NO. However, differences in factors known to regulate neutrophil migration, such as TNF-{alpha} and NO, were detected in pouch exudates from DA and F344.

Our data provide important clues to the mechanism of action of the Cia4 and Cia6 neutrophil migration regulatory effects. Specifically, levels of TNF-{alpha} were 2.5-fold higher in DA, compared with F344, and that difference was accounted for by Cia4, as DA.F344(Cia4) had levels similar to F344. To our knowledge this is the first description of a non-MHC arthritis locus, Cia4, which regulates levels of TNF-{alpha} in a synovial-like cavity, thus making it even more relevant to disease. One previous study implicated non-MHC loci on rat chromosomes 1 and 2 in the regulation of serum levels of TNF-{alpha} in response to LPS in a different intercross (41). TNF-{alpha} is a key mediator in the pathogenesis of rheumatoid arthritis and experimental models of arthritis in rodents. TNF-{alpha} induces several proinflammatory cytokines, chemokines, angiogenesis and matrix metalloproteinases that will ultimately lead to cartilage and bone destruction (42). Transgenic overexpression in mice induces spontaneous arthritis (43), and TNF antagonism has been one of the most effective strategies recently developed to treat RA (44, 45). TNF-{alpha} is also a known chemotactic factor for neutrophils (46, 47), and both activates endothelial cells and increases the expression of adhesion molecules (48, 49) further promoting leukocyte transmigration. Increased levels of TNF-{alpha} can be detected as early as 2h after the carrageenan injection in air pouches and precede the neutrophilic infiltration (50), supporting the concept of a key role in chemoattraction of these cells (47). Pouch lining macrophages, mast cells and fibroblasts contribute to the early levels of TNF-{alpha}.

It remains unclear whether Cia4 regulates levels of TNF-{alpha} directly, via the regulation of its transcription, or protein or message degradation, or indirectly. In either case, the identification of the Cia4 gene will reveal a novel and important regulator of this key cytokine, and will be relevant to other TNF-{alpha}-mediated diseases, such as colitis, as well as to susceptibility to bacterial infections. The Cia4 interval is long (40.6 Mb), contains 427 genes and at this point there is no obvious candidate to account for the differences in levels of TNF-{alpha}.

NO levels were unexpectedly higher in F344, compared with DA, and Cia6 accounted for that in vivo difference. NO (and SOD) production by neutrophils in vitro was similar in DA and F344, and therefore, pouch cells mediated the in vivo difference. NO is produced by several synovial cells, but most significantly by macrophages (51, 52). Although NO tends to be associated with tissue damage and proinflammatory properties (53), it also has anti-inflammatory properties. Specifically, NO inhibits several neutrophil functions such as expression of adhesion molecules, adhesion to endothelial cells, and migration into inflammatory sites (54, 55). NO can also induce neutrophil apoptosis (54). NO inhibitors up-regulate the expression of adhesion molecules and exacerbate several models of inflammation (54, 56), including chronic arthritis (57). There is evidence for a strong genetic contribution to the regulation of levels of NO in humans (58), but this is the first study to identify a QTL that regulates these levels. Additionally, the fact that Cia6 regulates arthritis provides a possible mechanistic explanation for its mode of action. The 13Mb interval of Cia6 contains 133 genes, however, similar to Cia4, no obvious candidate stands out.

We examined the levels of other neutrophil chemotaxis mediators while trying to explain the air pouch exudates differences in neutrophil numbers. We specifically measured levels of CINC1, fractalkine, LTB4 and LXA4 as these are the major neutrophil chemotaxis mediators. Exudate levels of CINC1, the rat equivalent of GRO1{alpha}, were not significantly different between DA and F344, and levels of fractalkine were very low in DA, and not detectable in F344. Levels of LTB4 were higher in F344 compared with DA. However, levels of the LTB4 inhibitor LXA4 were also higher in F344, making similar LTB4:LXA4 ratios on both strains. Additionally, levels of both LTB4 and LXA4 in DA.F344(Cia4) and DA.F344(Cia6) were similar to DA, demonstrating that these loci did not regulate neutrophil migration into the air pouch via the regulation of these two lipids. These data, combined with the in vitro chemotaxis data suggest that neutrophils from all studied strains were responsive to chemotactic agents, and neither one of these four factors accounted for the differences seen in exudates neutrophil numbers. Although we looked for the major chemoattractants for neutrophils, others have not been studied yet, and could conceivably be regulated by either Cia4 or Cia6.

In conclusion, we have determined that the arthritis severity regulatory QTLs Cia4 and Cia6 regulate neutrophil migration into a synovial-like inflammatory site, and regulate exudate levels of TNF-{alpha} and NO. This is the first time that non-MHC arthritis loci are implicated in the regulation of these two inflammatory mediators and neutrophil migration, central components of innate immune responses. The identification of the specific genes is anticipated to increase our understanding of the regulation of two major pathways involved in the pathogenesis of arthritis and other autoimmune disease, as well as responses to infections, and has the potential to generate novel targets for drug development for arthritis and other inflammatory diseases.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Funded by National Institutes of Health Grants R01-AR46213, R01-AR052439 (National Institute of Arthritis and Musculoskeletal and Skin Diseases) and R01-AI54348 (National Institute of Allergy and Infectious Diseases) to P.S.G. Back

2 Address correspondence and reprint requests to Dr. Pércio S. Gulko, Laboratory of Experimental Rheumatology, The Robert S. Boas Center for Genomics and Human Genetics, Feinstein Institute for Medical Research at North Shore-LIJ, 350 Community Drive, Room 139, Manhasset, NY 11030. E-mail address: pgulko{at}nshs.edu Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; QTL, quantitative trait loci; SOD, superoxide dismutase; LXA4, lipoxin A4; LTB4, leukotriene B4. Back

Received for publication July 18, 2006. Accepted for publication November 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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