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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

Low-dose methotrexate (MTX), a mainstay in the treatment of rheumatoid arthritis, is effective in only 60–70% of patients, a finding mirrored by poor antiinflammatory efficacy in some animal models, most notably collagen-induced arthritis. To determine whether genetic factors or the model itself is responsible for the poor response to MTX, we directly compared the responses of 4 inbred mouse strains to MTX in the air-pouch model of acute inflammation.

Methods

The exudate leukocyte count and adenosine concentration were determined in inbred mice treated with MTX (0.75 mg/kg intraperitoneally every week for 4 weeks) or vehicle 4 hours after injection of carrageenan into the air pouch using previously described methods. Quantitative trait locus mapping was performed using an in silico, or computer-based, method to identify loci potentially associated with each phenotype.

Results

MTX significantly reduced the exudate leukocyte count in C57BL/6J and BALB/cJ mice, but not DBA/1J (the strain used in the collagen-induced arthritis model) or DBA/2J mice. In a parallel manner, MTX increased adenosine concentration in inflammatory exudates of C57BL/6J and BALB/cJ mice, but not DBA/1J or DBA/2J mice. Antiinflammatory and adenosine responses to MTX in DBA/1J × C57BL/6J F1 and F2 offspring were most consistent with single genetic loci being responsible for each phenotype. In silico mapping identified partially overlapping loci containing candidate genes involved in both responses.

Conclusion

Genetic factors contribute to the antiinflammatory efficacy of MTX, and a single locus involved in MTX-induced adenosine up-regulation is likely responsible for the observed resistance to MTX in DBA/1J mice.

Low-dose methotrexate (MTX), the most commonly administered disease-modifying therapy for rheumatoid arthritis (RA), is effective in only 60–75% of patients (1). While much effort has gone into identifying risk factors for a poor and/or toxic response (2–4), the majority of MTX failures remain unexplained. A number of recent studies have addressed the potential role of genetic background in determining response to MTX (3, 5–7), focusing on the impact of single-nucleotide polymorphisms (SNPs) in genes of the folate uptake and metabolism pathways on the propensity toward MTX toxicity and/or poor efficacy. While these data have offered some evidence for the association of SNPs with toxicity and, to a lesser degree, efficacy, no clear demonstration of genetically based resistance to the antiinflammatory properties of MTX has been offered thus far.

The demonstration of inbred murine strain–specific resistance to the antiinflammatory effects of MTX would establish the principle that genetic factors affect the efficacy of MTX. At pharmacologically relevant doses (<1 mg/kg/week), MTX suppresses inflammation in the murine air-pouch model of inflammation and the adjuvant arthritis model of RA by increasing extracellular concentrations of the antiinflammatory autocoid adenosine (8–13). In contrast, we (Cronstein BN, et al: unpublished observations) and other investigators (14–16) have observed that pharmacologically relevant doses of MTX do not suppress collagen-induced arthritis in the DBA/1 inbred mouse strain.

To determine whether the antiinflammatory response to MTX has a genetic basis, we directly compared the capacity of low-dose MTX to suppress air-pouch inflammation in several inbred mouse strains. We report here that a pharmacologically relevant dose of MTX fails to suppress air-pouch inflammation or to up-regulate extracellular adenosine in DBA/1J, but not C57BL/6J, mice. Moreover, we report evidence that both phenotypes are due to genetic differences at one or a small number of loci. In addition, we used SNP allele data for 4 inbred strains, including DBA/1J and C57BL/6J, to apply a recently developed in silico mapping method in order to identify quantitative trait loci (QTLs) that are likely to be responsible for the observed differences in phenotype.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Materials

Carrageenan (type I) was obtained from Sigma (St. Louis, MO). MTX was purchased from Immunex (San Juan, PR). High-performance liquid chromatography–grade methanol was purchased from Fisher Scientific (Hampton, NH). All other materials were of the highest quality that could be obtained.

Induction of air pouches and carrageenan-induced inflammation

C57BL/6J, DBA/1J, and DBA/2J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in the New York University (NYU) animal facility, fed regular mouse chow, and given access to drinking water ad libitum. Mice in each experiment were matched in age, weight, and sex. All procedures described below were reviewed and approved by the Institutional Animal Care and Use Committee of NYU Medical Center and were performed under the supervision of the facility veterinary staff.

To induce air pouches, 10–15-week-old mice were injected subcutaneously on the back with 3 ml of air. After 2 days, the pouches were reinflated with 1.5 ml of air. On day 6, inflammation was induced by injecting 1 ml of a suspension of carrageenan (2% weight/volume in calcium- and magnesium-free phosphate buffered saline [PBS] solution) into the air pouch, as we have previously described (9). After 4 hours, the mice were killed by inducing CO2 narcosis, the pouches were flushed with 2 ml of PBS, and exudates were harvested. Aliquots were diluted 1:1 with methylene blue (0.01% w/v in PBS), and cells were counted in a standard hemocytometer chamber (American Optical, Buffalo, NY).

Treatment with MTX or vehicle

Animals were given weekly intraperitoneal injections of either MTX (0.75 mg/kg) or vehicle (0.9% saline) for 4 weeks. Experiments were performed within 3 days of the last dose of MTX or vehicle.

Quantitation of adenosine in inflammatory exudates

Aliquots of inflammatory exudates were added to an equal volume of 10% (w/v) trichloroacetic acid, maintained on ice, and then stored at −20°C until analyzed. The organic phase was extracted with freon:trioctylamine (31:9). The aqueous phase was applied to a C-18 Sep-Pak cartridge (Waters, Milford, MA) and eluted off with methanol. After evaporation of the methanol, the samples were reconstituted in water, and the adenosine concentration was determined by reverse-phase high-performance liquid chromatography, as previously described (2). Samples were applied to a Bondapack C-18 column (Waters, Milford, MA) and eluted with a linear 0–40% gradient of 0.01M ammonium phosphate (pH 5.5) and methanol formed over 70 minutes with a flow rate of 1.5 ml/minute. Adenosine was identified by its retention time and by the characteristic ultraviolet absorption spectrum, and the concentration was calculated by comparison with standards, as previously described (9).

Generation and phenotyping of C57BL/6J × DBA/1J F1 and F2 progeny

To further examine the potential genetic component of the MTX resistance observed in the DBA/1J strain, we used an intercross strategy in the manner described by Reeves and D'Eustachio (17), phenotyped the F1 and F2 progeny for both leukocyte counts and exudate adenosine concentrations with and without MTX treatment using the methods described (17), and then analyzed the phenotype data using methods described by Lander and Botstein (18). MTX-responsive (C57BL/6J) and MTX-resistant (DBA/1J) mice were grouped in mating trios, consisting of either 2 C57BL/6J females and 1 DBA/1J male or 2 DBA/1J females and 1 C57BL/6J male. The parental mating cage of each F1 mouse was recorded in order to allow for detection of any X chromosome–linked effect. Sex-matched F1 progeny from each mating cage were phenotyped starting at ∼6 weeks after birth, using the methods described above. Male and female F1 progeny were then intercrossed, and F2 progeny were phenotyped as described above.

Estimation of the number of loci responsible for MTX response phenotypes.

Two methods were used to estimate the number of QTLs responsible for conferring the observed phenotypes. Genetic variance, or the difference between the variances (squares of standard deviations) of the means of the genetically unique F2 and the genetically identical F1 mouse phenotypes, represents the portion of the variance that is due to genetic factors rather than environmental variance or experimental error (18). This value was determined for both the MTX-induced antiinflammatory response and the adenosine up-regulation phenotypes. The Castle-Wright formula, which estimates the number of segregating loci contributing to a trait based on the genetic variance and degree of phenotypic difference between strains, was then applied as described by Lander and Botstein (18).

In the second method, the number of F2 progeny exhibiting parental phenotypes was determined. The proportion of F2 progeny exhibiting a parental phenotype is an indicator of the number of QTLs responsible for the trait, since this proportion reaches a maximum if only 1 locus is responsible, and it decreases as the number of QTLs increases. Because for both of the phenotypes tested in the current study, the ranges of parental phenotypes overlapped, and thus the total number of F2 mice exhibiting parental phenotypes could not be directly assessed, we determined the proportion of the F2 population falling ≥2 SD below and above the mean parental values. For the leukocyte count, this represents progeny falling below the parental mean of the responsive strain (C57BL/6J) and above the parental mean of the resistant strain (DBA/1J). For the adenosine up-regulation phenotype, this represents progeny falling ≥2 SD below the parental mean of the resistant strain and above the parental mean of the responsive strain. The number of F2 progeny meeting these criteria was compared with the number that would be expected for the given sample size if 2 loci (i.e., >1 locus) were responsible for conferring the observed phenotypic difference.

In silico QTL mapping

In separate analyses, we screened for QTLs associated with response to MTX (percentage change in leukocyte counts) and with an MTX-induced increase in extracellular adenosine, using an in silico method based on that previously described by Grupe et al (19) and modified by Smith et al (20). The screen was performed on the C57BL/6J and DBA/1J strains as well as the DBA/2J and BALB/cJ strains, for which the phenotypes of interest had previously been measured in our laboratory.

First, a series of 5,268 SNPs for which genotype information for at least 2 of the 4 strains was known was partitioned into blocks based on genetic distance. Because the SNPs were originally annotated by physical and not genetic position, markers at the desired genetic distance intervals were identified in the Roche Mouse Database (available at http://mousesnp.roche.com), and the physical positions of these markers were determined using the Ensemble Mouse Database (available at http://www.ensembl.org/Mus_musculus), by entering either the marker names or the flanking sequence as query terms. A set of 131 blocks covering the genome resulted, averaging 12 cM each (39.7 cM being the largest). The SNPs were then sorted based on their physical positions relative to the marker locations, and adjacent blocks were combined to form 110 overlapping bins, which consisted of an average of 71 SNPs per bin, with a low of 17 and a high of 146. For each bin and for each strain pair, all SNPs for which genotype information was available were classified as either synonymous or polymorphic. The proportion of all SNPs within each overlapping bin that were polymorphic between each strain pair was determined for each of the 6 pairs, which resulted in a set of 6 values representing the relative genetic divergences among each of the 6 strain combinations for each of the 110 overlapping bin sets.

In order to compare the fold differences in MTX response, the values for the percentage change in leukocyte counts (described above) were converted to log values, and the difference between logs was calculated for each of the 6 strain comparisons (20). For the adenosine up-regulation phenotype, log values of the percentage increase in adenosine concentrations could not be determined since the change was slightly, although not significantly, negative for 2 of the strains. Thus, this phenotype was classified based on whether or not a significant change in adenosine concentration was observed in MTX-treated mice relative to vehicle-treated mice. Strains for which a change resulted were assigned a value of 1, and those for which there was no change were assigned a value of 0, such that a comparison between a strain in which a change was observed and one in which a change was not observed would result in a phenotypic difference of 1 – 0, or 1, whereas a comparison between 2 strains in which MTX had a similar effect would result in a net difference of 0. The resulting set of 6 values of 1 or 0 was then correlated with the proportional genetic divergence values as described above.

A correlation coefficient was calculated for the resulting sets of 6 phenotypic difference values, 1 for each phenotype, against each of the 110 sets of 6 proportional genetic divergence values described above. The values were scaled and R values determined as described by Smith et al (20). Based on the findings by Grupe et al (19), QTL identification was based on the R value cutoff that eliminated the highest percentage of the genome without surpassing 90%.

Statistical analysis

Overall differences between groups were analyzed by Mann-Whitney rank sum tests. Determination of the number of F2 mice categorized as having parental phenotypes was performed using chi-square tests. All statistical analyses were performed using Microsoft Excel (Microsoft, Redmond, WA) and SigmaStat software (SPSS, Chicago, IL).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

MTX suppression of inflammation in air pouches of C57BL/6J, but not DBA/1J, mice. The degree of inflammation induced by carrageenan, as measured by leukocyte counts in the air pouch exudates, was determined in MTX-treated and vehicle-treated mice (Table 1). In C57BL/6J mice, MTX treatment reduced leukocyte levels in air pouch exudates by 50% compared with vehicle-treated mice. In contrast, exudate leukocyte levels did not fall significantly in MTX-treated DBA/1J mice relative to that in the vehicle-treated controls.

Table 1. MTX-induced reduction in leukocyte counts in exudates of inbred mouse strains*
StrainExudate leukocyte count, ×106/ml% decreaseLog change in leukocyte count
Untreated miceMTX-treated mice
  • *

    Leukocyte counts in the inflammatory air pouch exudates of methotrexate (MTX)–treated and untreated mice were determined as described in Materials and Methods. Four inbred strains plus F1 progeny from C57BL/6J × DBA/1J crosses were tested. For the in silico analysis, values for the percentage reduction in the exudate leukocyte concentration induced by MTX were converted to log change in leukocyte counts in order to express relative changes as fold differences. The n values are the number of mice per group. Values are the mean ± SEM. NA = not applicable.

  • P < 0.001 versus untreated mice, by Mann-Whitney rank sum test.

C57BL/6J3.4 ± 0.4 (n = 23)1.7 ± 0.2 (n = 25)501.70
DBA/1J2.0 ± 0.1 (n = 12)1.8 ± 0.2 (n = 14)80.90
DBA/2J2.7 ± 0.2 (n = 9)1.9 ± 0.3 (n = 9)291.46
BALB/cJ4.0 ± 0.4 (n = 18)1.5 ± 0.1 (n = 18)621.79
C57BL/6J × DBA/1J F12.0 ± 0.4 (n = 13)1.7 ± 0.2 (n = 18)16NA

To determine whether the observed differences in antiinflammatory efficacy across strains were specific to MTX treatment and were not simply due to strain-specific variations in the severity of the induced inflammation, we tested the strains that were resistant to MTX for their response to dexamethasone (1 mg/kg). Dexamethasone significantly reduced air pouch leukocyte counts in DBA/1J mice (42% reduction relative to vehicle-treated controls [n = 11 mice per group]; P < 0.05), a response similar to those previously reported in MTX-responsive strains (9, 21). Dexamethasone also induced a significant reduction in leukocyte counts in DBA/2J mice (46% reduction relative to vehicle-treated controls [n = 8 mice per group]; P < 0.05), a strain closely related to DBA/1J that is also resistant to MTX (see below).

MTX-induced increases in adenosine concentrations in air pouch exudates of C57BL/6J, but not DBA/1J, mice. Because we have previously demonstrated that MTX-mediated suppression of leukocyte accumulation in the air pouch is due to increased adenosine concentrations in the air pouch exudates, we determined adenosine concentrations in the air pouch exudates from vehicle-treated control and MTX-treated mice of both the C57BL/6J and DBA/1J strains (Table 2). In MTX-treated C57BL/6J mice, adenosine levels in the exudates increased by 75% over the levels in vehicle-treated controls. In contrast, MTX treatment did not affect the adenosine levels in air pouch exudates from DBA/1J mice.

Table 2. MTX-induced increases in adenosine concentrations in exudates of inbred mouse strains*
StrainExudate adenosine concentration, nM% changeIncrease
Untreated miceMTX-treated mice
  • *

    Adenosine concentrations in inflammatory air pouch exudates of methotrexate (MTX)–treated and untreated mice were determined as described in Materials and Methods. Four inbred strains plus F1 progeny from C57BL/6J × DBA/1J crosses were tested. Because log values could not be determined for MTX-induced adenosine up-regulation in all strains tested, each strain was categorized based on the presence or absence of a statistically significant change (see Materials and Methods). The n values are the number of mice per group. Values are the mean ± SEM. NA = not applicable.

  • P < 0.05 versus untreated mice, by Mann-Whitney rank sum test.

C57BL/6J124 ± 27 (n = 11)216 ± 25 (n = 15)75+
DBA/1J174 ± 29 (n = 8)156 ± 19 (n = 6)−11
DBA/2J158 ± 50 (n = 5)145 ± 31 (n = 7)−8
BALB/cJ570 ± 90 (n = 16)1,110 ± 190 (n = 16)95+
C57BL/6J × DBA/1J F1230 ± 45 (n = 7)258 ± 26 (n = 12)13NA

Distributions of both MTX-response–related phenotypes in F2progeny from a C57BL/6J × DBA/1J cross suggest single loci responsible for each. In C57BL/6J × DBA/1J F1 mice, MTX treatment induced a slight, but not significant, reduction in leukocyte accumulation in air pouches. There was no significant difference between male F1 mice derived from mating trios with a DBA/1J male relative to the C57BL/6J male parents, suggesting that there is no X-linked effect. The variance (square of the standard deviation) in the F1 progeny was 194, whereas the variance in the F2 progeny was greater at 2,461, resulting in a genetic variance (σG) of 2,267. The Castle-Wright estimate (D; calculated as follows: [distance between means]2/8 × σG) based on the mean leukocyte counts in the C57BL/6J versus DBA/1J mice was 272/8 × 2,267 = 0.04. Among the F2 progeny, the number of individuals exhibiting leukocyte counts that were 2 SD below the mean value for C57BL/6J mice and above the mean for DBA/1J mice was significantly greater (P < 0.05) than would be expected if more than 1 locus were responsible for conferring the observed interstrain difference (data not shown).

The adenosine concentration in the exudates was not significantly higher in MTX-treated F1 mice than in saline-treated controls (Table 2). The variance in the F1 progeny was 8,259, whereas that in the F2 progeny was greater at 9,763, resulting in a genetic variance of 1,504. The Castle-Wright estimate based on the mean phenotype adenosine concentrations in C57BL/6J versus DBA/1J MTX-treated mice was therefore 932/8 × 1,504 = 0.72. The number of F2 progeny with exudate adenosine concentrations that were 2 SD below and above the DBA/1J and C57BL/6J means, respectively, was significantly greater (P < 0.05) than would be expected if more than 1 locus were responsible for the observed interstrain difference (data not shown).

Correlation of MTX efficacy with increase in exudate adenosine concentrations in the air-pouch model of acute inflammation. Because both the suppression of leukocyte accumulation and the increase in adenosine concentration induced by MTX varied in 2 strains of mice and given the previously demonstrated role of adenosine in modulating the antiinflammatory properties of MTX in this model, we sought to determine whether the 2 phenotypes were correlated. In order to perform this analysis, data from additional inbred strains were required.

We examined the effect of MTX treatment on the adenosine concentrations and leukocyte counts in air pouch exudates from DBA/2J mice, a strain closely related to DBA/1J. While there appeared to be a slight antiinflammatory response to MTX in DBA/2J mice (29% reduction in leukocyte count relative to vehicle-treated controls), the change did not reach significance (Table 1). Moreover, there was no MTX-induced up-regulation of adenosine (Table 2). In addition, results from previously published studies in another strain of inbred mice, BALB/cJ, which were performed under identical experimental conditions, revealed a marked MTX-induced decrease in leukocyte counts and increase in adenosine concentrations in the exudates. In the tested strains, the MTX-induced reduction in air pouch exudate leukocyte counts was highly correlated with the increase in adenosine concentrations (R = 0.927, P = 0.01) (Figure 1).

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Figure 1. Correlation of the degree of reduction in leukocyte counts and the up-regulation of adenosine concentrations in inflammatory air pouch exudates from methotrexate-treated mice across inbred strains. The correlation coefficient and P value were determined by Pearson's product-moment correlation.

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Partly overlapping loci identified byin silicoQTL mapping for antiinflammatory response to MTX and MTX-induced increase in exudate adenosine concentrations. We performed QTL mapping by separate computerized analysis, based on the identification of loci at which the pattern of genotypic divergence among the tested strains is most consistent with the observed differences for 2 phenotypes: the antiinflammatory efficacy of MTX, as measured by a reduction in the exudate leukocyte count, and the degree of up-regulation in the exudate adenosine concentration induced in MTX-treated mice relative to vehicle-treated control mice. The phenotypic differences between strains (difference in log values for leukocyte count reduction and the presence or absence of adenosine up-regulation, respectively) were calculated (Tables 1 and 2).

The analysis for antiinflammatory efficacy identified 10 bins, grouped into 7 loci, that met the required threshold (R > 1.5) (Figure 2). These loci were at 8.7–21.1 cM (R = 1.99) and 40–87 cM on chromosome 1 (3 bins: R = 2.16, R = 2.15, and R = 1.77), 12.9–49.8 cM (2 bins: R = 1.80 and R = 2.29) and 60.1–94 cM on chromosome 2 (R = 1.66), 0 to 15.3 cM on chromosome 3 (R = 1.99), 0–21 cM on chromosome 14 (R = 1.58), and 0–17 cM on chromosome 15 (R = 2.32).

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Figure 2. Quantitative trait loci potentially associated with the degree of reduction in leukocyte concentrations in inflammatory exudates from methotrexate-treated mice. Each of 110 vertical bars represents a genetic “bin” of an average of 17 cM of genetic distance (see Materials and Methods). Divisions between bins from different chromosomes are indicated. The R value is equal to the number of standard deviations above the normalized mean correlation value of genetic divergence and phenotypic divergence across the genome for the tested strains (19, 20). The horizontal broken line at R = 1.5 corresponds to the cutoff value that eliminated the highest percentage of the genome without surpassing 90% (19).

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The adenosine up-regulation analysis identified 9 bins, grouped into 6 loci, that met the required threshold (R > 1.4) (Figure 3). These loci were at 8.7–21.1 (R = 1.46) and 40–87 cM on chromosome 1 (3 bins: R = 1.54, R = 1.56, and R = 1.42), 12.9–49.8 cM (2 bins: R = 1.48 and R = 1.70) and 60.1–94 on chromosome 2 (R = 1.51), 21–53 cM on chromosome 14 (R = 1.52), and 0–25 cM on chromosome 16 (R = 1.47). Thus, at least 4 of the same and/or overlapping loci were identified in the 2 screens.

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Figure 3. Quantitative trait loci potentially associated with the degree of increase in adenosine concentrations in inflammatory exudates from methotrexate-treated mice. Each of 110 vertical bars represents a genetic “bin” of an average of 17 cM of genetic distance (see Materials and Methods). Divisions between bins from different chromosomes are indicated. The R value is equal to the number of standard deviations above the normalized mean correlation value of genetic divergence and phenotypic divergence across the genome for the tested strains (19, 20). The horizontal broken line at R = 1.4 corresponds to the cutoff value that eliminated the highest percentage of the genome without surpassing 90% (22).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In the current study, we found that DBA/1J mice, in contrast to C57BL/6J mice, are resistant to the antiinflammatory effects of MTX in the air-pouch model of acute inflammation. We further found that in DBA/1J mice but not C57BL/6J mice, MTX fails to induce an increase in exudate concentrations of adenosine, a potent endogenous antiinflammatory agent that has previously been shown to mediate the antiinflammatory effects of MTX in this model (9, 10, 22, 23). Genetic analysis based on the phenotypes of parental, F1, and F2 mice from a C57BL/6J × DBA/1J cross suggest that single loci are responsible for both the differences in MTX-induced reduction of exudate leukocyte counts and the up-regulation of adenosine concentrations observed between the 2 strains and, together, are consistent with the hypothesis that these traits are conferred by the same locus.

The MTX resistance observed in DBA/1J mice is strain-specific and, therefore, genetically based, so phenotype analyses of F1 and F2 intercross progeny were performed to further elucidate the nature of the effect. The low Castle-Wright estimates for the 2 phenotypes studied indicate that genetic variations at single loci best explain each of the observed differences in the DBA/1J mice. However, the Castle-Wright value is only an estimate and is based on certain assumptions: the QTLs are unlinked, the genes from both strains have effects of equal magnitude, and all alleles in the “high” and “low” strains increase and decrease the phenotype, respectively (18). Partly because of these limitations, the calculated value tends to underestimate the actual number of responsible loci (18). However, the proportion of F2 progeny exhibiting values within the ranges of the parental phenotypes is, in both cases, consistent with the Castle-Wright estimates, and these data are, together, most consistent with a single locus being responsible for each of the observed phenotypes.

The results reported here strongly support the hypothesis that, in the air-pouch model of inflammation, genetic factors influence MTX efficacy, a phenomenon not clearly established in the clinical setting. Recently, much attention has been paid to the importance of genetic background, particularly the utility of SNPs, in predicting the susceptibility of individuals to heritable diseases as well as their responses to drug therapies (3, 24–26). The potential role of pharmacogenetics in identifying determinants of poor MTX response and/or toxicity was recently reviewed by Ranganathan et al (3) and Krajinovic and Moghrabi (27). A number of small studies have suggested that SNPs in genes involved in folate metabolism are associated with MTX toxicity and/or efficacy (5–7, 28).

These studies, however, have been limited by small patient populations and inconsistent definitions of MTX efficacy in RA. Moreover, the poor efficacy of MTX in any given individual with RA may result from such non–drug-related factors as increased disease activity, caffeine consumption, or initiation of MTX therapy late in the course of RA, when the disease is less amenable to any therapy. Studies in large patient cohorts with well-defined criteria for MTX response will therefore be required to identify any genetic factors associated with resistance to MTX in the clinical setting. In contrast, in the current study, we used a murine model to establish evidence for the principle of genetically based resistance to the antiinflammatory properties of MTX in the absence of such variables. While no true animal model of RA is available, the air-pouch model was chosen because of its inducibility in a number of inbred strains as well as its established utility as a means of screening agents for potential efficacy in acute and chronic inflammation (29–33).

The correlation between the quality of response to MTX and its ability to up-regulate adenosine concentrations in exudates is consistent with previous findings in this laboratory. Through the use of models of both acute and chronic inflammation, a role of adenosine, acting through its cell surface receptors, has been demonstrated as a primary mediator of the antiinflammatory effects of MTX (9, 10, 22, 34). The most recent evidence supporting this hypothesis includes the observations that MTX failed to suppress air pouch inflammation in adenosine A2A receptor–knockout mice (22), that adenosine deaminase or adenosine A2A receptor antagonist added to inflamed air pouches attenuated the antiinflammatory effect of MTX treatment (9), and that MTX does not suppress inflammation in animals lacking ecto-5′-nucleotidase, an enzyme responsible for the generation of extracellular adenosine from AMP (Montesinos MC, Cronstein BN, and Thompson L: unpublished observations). In contrast, Andersson et al (35) reported that the ability of MTX to suppress antigen-induced arthritis in rats was not inhibited by adenosine antagonists. In that study, however, the MTX dosage (0.2–0.3 mg/kg/day) was substantially higher than the range commonly used to treat RA, and unlike in RA patients, the antiinflammatory effect was completely reversed by folic acid (35).

Importantly, evidence for the role of adenosine in the antiinflammatory properties of MTX has been reported in more than one animal model (10, 22) as well as in RA patients (36) and is thus not an air-pouch model–specific phenomenon. Interestingly, there was no observed correlation between the exudate adenosine concentrations reached in vehicle-treated mice after carrageenan administration and the degree of inflammation reached across strains, a finding which suggests that relative changes in adenosine concentrations, rather than absolute concentrations, may influence the efficacy of MTX. The data from the current study thus lend further support to the hypothesis that the antiinflammatory efficacy of MTX depends at least in part on its ability to up-regulate extracellular levels of adenosine.

Taken together, the strong correlation between the antiinflammatory efficacy of MTX and the MTX-induced increase in adenosine concentrations in air pouch exudates across inbred strains, the previous demonstration that adenosine is an important mediator of the antiinflammatory properties of MTX, the similarity of the calculated estimates of the number of loci responsible for resistance to both effects in DBA/1J mice, and the overlapping loci identified by independent in silico analyses of each phenotype are consistent with the hypothesis that the two DBA/1J-specific MTX resistance phenotypes share a genetic basis. It is likely, based on this and previous studies, that in such a case, the gene responsible for both phenotypes would be involved in MTX metabolism, MTX-induced adenosine up-regulation (modulation, catabolism, or uptake), or adenosine response. Overlapping loci identified by in silico QTL screens based on both the MTX-induced decrease in leukocyte counts and the MTX-induced increase in adenosine concentrations include CD26 (dipeptidylpeptidase 4 [DPP-4], or adenosine deaminase complexing protein 2), adenosine deaminase (ADA), and folylpolyglutamate synthetase (FPGS) (Table 3). ADA and DPP-4 are directly involved in adenosine metabolism (37–40), whereas FPGS is known to add glutamate residues to MTX, thereby increasing its intracellular retention and, in turn, its ability to up-regulate adenosine (41, 42). Thus, the in silico screens identified loci containing candidate genes for which alleles altering regulation or function could directly explain both of the observed phenotypes.

Table 3. Genes within identified loci relevant to methotrexate-induced adenosine up-regulation and antiinflammatory efficacy*
ChromosomePosition, cMR for response and adenosine releasePutative candidate gene
  • *

    Position indicates the genetic bins selected by each analysis in which the indicated candidate genes are located. Neighboring, overlapping bins that were both selected are grouped as a single locus. The R values are for each analysis (response or reduction in exudate leukocyte concentration and adenosine release due to methotrexate treatment) and each selected bin. Identification of candidate genes mapping within selected loci was performed using the Mouse Genome Informatics database available at The Jackson Laboratory Web site (www.informatics.jax.org). DPP-4 = dipeptidyldipeptidase 4; FPGS = folylpolyglutamate synthetase; ADA = adenosine deaminase.

212.9–34.5; 23.5–49.81.80 and 1.48; 2.29 and 1.70DPP-4 (CD26); FPGS
260.1–941.66 and 1.53ADA

Interestingly, the loci identified by this analysis did not include the folate metabolism–related genes on which patient-based association studies have focused, but the genetic basis for MTX resistance in DBA/1J mice is likely to represent only 1 of a number of mechanisms by which MTX resistance could occur in patients. In DBA/2 mice, the marginal MTX-induced reduction in exudate leukocyte counts in the absence of an increase in exudate adenosine concentrations seems inconsistent with an adenosine-dependent antiinflammatory mechanism for MTX. However, the change observed in the MTX-treated DBA/2 mice did not achieve statistical significance, and the magnitude of the change was less than that observed in the strains of mice in which up-regulation of exudate adenosine levels accompanied the antiinflammatory effects of MTX.

The in silico method applied in our studies has been demonstrated to be effective in identifying the regions of the genome most likely to be responsible for observed phenotype differences without missing loci previously identified by traditional QTL screens, while requiring far less time and fewer animals (19, 20). This method, like traditional QTL mapping, cannot by itself establish associations between particular genes and phenotypes. However, because the mechanisms of MTX-induced adenosine up-regulation, adenosine metabolism, and adenosine reuptake are well-elucidated and the involved genes have been identified and characterized, we have simply identified which of these genes fall within the identified loci. These candidates warrant particular attention in the context of future experiments using methods commonly employed to build upon QTL data, such as comparative, microarray-based expression assays (43–45).

Data from the current study demonstrate that genetic factors influence the efficacy of low-dose MTX in the air-pouch model of inflammation and are consistent with the role of adenosine as an important mediator in the antiinflammatory mechanism of action of MTX in this model. Moreover, the data reinforce the potential for identifying genetic risk factors that contribute to MTX failure in humans.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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