Background/Purpose: Methotrexate inhibits the production of inflammatory cytokines, but is also an antifolate.  The activity of MTX is thought to depend on the formation of polyglutamate metabolites (MTXGlu_n) traditionally measured in erythrocytes (RBCs).  Therefore, this study evaluates the relationship between cytokines, folate homeostasis and methotrexate polyglutamates in a cohort of JIA patients treated with low-dose MTX.

Methods: Blood samples from JIA patients between 3 and 17 years of age (n=32) were evaluated.  Samples were collected prior to and 3 months after the initiation of MTX therapy.  IL-1α, IL-1β, IL-1RA, IL-6, TNFα and NAMPT in the plasma were measured by immunoassay.  Concentrations of MTX and its polyglutamate metabolites were measured in patient RBCs and a subset of patient PBMCs (n=10).  Whole blood, plasma and RBC levels of folic acid, tetrahydrofolate, 5-formyl tetrahydrofolate and 5-methyl tetrahydrofolate (5mTHF) were measured.   MTX and folate analyses were conducted by previously established UPLC/MS/MS methods.  Statistical analyses were conducted by simple linear regression and Wilcoxon signed rank tests using analyte measurements at each time point (0 and 3 month) and the change in analyte from 0-3 months.

Results:

Consistent with folic acid supplementation and the inhibition of intracellular reduced folate formation by MTX, post-treatment concentrations [median (IQR)] of plasma folic acid increased [0.85 (0.6, 2.35) to 27 (0.83, 58.2) nM (p<0.01)] and RBC 5mTHF levels decreased [750.1 (513.7, 968.8) to 560.8 (376.3, 707.2) nM, (p<0.001)].  An increase in plasma folic acid from 0-3 months was associated with a reduction in IL-1α (p<0.01), IL-1β (p<0.05), IL-1RA (p<0.01) and IL-6 (p<0.01), and a reduction in RBC 5mTHF concentrations from 0-3 months was associated with a reduction in TNFα (p<0.05). 

Reduced concentrations of RBC 5mTHF at 3 months were associated with an increase in short chain RBC MTXGlu_1-2 (p<0.05), and a reduction in long chain RBC MTX_3-5 (p<0.05).  Accumulation of RBC MTXGlu_3-5 was associated with increased IL-1β (p<0.05), IL-6 (p=0.05) and NAMPT (p<0.01) concentrations at 3 months.  There were no changes in cytokine concentrations associated with short chain RBC MTXGlu_1-2 accumulation at 3 months.

In contrast, short chain PBMC MTXGlu₁ (p<0.05) and PMBC MTXGlu₂ (p<0.05) were inversely associated with 3 month IL-1α concentrations, and PBMC MTXGlu₄ (p<0.01) was inversely associated with 3 month TNFα concentrations.

Conclusion: The accumulation of folic acid and depletion of RBC 5mTHF following the initiation of low-dose MTX therapy are associated with reduced concentrations of several cytokines implicated in inflammatory arthritis.  Furthermore, an accumulation of short-chain, but not long-chain, MTX polyglutamates in RBCs was observed to associate with the depletion of RBC 5mTHF at 3 months after therapy initiation.  At this early time point after MTX initiation, long-chain RBC MTXGlu_3-5 were associated with higher concentrations of inflammatory cytokines, while  short chain PBMCMTXGlu₁₊₂  were associated with reduction of cytokines, suggesting perhaps PBMCs may be a more reliable cell type to investigate MTXGlu_n.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/plasma-cytokine-concentrations-are-associated-with-folate-perturbations-and-methotrexate-polyglutamate-accumulation-in-the-peripheral-blood-of-juvenile-idiopathic-arthritis-patients-treated-with-low-d/