Lymphocyte proliferation in draining lymph nodes and spleen after intravitreal triamcinolone acetonide injection

Abstract

 

Purpose

 

Intravitreal (IVT) corticosteroid administration is a common and effective treatment for ocular inflammation. However, it is unclear whether the anti-inflammatory effects of IVT corticosteroid are limited to the eye. Previously, we found that triamcinolone acetonide (TA) drained to local lymph nodes (LN) and spleen differentially in naïve, acutely inflamed, and chronically inflamed animals. In this study, we tested the hypothesis that TA draining to local LN and spleen in chronic uveitis is able to inhibit lymphocyte proliferation beyond the local environment of the eye.

 

Methods

 

Female New Zealand white rabbits were injected intramuscularly with an emulsion of heat-killed Mycobacterium butyricum (Mb) in incomplete Freund’s adjuvant and injected IVT in the right eye with Mb 3 weeks later to induce a chronic uveitis. TA (2 mg) or saline was administered IVT into the right eye one day after the IVT Mb challenge. Bilateral submandibular (SM), deep cervical (DC), and superficial cervical (SC) LN, vitreous and aqueous humor, and spleen specimens were obtained 7 or 28 days after uveitis induction. Lymphocyte proliferation was measured in LNs and spleen after activation by phorbol myristate acetate (PMA) and ionomycin by BrdU incorporation proliferation assay. TA quantitation was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on homogenized samples. Lymphocytes cultured in the presence of increasing concentrations of TA were also assayed for proliferation.

 

Results

 

TA was detected in almost all LNs and spleen of TA-treated animals by d7, and in approximately half of LNs at d28 (Fig. 1). Even very low doses of TA were found to decrease lymphocyte proliferation in a dose-dependent manner (p<0.05 for all TA doses > 0.1 ng/mL), suggesting the TA doses detected in the LNs and spleen are sufficient to inhibit lymphocyte proliferation. Additionally, a significant negative correlation was found in day 28 LN samples between TA concentration and lymphocyte proliferation (Fig. 2, r_s=-0.45, p = 0.01).

 

Conclusions

 

Our data suggest that IVT administered TA may not only travel to draining LNs and spleen, but may modulate lymphocyte proliferation and activation at these sites.  

 

Figure 1. TA concentration as measured by LC-MS/MS in (A) LN and spleen, and (B) aqueous and vitreous humor on d28.

 

 

Figure 2. Lymphocyte proliferation compared to TA concentration from d28 LN.