Differential Expression and Signaling of IFN‐γ in the Conjunctiva between Lewis and Brown Norway Rats

Abstract

Genetic background determines the histological features of experimental immune‐mediated blepharoconjunctivitis (EC) in rats, which is a model for human allergic conjunctivitis (AC). A great number of lymphocytes predominate in EC of Lewis rats, while less lymphocytes and more eosinophils are present in that of Brown Norway (BN) rats. Although this difference could be attributed to their systemic Th1/Th2 dominancy, it remains unclear whether some regulatory mechanisms may exist in the inflammatory site in the conjunctiva. Here, we aim to investigate this hypothesis by comparing the expression levels of inflammatory mediators in the conjunctiva between the two strains. EC was induced in Lewis and BN rats by transfer of ovalbumin (OVA)‐specific CD4+ T‐cell lines followed by eye drops of OVA as antigen challenge, and then was clinically and histologically evaluated. Reverse‐transcription (RT)‐PCR was performed to compare the expressions of cytokines and cytokine receptors (Rs) in conjunctivas of both strains of rats either with or without EC. To confirm the biological significance of interferon (IFN)‐γ R expression, phosphorylation of signal transducers and activators of transcription (STAT)‐1 was examined in the conjunctivas, followed by subconjunctival injection of IFN‐γ. BN T cells contained interleukin (IL)‐4 and IFN‐γ, while Lewis T cells expressed no IL‐4. Transfer of those cells induced more severe EC in Lewis rats. RT‐PCR using naive conjunctivas detected more IL‐4, IFN‐γ, and IFN‐γR β‐chain RNA expression in BN rats. After the EC induction, BN rats expressed significantly higher amounts of IFN‐γR β‐chain, and upregulation of interferon regulatory factor (IRF)‐1 was observed. Phosphorylation of STAT‐1 was more remarkable in BN rats. The findings demonstrate differential expression of IFN‐γR and signaling through IFN‐γ in the conjunctiva between the two strains. This may be due to differences in histopathological character between the two strains.

Cervical sympathectomy causes alveolar bone loss in an experimental rat model

“…cDNA was reverse transcribed and amplified using the SuperScript ™ III One‐Step RT‐PCR System with Platinum ® Taq DNA Polymerase (Invitrogen Corporation, San Diego, CA, USA). The following primer pairs (including the PCR product size in parentheses) were synthesized by Invitrogen (18–20): interleukin‐1alfa, 5′‐CTA AGA ACT ACT TCA CAT CCG CAG C‐3′ and 5′‐CTG GAA TAA AAC CCA CTG AGG TAG G‐3′ (623 bp); tumor necrosis factor‐alfa, 5′‐CAC GCT CTT CTG TCT ACT GA‐3′ and 5′‐GGA CTC CGT GAT GTC TAA GT‐3′ (616 bp); interleukin‐6, 5′‐CAA GAG ACT TCC AGC CAG TTG C‐3′ and 5′‐GGA GAA AGC TTC CCA ACT TTT G‐3′ (335 bp); and beta‐actin, 5′‐TTG TAA CCA ACT GGG ACG ATA TGG‐3′ and 5′‐GAT CTT GAT CTT CAT GGT GCT AGG‐3′ (759 bp). Cycling conditions for denaturation, annealing and extension were as follows.…”

Restraint stress enhances alveolar bone loss in an experimental rat model

“…cDNA was reverse transcribed and amplified using the SuperScript™ III One‐Step reverse transcription–polymerase chain reaction (RT–PCR) System with Platinum® Taq DNA Polymerase (Invitrogen). Based on previous reports (20,21), the following primer pairs (including PCR product size in parentheses) were synthesized by Invitrogen: interleukin‐1β, 5′‐AAG CTC TCC ACC TCA ATG GAC AG‐3′ and 5′‐CTC AAA CTC CAC TTT GCT CTT GA‐3′(260 bp); tumor necrosis factor‐α (TNF‐α), 5′‐CAC GCT CTT CTG TCT ACT GA‐3′ and 5′‐GGA CTC CGT GAT GTC TAA GT‐3′ (616 bp); IFN‐γ, 5′‐ATC TGG AGG AAC TGG CAA AAG GAC G‐3′ and 5′‐CCT TAG GCT AGA TTC TGG TGA CAG C‐3′ (288 bp); and β‐actin, 5′‐TTG TAA CCA ACT GGG ACG ATA TGG‐3′ and 5′‐GAT CTT GAT CTT CAT GGT GCT AGG‐3′ (759 bp). Cycling conditions for denaturation, annealing and extension were as follows: IL‐1β, 35 cycles of 94°C for 60 s, 58°C for 45 s and 72°C for 45 s; TNF‐α, 35 cycles of 94°C for 60 s, 58°C for 45 s and 72°C for 45 s; IFN‐γ, 40 cycles of 94°C for 60 s, 58°C for 45 s and 72°C for 45 s; and β‐actin, 35 cycles of 94°C for 60 s, 58°C for 45 s and 72°C for 45 s. PCR was performed with an iCycler™ (Bio‐Rad).…”

Thymus-Derived CD4+ CD25+ T Cells Suppress the Development of Murine Allergic Conjunctivitis

“…Therefore, by using experimental AC (EC) in rats [2,3,4] and mice [5, 6], we previously demonstrated that antigen (Ag)-primed CD4+ T cells play a crucial role in the infiltration of eosinophils into the conjunctiva. Although CD4+ T cells play the most prominent role in the development of EC, other types of immunocompetent cells also appear to participate.…”