The immunization protocol determines whether endogenous interferon-γ suppresses the infiltration of eosinophils into the conjunctiva

Abstract

Background: Studies with interferon-γ knockout (GKO) mice showed that endogenous IFN-γ suppresses the infiltration of inflammatory cells into the conjunctiva. To examine whether this phenomenon is universally true, we induced conjunctival inflammation by four different immunization protocols. Methods: Both wild type (WT) and GKO mice (C57BL/6 background) were immunized with ragweed (RW) in aluminum hydroxide (alum). Two different immunization protocols were used: either the emulsion was injected into only the left hind footpad or it was also injected into the tail base (50 μg RW in 2 mg alum per injection site). In addition, to compare the effects of the immunization dose of RW and the immunization site, 100 μg RW in 2 mg alum was injected into only the left hind footpad and 25 μg RW in 2 mg alum per injection site was injected into both the left hind footpad and the tail base. Ten days later, the mice were challenged with 2 mg RW in 10 μl PBS. Twenty-four hours later, the conjunctivas were analyzed histologically, and the cellular and humoral immune responses in the spleens and sera were determined, respectively. Results: Similar to a previous report, GKO mice showed significant eosinophilic infiltration into the conjunctiva after the footpad only injection of 50 μg RW. However, injection of 50 μg RW per injection site into the footpad plus the tail base resulted in comparable levels of eosinophilic infiltration in WT and GKO mice. On the contrary, either immunization of 100 μg RW in 2 mg alum into only the left hind footpad or that of 25 μg RW in 2 mg alum into both the left hind footpad and the tail base induced significant infiltration of eosinophils into the conjunctiva of GKO mice, compared to WT mice. Conclusions: These results show that the immunization protocol employed has a marked effect on the severity of eosinophilic infiltration. These observations indicate that in interpreting experimental results in the study of EC, the immunization protocol employed must be considered.