The immunization protocol determines whether endogenous interferon-gamma suppresses the infiltration of eosinophils into the conjunctiva.
BACKGROUND
Studies with interferon-gamma knockout (GKO) mice showed that endogenous IFN-gamma 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 microg RW in 2mg alum per injection site). In addition, to compare the effects of the immunization dose of RW and the immunization site, 100 microg RW in 2mg alum was injected into only the left hind footpad and 25 microg RW in 2mg alum per injection site was injected into both the left hind footpad and the tail base. Ten days later, the mice were challenged with 2mg RW in 10 microl 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 microg RW. However, injection of 50 microg 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 microg RW in 2mg alum into only the left hind footpad or that of 25 microg RW in 2mg 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.
Fukushima
Atsuki
A
Department of Ophthalmology, Kochi Medical School, Kochi University, Kohasu, Oko-cho, Nankoku-city 783-8505, Japan. fukusima@med.kochi-u.ac.jp
Yamaguchi
Tomoko
T
Ishida
Waka
W
Fukata
Kazuyo
K
Ozaki
Akemi
A
Ueno
Hisayuki
H
D000485:Allergens / Q000276:immunology*; D031164:Ambrosia; D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D003233:Conjunctivitis, Allergic / Q000209:etiology / Q000276:immunology* / Q000473:pathology; D004306:Dose-Response Relationship, Immunologic; D004804:Eosinophils / Q000276:immunology; D007114:Immunization / Q000379:methods; D007073:Immunoglobulin E / Q000097:blood; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology*; D015847:Interleukin-4 / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011058:Pollen / Q000276:immunology*; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000378:metabolism
D000485:Allergens; D015847:Interleukin-4; D007073:Immunoglobulin E; D007371:Interferon-gamma