Joan M. Hall

Specificity of antibody formation after intravitreal immunization with bovine gamma globulin and ovalbumin: II. Nonspecific enhancement of the secondary response☆

Abstract Rabbits immunized intravitreally with bovine gamma globulin and ovalbumin were challenged intravitreally with both bovine gamma globulin and ovalbumin, with bovine gamma globulin and human serum albumin, or with bovine gamma globulin alone. The specificity demonstrated in the primary response did not appear in rabbits challenged with both antigens; antibody-producing cells of both specificities were found in the uveal tracts of both eyes. Anti-ovalbumin-producing cells were found in the uveal tracts of the rabbits challenged with bovine gamma globulin alone, or with bovine gamma globulin and human serum albumin. Anti-bovine gamma globulin producing cells were also present. No antibody-producing cells were found in the unchallenged eyes of rabbits challenged with bovine gamma globulin alone. The action of a nonspecific soluble enhancing factor is postulated to explain the enhanced response to ovalbumin that resulted when rabbits were challenged with bovine gamma globulin alone.

Indomethacin and the corneal immune response.

To study the effect of treating rabbits with a locally administered noncorticosteroid anti-inflammatory drug, we gave 26 rabbits unilateral subconjunctival injections of 15 mg of indomethacin suspension daily for 16 days, starting one day before the intracorneal injection of the same eye with bovine gamma globulin. We graded the eyes clinically and killed groups of rabbits on postimmunization days 6, 9, 12, and 15. We determined the number of antibody-forming cells in the homolateral cervical lymph nodes, corneas, and uveal tracts and tested the sera, aqueous humor, and vitreous humor for antibodies. Indomethacin-treated eyes showed significantly less inflammatory response, and an insignificantly greater number of antibody-forming cells, than the controls.

Ocular immunization of guinea pigs.

The experiments described were undertaken to determine which cells of guinea pigs immunized by different ocular routes produce the IgG1 and IgG2 antibodies detected in the serum. Guinea pigs were immunized intravitreally, topically, or by intravitreal immunization followed by topical conjunctival challenge. An indirect plaque assay was used to detect antibody producing cells in the cervical lymph nodes and ocular tissues. Passive hemagglutination, passive cutaneous anaphylaxis and ELISA assays were used to detect serum antibody. Both the topical and intravitreal methods initiated a primary antibody response. The guinea pigs developed IgG1 and IgG2 serum antibodies, but IgE and IgA antibodies were not detected. IgG1 and IgG2 plaque forming cells (PFC) were found in the lymph node and uveal tissues of the intravitreally immunized guinea pigs, and in the lymph node and conjunctival tissues of the topically immunized animals. IgA plaque forming cells were not detected in topically immunized animals. No antibody producing cells were found in intravitreally immunized guinea pigs sacrificed after the first conjunctival challenge (two months after sensitization). The highest numbers of lymph node and conjunctival PFC were found in the animals sacrificed three days after the second or third topical challenge. The numbers of IgG1 antibody producing cells in this group of guinea pigs were usually higher than the numbers of IgG2 PFC. Serum antibody levels, undetectable before challenge, increased after the second challenge. We conclude that both lymph node and ocular cells produce antibody in guinea pigs immunized by ocular routes.

An ELISA test to detect IgG antibody production by rabbit ocular cells

An ELISA method has been used to document antibody production by the ocular and lymph node cells of rabbits immunized intravitreally with ovalbumin. Simultaneous plaque assays were performed on many of the tissues. We found plaque forming cells in the ocular tissues by day 7. IgG producing cells, as detected by the ELISA assay, were not detected until day 10. Both IgG and IgM producing cells were present in the corneal and uveal tract suspensions throughout the remainder of the observation period. Lymph node and spleen IgG production was usually only detected early in the antibody response.

Effects of conjunctival resection on the corneal immune response

In order to evaluate the effect of perilimbal conjunctival resection on the corneal immune response, we immunized rabbits with either intravenous or topical administration of bovine gamma globulin. After unilateral perilimbal conjunctival resection, the corneas were challenged with intracorneal injections of bovine gamma globulin. Perilimbal conjunctival resection prevented the development of peripheral corneal infiltrates in the rabbits immunized intravenously (P less than .0016) but not in those immunized topically. All the animals developed antibody titers to bovine gamma globulin. Similarly, perilimbal conjunctival resection did not prevent corneal graft rejection in rabbits sensitized to their donors by previous skin grafting.