How Antibodies are made

The adaptive immune response requires a lag phase, in which chicken RBC antigen-specific lymphocytes begin to divide, about 4-5 days after immunization of a mouse/animal with chicken erythrocytes which are seen as foreign. After this lag phase, antibody appears. Therefore, after 7 days, the immunized mouse possessed antibodies against the chicken erythrocytes in its circulation and at sites of inflammation, with IgG is predominant extracellularly and in tissues. The control mouse did not possess antibodies to the chicken RBC antigens. Therefore, the higher percentage of RBCs attached and engulfed by mouse tissue macrophages was observed in immunized mouse versus the lower values in the non-immunized, control mouse, because antibodies were present in the immunized mouse that acted as opsonins, binding to and altering the chicken RBCs surface and thereby enhancing phagocytosis by the macrophages which possess Fc receptors on their surfaces. These Fc receptors enabled for the recognition of the coated antibodies and also for the activation of their engulfment, in addition to any complement proteins that are present in plasma, but may have spread into the peritoneum when a vessel was pierced during preparation of slides. Even though low in number, complement can also enhance opsonization (however to a lower extent and even lower due to use of BSA, but still worthy of mentioning). The control mouse possessed no antibodies against the chicken RBCs, but a small amount of complement from an unexperienced preparation, which could have pierced a blood vessel (1).

The spleen of a mouse, one week previously infected by a virus, will have numerous visible nodules, far more than a non-immunized mouse (due to some environmental antigens). Nodules resemble protruding balls from the surface of the spleen that are rapid proliferating clones specific to the viral antigens that have been stimulated to expand, known as germinal centers. The spleen is simply enlarged due to the large increase in lymphocytes through replication. Lymphocytes, before infection, are in the resting state and are small, with a small amount of cytoplasm, absence of rough endoplasmic reticulum, and condensed chromatin, which when stimulated by antigen, eventually results in effector cells, which have a large amount of cytoplasm, a nucleus with prominent nucleoli, abundant mitochondria and the presence of rough endoplasmic reticulum. Lymphocytes (antigen-specific) numbers increase, through clonal expansion, proliferation, after an infection because of stimulation and engagement of receptors (1,4).

The macrophages do not recognize the chicken erythrocytes immediately as foreign, but require them to be opsonized. In the immunized mouse, we saw phagocytosis at both incubation times, but a higher level at the 30 minutes, probably due to the macrophages having more time to engulf its' surroundings, antibody opsonized chicken RBCs, as well as there being more time for antibodies to coat the chicken RBCs. However, we saw no phagocytosis in the 5 minute control mouse, which is expected as no antibodies would have been present. Moreover, we saw a level of phagocytosis in the 30 minute mouse that is comparable to the 5 minute immunized mouse. To accound for this non-antibody phagocytosis, complement proteins that were released from a pierced vessel (alternative pathway) could account for the processes, even though BSA could have decreased their entrance into the peritoneum. More importantly, is the idea that macrophages always engulf particles from their surroundings, foreign and non-foreign, and process these particles for presentation in the lymph nodes. Macrophages 'sample' their surroundings, in a process of looking for hidden invaders, that only lymphocytes can recognize. It is probable, that the low percentage of phagocytosis, seen in the 30 minute control mouse was this 'sampling' of surroundings, a constitutive phagocytosis process. This small precentage would also be evident in the immunized mouse. Moreover, the 0% found in the 5 minute control mouse was probably due to the macrophages not stabilizing themselves on the glass slide quickly enough to phagocytose. The immunized 5 minute mouse, however, would have had enhanced and directed phagocytosis due to antibody opsonization, that could account for it not needed to be highly stabilized on the glass matrix due to a higher attraction to the opsonized chicken RBCs (1,3).

References:

1. Janeway, C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA, pages 1-40, 2.5-2.22, 3.1-3.12

2. DeFranco, A.I. 1987. Molecular aspects of B-lymphocyte activation. Annu. Rev. Cell Biol. 3:143-178

3. MacLennan, I.C.M. 1994. Germinal centers. Annu. Rev. Immunol. 3:117-139

4. Ravetch, J.V. and Kinet, J. 1993. Fc receptors. Annu. Rev. Immunol. 9:457-492