The site of antibody formation, from the points of the organs where it takes place and the cell type responsible for its synthesis, has been to subject of several exhaustive reviews in recent years.
The evidence bearing on the tissue in the body where antibody formation or storage takes place is described by Coons et al.(1955). McMaster and Hudack(1935) first demonstrated in a convincing way that the regional lymph nodes draining the injection site of an antigenic stimulus were site of formation or storage of antibody by finding higher titer than in the blood. Ehrich(1942, 1946) and Harris(1945) confirmed and extended this observation in their studies of the popliteal lymph nodes of the rabbit. They found that antibody first appeared in the node on th 4th day after the first injection of antigen into the foot-pad.
Topley(1930) first demonstrated that cells transfered from the spleen of a stimulated animal survived and synthesized antibody in a recipient animal into which they were injected.
The germinal centers in lymph nodes undergo a regular sequence of changes following the injection of antigenic material(Marshall et al. 1950, Ringertz et al.
1950), which indicated that they are involved in some way in the response to such materials. Reiss et al. (1950), and Moeschlin et al.(1954) found that antibody is present in both immature and mature plasma cells. Hayes et al. (1954), however, carrying out the same reaction on stimulated subcutaneous tissue, foune adherence of the organisms to small lymphocytes. Easty et al.(1958) and Magan de Barniner (1958) reported the role of the antibody production by the numerous antigenic injections. Since 1960, many workers proved that the plasma cell is the antibody
Ilhan et al.(1965) found by repeated injection of Ferritin plasma cells which contained antibody in the endoplasmic reticulum in rats or rabbits lymph nodes. The present attempt is to study the regional differences of antibody production with
fluorescent-microscopy following antigenic stimulation through a different route, namely local or systemic injections.
Materials and Methods
Male albino rabbits, a total of 114, weighing about 2.0kg were used, divided into six groups, and treated as follows.
Group Ⅰ : Intravenous injection of saline as saline control.
Group Ⅱ : Intravenous injection of antigen solution, once only.
Group Ⅲ : Intravenous injection of antigen solution, twice, two weeks apart each.
Group Ⅳ : Subcutaneous injection of saline alone as saline control.
Group Ⅴ : Subcutaneous injection of antigen solution once only.
Group Ⅵ : Subcutaneous injection of antigen, twice, two weeks apart each.
Antigens were prepared with purified horse gamma globulin dissolved in saline. They ear given in a dose of 20mg per animal either intravenously through the marginal ear vein or subcutaneously in the sole of the foor.
Following the last injection of antigens, animals in each group were killed at the 1st, 2and, 3rd, 4th, 5th, 7th, 9th, 10th, 14th. 17th and 40th day by air embolism and necropsied. Prior to the killing of the animals a blood sample was obtained to estimate antibody titers. The antibody titration was made by the
passive hemagglutination method of Carpenter91965).
Lymph nodes from various sites of each animals were removed quickly and stored frozen at-30 degree centigrade until used. Fluorescent antigens were prepared by the fluorescence isothiocyanate coupling method of Coons et al.(1942, 1950, 1951).
Then nonspecific portions of fluorescent antigen were removed by acetone-dried tissue powder.
Fluorescent stainings were carried out on the frozen sections of various lymph nodes prepared with cyrostat by applying a direct Coomb's technique. Specificity of direc Commb's test was checked by blocking test. A set of frozen sectional and a permanent paraffin section from the tissue left after frozen sections were stained with hematoxylin and eosin, and methlgreen pyronin, and compared with the findings of fluorescent conjugate stained sections.
Results and Discussion
A. Serum antibody titers:
There was no demonstrable antibody in groups Ⅰ and Ⅳ. In group Ⅱ, serum antibody titer against horse gamma globulin was 1: 20 at 24 hours after the antigen injection but rose suddenly at the 3rd day and reached the peak, 1:280, on the 5th day and gradually declined from the 7th day.
In group Ⅲ, antibody titer on the 1st day after the 2nd antigen injection was higher (1:320) than that of group Ⅱ, and reached a peak on the 3rd day, remaining so till the 7th day.
In group Ⅴ, the serum antibody titer did not rise on the 1st or 2nd day, but from the 3rd day the antibody titer begun to rise suddenly and reached a peak(1:2,560) on the 7th day followed by gradual decline thereafter. In group Ⅵ, the serum antibody rose to 1 : 320 even on the 1st day after the 2nd antigen injection, and reached the peak on the 7th day. This remained until the 17th day.
B. Histologic findings:
In the groups in which antigenic stimulation was given through the intravenous route, the lymph nodes from the various sites showed histologic changes characterized by activation and proliferation of reticulum cells in the germinal center of cortical follicles. There was concomitant decrease of mature lymphocytes, from the 2nd to the 10th day in group Ⅱ, and the 1st to 10th day in group Ⅲ. The spleen also showed similar histologic alterations. Newly formed and activated reticulum cells showed large polygonal, round to oval shape, with rather abundant cytoplasm which stained strongly positive with pyronin. There was also increased numerous of mitotic activity. These changes ware more marked and approach slightly ealier in group Ⅲ than in group Ⅱ. There was no notable difference among lymph
holdes at different locations, Histologic changes gradually returned to original normal picture about the 14th to 17th day after the antigen stimulation.
In the groups in which antigenic stimulations were given locally by subcutaneous injection, the histologic alterations of the lymph nodes appeared one to two days late than in the groups stimulated systematically by intravenous antigen injections. However, the histologic changes were essentially similar in quality to those found in the groups stimulated systematically, except that the alterations were marked in the lymph nodes near the antigen injection site, namely popliteal, inguinal, iliac and mesenteric in order. No appreciable histologic changes were found in the cervical, retroauricular and mesentric nodes. The spleen showed similar histologic alterations to those seen in group Ⅱ and Ⅲ. The difference between group Ⅴ and Ⅵ, namely one or two stimulations, were a little earlier appearance and more severe changes in group Ⅵ. These changes also returned to normal around the 14th and 17th day after the antigenic stimulations.
C. Fluorescent microscopy findings:
Fluorescent positive cells were found parallel to the histologic alteration of the lym[h nodes and spleen. They correponded to the appearance of phronin positive activated reticulum cells which gradually matured toward plasma cells. They were more numerous at the cortical follicles in early stage in which antigenic stimulations were given systematically, fluorescent positive cells were found in the lymph nodes located at various sites. However, in the groups in which antigenic stimulations were made locally, fluorescent positive cells were found more numerous in the lymphnodes located close to the site of antigen injections. There correlated well with the histologic alterations and pyronin staining findings.
Serum antibody rose earlier in the groups stimulated systematically with antigen, but a higher titer was obtained by local stimulation. The lymph nodes showed histologic alternations at all locations in systemic stimulation groups while more intense alterations were noted at regional nodes in locally stimulated group. The nature of histologic alternations was identical. Fluorescent microscopy findings after direct Coomb's technique followed essentially the same pettern as histologic alterations indicating the histologic changes in the lymph nodes. These changes represe ntreaction between antigen stimulation and antibody formation.