Quantitative Studies of the Cellular Responses to Antigen Injections in Normal Mice : Technic for Determining Cells in the Peritoneal Fluid

This paper has presented a procedure for collecting undiluted peritoneal fluid its quantities sufficient for total and eosinophil cell chamber counts as well as films for differential counts. Diluents and staining procedures were described for both blood and peritoneal fluid. These included a modified Wright-Giemsa stain for bulk staining which was found to give consistent results equivalent to the best slides made in our laboratory by other Romanowski stains.

These modified hematologic technics were applied to blood and peritoneal fluid of over 200 mice which had received intraperitoneal injections of various antigens. Time following observations were made:

1. The blood leukocytes showed few characteristic changes following an antigen injection into sensitized as compared with non-sensitized mice. In both groups of animals, there was a temporary neutrophilia and a slight increase in both lymphocytes and monocytes, which lasted throughout the experiment. A significant increase in blood eosinophils occurred 7 days after the injection of pollen into pollen-sensitized mice.

2. The cells of the peritoneal fluid showed very marked changes following the intraperitoneal injection of antigen:

a) The neutrophils were rarely present in normal peritoneal fluid, but within one hour after intraperitoneal injection these cells were found to accumulate in great numbers in the peritoneal fluid. They disappeared almost completely by the 2nd day.

b) The mononuclear cells showed very little change in number during the first 24 hour period after the injection of either pollen or albumin. They increased in both groups of animals by the 2nd day after injections, but continued to increase over a 10 day period in the animals which had been both sensitized and reinjected with pollen.

c) The eosinophil response to antigen injection was significantly greater its animals which had been previously sensitized to the antigen injected. Albumin injections into pollen-sensitized mice did not produce any significant changes in the number of eosinophils in the peritoneal fluid, over a 42 day period. However, the injection of pollen into animals which had received prior injections of pollen, was followed by a progressive increase in the number of eosinophils within the first 12 hours after injection. By the 4th day, these cells averaged 45,000 cells per cu. mm. and accounted for 25 per cent of the total peritoneal cells. The number of eosinophils returned to the original levels by the l0th day and averaged about 5,000 cells per cu. mm. for the remainder of the experiment. The cosinophil response at 48 hours was found to be specific. Only a slight eosinophilia occurred following single intraperitoneal injections of pollen, bovine albumin, horse Serum, ascaris extract, or keratin into non-sensitized mice. However, marked eosinophilia occurred in all animals which had been sensitized by repeated injections of the same antigen.

In conclusion, these experiments indicate that mice respond in a specific manner to the antigen with which they were sensitized. There is a quantitative increase in the mononuclear and eosinophil cells at the site of injection.