A Quantitative Procedure for the Study of Cells in Experimentally Induced Granulomas

Abstract

Granuloma formation has been the concern of many investigators at both the research and clinical levels. The procedures used to study granulomatous inflammation have included morphological observation ( 1 ), analysis of cell migration patterns (2-4), and measurement of granuloma size in terms of granuloma diameter ( S ) , volume ( 6 ) , and weight ( 7 ). These methods, however, lack the capacity to quantitate the actual number of each cell type involved in the formation of a granuloma.Our laboratory has been particularly concerned with quantitation procedures for the study of the inflammatory exudate in the peritoneal cavity (8). In the course of these studies, it was noted that granulomatous tissue was subsequegtly formed at the site of inflammation (9, lo). Our investigations were then extended to determine if it would be possible to develop a technique for quantitating the cells in the granuloma. This study describes a procedure for dispersing experimentally induced granulomas into their cellular components. Also presented are the results obtained when this procedure was applied at different stages of granuloma formation following the injection of antigenic and nonantigenic substances.ulomas. In this investigation, 3 types of granulomas were induced by subcutaneous injections into the inguinal region of adult female BDFl hybrid mice. Type I (primary granuloma) consisted of granulomas formed in response to a priming injection of 0.4 ml of tetanus toxoid adsorbed onto aluminum phosphate (APTT) (Parke Davis). Type I1 (secondary granuloma) consisted of granulomas formed in response to a challenging injection of 0.4 ml of APTT in mice which had been primed subcutaneously in the dorsal neck 5 weeks previously with 0.2 ml of APTT mixed with 0.2 ml of pertussis vaccine (PV) (Eli Lilly) adjuvant diluted 1 : 10 with saline. Type I11 (alum granuloma) consisted of granulomas formed in response to an injection of 0.4 ml of a nonantigenic substance, aluminum phosphate (AP), ( 5 mg/ml of saline) into mice which had been primed with PV-APTT as above. In order to facilitate location of the granulomas, activated sterile carbon (C) (Merck and Co.) was mixed with AP, APTT and PV-APTT solutions in a concentration of 2 mg/ml.Processing of granulomas. Mice were killed by cervical dislocation, dipped quickly into a solution of pHisoHex (Winthrop Laboratory, New York), and the skin was peeled back to reveal the subcutaneous granulomas.Each granuloma was freed of any adherent connective tissue and then subjected to dispersion and digestion procedures in a siliconized tube containing 1 ml of a cold, freshly prepared solution of collagenase.Collagenase, from Clostridium histo2yticum (General Biochemical Corp.) was prepared at a concentration of 4 mg of collagenase/ml of Hanks' balanced salt solution (pH 7.1-7.2). After 1-3 hr of incubation at 37", the stroma dissolved; and the granuloma fell 1090

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“…7), alum plus a primary exposure to tetanus toxoid (middle panels), and alum plus a secondary injection of tetanus toxoid (right panels) were compared. The greatest numbers of eosinophils and plasma cells were found in mice that received a second injection of specific antigen [45].…”

“…The accumulation of eosinophils and the release of their diverse eokines during the inflammatory process suggests that they must have an immunoregulatory function [18,23,33,[44][45][46][47]. The eosinophil has been referred to as a "pleiotropic multifunctional leukocyte" acting as a modulator of innate and adaptive immunity [48].…”

“…The inflammatory exudate, with its burden of dendritic cells, eosinophils, T and B memory cells, as well as cellular debris and a broad assortment of enzymes, flows into the afferent lymphatics and into the regional lymph node(s) [4,17,[138][139][140]. Subsequent reactions lead to the transformation of B memory cells into plasma cells both locally in the granulomatous tissue and in the regional lymph nodes [45,83,141,142]. Some of the T and B memory cells pass via the thoracic duct to the blood and to lymphatic and myeloid tissues.…”

“…6). This involved the use of recipient TIR mice that were Thymectomized, lethally Irradiated (1100r) and Reconstituted with bone marrow or fetal liver cells [45,65,143,145,148]. Anamnestic eosinophil and antibody responses to tetanus toxoid were obtained in these mice only when viable lymphoid cells from immunized mice were injected intravenously after irradiation.…”

“…Irradiated, syngeneic hosts receiving a granuloma transplant acquired the ability to synthesize antibody, as well as the capability to mount an anamnestic response when challenged with specific antigen [45,83,169]. These granulomas are therefore a local manifestation of the cells responsible for humoral immunity.…”