Graham Mayrhofer

Fixation and staining of granules in mucosal mast cells and intraepithelial lymphocytes in the rat jejunum, with special reference to the relationship between the acid glycosaminoglycans in the two cell types.

SummaryVarious fixation and staining procedures have been examined in order to obtain optimal numbers and acceptable morphology of the mucosal mast cells and granular intraepithelial cells in the rat jejunum. For subsequent staining with Alcian Blue, the best fixation of the jejunum was obtained with a methanol-formaldehyde-acetic acid mixture. Specific staining of the granules of these cells has been obtained using Alcian Blue at pH 5.8, at which hydrogen ion concentration more cells stain than in the usual very acid conditions. Specificity is achieved by the use of magnesium chloride concentrations above the critical electrolyte concentrations for staining of protein and nucleic acid by Alcian Blue, and by the use of Safranin O as a competitive counterstain.The critical electrolyte concentration technique has also been applied to a comparative study of the glycosaminoglycan in the two cell types. Evidence is presented that the glycosaminoglycan in the granular intraepithelial cell has either a lower degree of sulphation or a lower molecular weight or both than the material in mucosal mast cells. This finding may support the possibility that the granular intraepithelial lymphocyte is a precursor of the mucosal mast cell.

Nature of the Thymus Dependency of Mucosal Mast-cells .3. Mucosal Mast-cells in Nude-mice and Nude Rats, in B-rats and in a Child With the Digeorge Syndrome

Mucosal mast cells have been examined in the small intestinal mucosae of nude mice and nude rats, B rats and a child with the DiGeorge syndrome. In all three species, mast cells were present in normal numbers despite the athymic status of the nude mice and nude rats, the vestigial nature of the thymus in the child, and the functionally T lymphocyte-deprived status of the B rats. Connective tissue mast cells were also plentiful in skins and tongues of the nude mice and the child with thymic aplasia. It is concluded that normally neither population of mast cells has a obligatory dependence on the thymus or T lymphocytes for its differentiation, but that mucosal mast cells, under certain conditions of rapid hyperplasia, require an inductive influence provided by T lymphocytes.

The nature of the thymus dependency of mucosal mast cells: II. The effect of thymectomy and of depleting recirculating lymphocytes on the response to Nippostrongylus brasiliensis

Abstract The effect of adult thymectomy and of lymphocyte depletion by thoracic duct drainage on the jejeunal mucosal mast cell (MMC) response to infestation with Nippostrongylus brasiliensis has been studied in rats. Adult thymectomy 2 to 4 weeks before infestation with the parasite had no effect on the production of MMC hyperplasia. However, long-term thymectomy (12–39 weeks) produced a progressive decline in the MMC response to infestation. When adult-thymectomized rats were depleted of recirculating lymphocytes by thoracic duct drainage, infestation either 2 weeks or 12 weeks after completion of drainage significantly reduced the MMC response. The MMC response was restored to normal when the animals washed thoracic duct lymphocytes were returned to it, and could be regenerated over the course of 12 weeks in the presence of thymus tissue. It is concluded that the thymus influences MMC indirectly, through the production of peripheral T lymphocytes, and that the T-lymphocyte subset involved belongs to the long-lived recirculating pool.

Granular Intraepithelial Lymphocytes of the Rat Small Intestine

Intraepithelial lymphocytes have been studied in the small intestinal epithelium of normal and thymus-deficient rats. A method is described that allows recovery of purified intraepithelial lymphocytes with high yield and viability. Granular intraepithelial lymphocytes were present in normal numbers in jejunal sections from nude rats and from adult thymectomized, irradiated, bone marrow-reconstituted rats. These probably corresponded to the cells with prominent cytoplasmic granules seen in smears of isolated intraepithelial lymphocytes. Studies in athymic chimeras indicated that both granular and nongranular intraepithelial cells differentiate from bone marrow without requirement for thymus processing and that they are relatively more radio-resistant than classical lymphocytes. It is suggested that all or most intraepithelial lymphocytes form a single cell lineage in which the degree of cytoplasmic differentiation is controlled by either the thymus or T lymphocytes. Intraepithelial lymphocytes are not postthymic T cells and their relationship to mucosal mast cells and to natural killer cells is discussed.

The nature of the thymus dependency of mucosal mast cells: I. An adaptive secondary response to challenge with Nippostrongylus brasiliensis

Abstract The kinetics of mucosal mast cell (MMC) hyperplasia in the jejeunum of rats following primary and secondary infestations with Nippostrongylus brasiliensis have been studied. Evidence of memory was found in the secondary MMC response, characterized by reduced latent period of the response, heightened magnitude, and increased local proliferation of MMC. It is suggested that the similarity to the secondary immune response may not be fortuitous, and that MMC hyperplasia may be a manifestation of the adaptive immune response to the parasite.

Macrophages as effectors of T suppression: T-lymphocyte-dependent macrophage-mediated suppression of mitogen-induced blastogenesis in the rat.

Abstract Small numbers of appropriately stimulated (but not resident) peritoneal macrophages in the rat are shown to inhibit mitogen induced lymphocyte proliferation. A variety of in vivo and in vitro cell selection/fractionation procedures applied to the indicator (lymphocyte) population indicates that the cytostatic potential of the macrophages is not expressed spontaneously in vitro, but is dependent upon the activity of a second “regulator” cell from the lymphocyte population. The latter cell is shown to be large, long lived, and recirculating, highly sensitive to anti-thymocyte serum and cyclophosphamide, insensitive to irradiation, indomethacin, and mitomycin C, adherent to glass wool, and to survive poorly in culture—it is most active in vivo in the early neonatal period, contributing significantly to the lack of demonstrable PHA-responsiveness in splenocyte cultures from new born rats. A similar cell (nominal suppressor T cell) is shown to appear in lymph nodes during primary immune responses to soluble antigen.

Kinetics of Intestinal Lamina propria Mast Cells, Globule Leucocytes, Intraepithelial Lymphocytes, Goblet Cells and Eosinophils in Murine Strongyloidiasis

The changes in numbers of 6 cell populations in the intestine of mice at various intervals after primary and challenge infections with Strongyloides ratti have been quantified. The number of lamina propria mast cells increased 8 days after primary infection and reached a peak at 12 days. After secondary infection, there was a transient fall in mast cell numbers followed by a slow increase. Globule leucocytes showed a similar trend early in the primary infection and had reached normal levels after 28 days. After challenge infection, there was an early and rapid increase in their numbers. Granular intraepithelial lymphocytes did not alter significantly during the first 14 days, but were significantly greater 28 days after primary infection; they did not vary significantly after challenge infection. However, numbers of non-granular intraepithelial lymphocytes increased 10 days after infection, were elevated prior to the secondary infection at 28 days, then declined in numbers nearly 2 weeks after challenge infection. Goblet cells increased significantly 12 days after primary infection then declined rapidly. After challenge infection, there was an accelerated increase in numbers. Eosinophil numbers increased 4 days after infection, reached a peak at 12 days and then declined. After challenge infection, there was an augmented and accelerated increase in eosinophil numbers followed by rapid decline. The role of the various cells types in host defences against worms or in containment of the inflammatory responses evoked by these parasites are discussed.

The role of the thymus in the maintenance of natural killer cells in vivo

This report describes a model for investigating the role of the thymus in regulating natural killer (NK) cell activity in vivo. Evidence is presented that the thymus can regulate NK cells, and that at least some NK cells can develop without thymic help. Marrow from thymectomized rats depleted of circulating T cells by thoracic duct cannulation was transplanted into rats without a thymus (1 degree ATX.BM). These 1 degree ATX.BM rats had NK cell levels above controls 3 months after reconstitution but markedly depressed NK cell levels by 9 months. When 1 degree ATX.BM marrow was used to reconstitute rats with or without a thymus, those without a thymus (2 degrees ATX.BM) exhibited low NK cell levels after 3 months, and a similar result was obtained when 2 degrees ATX.BM marrow was used to reconstitute 3 degrees ATX.BM rats. The low NK cell levels in 2 degrees and 3 degrees ATX.BM rats were due to a deficiency in spontaneously cytotoxic NK cells, as they had normal numbers of interferon-responsive pre-NK cells. Spleen cells from 2 degrees and 3 degrees ATX.BM rats produced less interferon than control spleen cells when cultured with P815 tumor cells in vitro. However, 2 degrees and 3 degrees ATX.BM rats had higher numbers of large granular lymphocytes than controls despite their low NK cell levels. In marked contrast to 2 degrees and 3 degrees ATX.BM rats, spleen cells from 4 degrees ATX.BM rats had higher levels of cytotoxicity and a higher frequency of both spontaneously cytotoxic and pre-NK cells than controls. The 4 degrees ATX.BM rats also had the highest frequency of large granular lymphocytes in the spleen.

Local and Systemic Factors Regulating Mucosal Mast Cells

The contributions of local and systemic factors to the regulation of mucosal mast cells and globule leukocytes have been examined in the rat. Nippostrongylus brasiliensis has been used to provide a potent immunological stimulus for mucosal mast cell hyperplasia and the roles of intestinal and extraintestinal sensitization observed by comparison of the gut mast cell responses to larval and adult worm infestations. Systemic effects of adult worm infestations have been examined in isolated Thiry-Vella loops of intestine. It is concluded that the extraintestinal phase of larval infestation is not obligatory for a gut mast cell response and that mast cell hyperplasia and globule leukocyte formation are not dependent on direct contact with the parasite or its products. The dissemination of the mast cell response and the general significance of the results are discussed.

Potentiated IgE Response in Nippostrongylus brasiliensis Infested Rats – Sites of Synthesis and Traffic of Cells Secreting Potentiated Antibody

The distribution and the kinetics of potentiated antibody synthesis have been studied at the cellular level in rats infested with Nippostrongylus brasiliensis using the homologous adoptive cutaneous anaphylaxis technique. In animals immunized in the hind footpads with alum-absorbed ovalbumin 10 days prior to infestation with the parasite, the major sites of potentiated anti-ovalbumin homocytotropic antibody synthesis were the regional lymph nodes of the gut and the lungs. Peyers patches were weakly active late in the response and the spleen produced considerable amounts of potentiated antibody. The regional lymph nodes of the ovalbumin immunization sites were the only organs in which specific homocytotropic antibody synthesis was detected in uninfested control rats. The kinetics of synthesis of the potentiated antibody by cells correlated well with the levels of anti-ovalbumin IgE antibodies in the sera of the infested rats. A traffic of cells secreting anti-ovalbumin homocytotropic antibody was detected in the thoracic duct lymph, but not the mesenteric lymph of immunized uninfested rats. After infestation, the mesenteric lymph also contained cells secreting potentiated antibody. The mesenteric lymph is a major route by which IgE and potentiated IgE antibodies reach the circulation in infested rats. The possible mechanisms responsible for the effects of the parasite on antibody secretion in distant lymphoid organs are discussed.