Delayed development of immunological responsiveness in neonatally thymectomized mice: a time-course study

Haemolysin responses to first injection of sheep erythrocytes in neonatally thymectomized, neonatally sham-thymectomized and intact Swiss albino mice were tested when the mice were 10 days, 4 weeks, 6–7 weeks and 6 months of age. The serum haemolysin activity was assessed at a number of times after injection of antigen (time-course study). Neonatal thymectomy of Swiss mice was followed by a decreased and delayed haemolysin response. These abnormalities in antibody response following neonatal thymectomy became less obvious when the age at which the mice were injected was increased, indicating that delayed development of immunological responsiveness had occurred in neonatally thymectomized Swiss mice.     

Delayed development of primary immunological responsiveness in neonatally thymectomized swiss albino mice

Neonatally thymectomized Swiss mice exhibit a delayed development of immunological responsiveness. The behaviour of 19S and 7S haemolysin during the response that eventually arises in thymectomized mice is consistent with that of a primary response with the major abnormality being the delayed production of maximal amounts of 7S haemolysin.     

The immunological responsiveness of germ-free mice thymectomized at birth. II. Lymphoid tissue and histopathology

The effect of neonatal thymectomy and antigenic stimulation on the lymphoid cell population has been studied in germ-free mice. Neither thymectomy nor injection of sheep erythrocytes induced any significant alteration in the blood lymphocyte levels. There was a clear-cut reduction in the cellularity of the periarteriolar lymphocyte sheaths of the spleen and of the paracortical regions of the lymph nodes in the thymectomized mice. Following stimulation with sheep erythrocytes, large pyroninophilic cells appeared in these areas in the intact germ-free controls but in only a few thymectomized mice and then in reduced numbers. Thymectomy did not influence the cellularity of the lymphoid follicles but less germinal centre and plasma cell activity occurred in response to an injection of sheep erythrocytes. Lesions suggestive of autoimmune reactivity were not found in lymphoid or nonlymphoid tissues of neonatally thymectomized germ-free mice. Lesions typical of viral infections were seen in some germ-free mice in both thymectomized and intact groups. It is concluded that the specific defect associated with the absence of the thymus is a reduction in a particular class of lymphocytes the development of which is under thymus control and the activities of which are to mediate certain defined immunological responses.     

The immunological responsiveness of germ-free mice thymectomized at birth. I. Antibody production and skin homograft rejection

The responses of conventional and germ-free mice thymectomized at birth to sheep erythrocytes and to skin homografts have been investigated. Antibody production to sheep erythrocytes was depressed to an equal extent in thymectomized germ-free and conventional mice. Skin graft rejection in thymectomized germ-free mice was impaired in some cases although the extent of the impairment was generally not as extreme as it was in thymectomized conventional mice. The implications of the results are discussed. It is concluded that a primary consequence of thymectomy is some degree of immunological impairment but that other factors operating in the conventional state, such as bacterial contamination, endotoxins and cross-reacting antigens, act to reduce even further the number of antigen-reactive cells available in an already limited pool of immunologically competent cells.     

The mechanism of reduction of cell-mediated cytotoxicity in neonatally thymectomized mice.

The effect of neonatal thymectomy at various times after birth (Tx-1, Tx-7) on effector and suppressor T cells responsible for cell-mediated cytotoxicity (CMC) for allogenic antigens was determined. Following in-vitro primary mixed lymphocyte cultures, in the absence of T-cell growth factor (TCGF), alloreactive CMC was not detected in spleen cells of Tx-1 mice, but was detected in spleen cells of Tx-7 mice at as high levels as in those of sham-operated mice. However, in the presence of TCGF, as much alloreactive CMC was detected in spleen cells of Tx-1 mice as in those of Tx-7 mice. Furthermore, TCGF production was not detected in spleen cells of Tx-1 mice but was detected in those of Tx-7 mice. In in-vivo experiments, inhibition of allogeneic tumour growth and CMC in spleen cells showed the same pattern as in in-vitro experiments. These results support the concept that the reduction of CMC in Tx-1 mice might be due to a defect in helper function (TCGF-producing capacity) rather than to a defect in cytotoxic T lymphocytes and/or cytotoxic T lymphocyte precursors. Alloreactive suppressor T cells could not be induced in spleen cells of Tx-1 mice but were induced in spleen cells of Tx-7 mice. Therefore, it was suggested that alloreactive suppressor T cells require the presence of the thymus for 7 days after birth in their development.     

Pattern of distribution of syngeneic spleen cells in the lymphoid organs of neonatally thymectomized mice

Newborn mice were thymectomized and intraperitoneally inoculated with syngeneic spleen cells on days 0, 3, 7 and 20 after birth. Donor cells were chromosomally distinguishable from host cells (T6). Cytological analysis performed between days 2 and 118 post-natal revealed greatly increased proportions of donor cells in host lymph nodes if comparison were made with similarly injected non-thymectomized hosts. The effect is highly specific, since the proportions of donor cells in the host spleen and bone marrow remained similar to those in control, non-thymectomized mice (syngeneic standard). The mechanism behind this phenomenon is unclear.     

Immune-mediated cholangiohepatitis in neonatally thymectomized mice: the role of T cells and analysis of T-cell receptor Vbeta usage

We have previously reported the induction of immune-mediated cholangiohepatitis following injection of a hybrid recombinant proteins containing the E2 of the pyruvate dehydrogenase (PDC-E2) and the branched-chain keto-acid dehydrogenase (BCOADC-E2) to neonatally thymectomized (Tx) A/J mice. Further, we demonstrated that intrahepatic infiltrating mononuclear cells could transfer pathology to other Tx mice. To further our observations, we examined intrahepatic infiltrating mononuclear cells by flow cytometry and used cell transfer experiments to identify the phenotype involved. Interestingly, following immunization of neonatally Tx A/J mice and immunization with the bihybrid molecule, the number of CD3+infiltrating mononuclear cells were significantly higher (77.8%) compared with the control group. There was a small although not significant increase among intrahepatic infiltrating mononuclear cells and splenic cells of Vbeta 5.1,5.2+, Vbeta7+and Vbeta17+. In addition, Vbeta14+cells accounted for 20.4% of the infiltrating T-cells (P<0.01 vs. the control group). In further experiments, CD3+, CD4+or CD8+cells were isolated and removed from intrahepatic infiltrating mononuclear cells and subpopulations of mononuclear cells transferred to Tx mice. Both CD3+CD4+cells and CD3+CD8+cells are required for development of the lesion, and the damage is mediated by CD3+Vbeta14+cells.     

Distribution of immunoglobulin-containing cells in normal and neonatally thymectomized rats

The distribution of immunoglobulin-containing cells (ICC) of the immunoglobulin A (IgA), IgG, and IgM isotypes was examined in various lymphoid and secretory tissues of rats. The effect of neonatal thymectomy of rats on T cells, B cells, and ICC in these tissues was determined by immunofluorescence. The results showed that although T cells were severely depleted in both lymphoid and secretory tissues of the thymectomized (Tx) rats, Tx and normal rats showed comparable numbers of B cells staining for IgA, IgG, and IgM. After neonatal thymectomy, IgA ICC in both lymphoid and secretory tissues were significantly decreased. However, the Tx rats exhibited a compensatory increase in IgM ICC in the identical tissues. Local injection of normal and Tx rats with Streptococcus mutans 6715 resulted in an increase in all isotypes of ICC in the secretory tissues. Although the primary increase in normal rats was due to IgA ICC, Tx rats exhibited the greatest change in the number of IgM ICC.     

Self-reactive T cells are activated by the 65-kDa mycobacterial heat-shock protein in neonatally thymectomized mice

To elucidate the mechanism of autoimmune disease in neonatally thymectomized (NTX) mice, we have investigated the responsiveness of the self-reactive T cells which have not undergone clonal deletion in such animals. Consistent with a recent report (Yuuki et al., Eur. J. Immunol. 1990. 20: 1475), T cells bearing V beta 11-gene products capable of recognizing I-E-encoded molecules were readily detected in the mature T cell pool of NTX BALB/c (I-Ed, Mls-2a) mice. The V beta 11-bearing T cells in NTX mice expressed interleukin 2 receptors and responded normally to signals delivered through the T cell receptor. Notably, these T cells in NTX mice proliferated significantly after culture with the 65-kDa mycobacterial heat-shock protein, whose amino acid sequence is highly homologous to that in eukaryotes. These results suggest that self-reactive T cells in NTX mice may be activated by heat-shock proteins derived from various pathogens and/or stressed autologous cells, resulting in the development of autoimmune diseases in such animals.     

Protective immunity to Listeria monocytogenes in neonatally thymectomized (NTx) mice: involvement of T cells distinct from those in sham-thymectomized mice.

Neonatally thymectomized (NTx) mice, whose ability to mount antigen-specific cell-mediated immunity is reported to be generally defective, were found to be capable of mounting a normal level of acquired cellular resistance (ACR) and delayed footpad reaction (DFR) to Listeria monocytogenes. The present study was done in order to determine the functional differences of T cells contributing to the protection against L. monocytogenes between NTx and sham-operated mice. In mice immunized with viable L. monocytogenes, the absolute number of splenic T cells was significantly lower in NTx mice compared with sham-operated mice. When the ability of immune T cells to transfer ACR and DFR was examined by passive transfer, lymphocytes from immune NTx mice conferred a higher level of ACR and DFR on naive recipient mice, despite the marked difference in total number of T cells compared with immune Sham mice. Antigen-specific proliferation and interleukin-2 (IL-2) production by splenic T cells from immune NTx mice were significantly lower than in those from immune Sham mice. The proliferative response of T cells to exogenous IL-2 was also lower in NTx group. These results suggest that the requirement for the IL-2-driven T-cell proliferation system is basically low in the generation of effector T cells specific for L. monocytogenes.     

Effects of whole-body irradiation on neonatally thymectomized mice. Incidence of benign and malignant tumors.

The individual and combined effects of neonatal thymectomy and whole-body irradiation on the prevalence of benign and malignant tumors in germ-free female mice of the Charles Rivers line were studied to determine if a portion of the tumorigenic effects of irradiation can be attributed to injury of the thymic-dependent component of the immune response. Neonatal thymectomy increased a) the incidence of benign and malignant tumors and b) the prevalence of multiple primary neoplasms in an individual mouse. Whole-body exposure to 700 rad at 6 weeks of age further increased th incidence of tumors, but the relative magnitude of this increase was less pronounced than in sham-operated controls. Thus, the cumulative effects of thymectomy plus irradiation are less pronounced than the sum of the individual effects. One of several possible explanations for this observation is that a portion of the carcinogenic effects of whole-body irradiation is mediated by suppression of the thymic-dependent component of the immune response.     

Generation of Listeria monocytogenes-specific T cells mediating delayed footpad reaction and protection in neonatally thymectomized mice but not in nude mice

Neonatally thymectomized (NTx) mice, sham-operated control mice and congenitally athymic nude mice were immunized with viable Listeria monocytogenes and their spleen cells examined for the capacity to transfer both delayed footbad reaction and protection against challenge at the site of local transfer. Cells from immune NTx mice conferred significant degrees of delayed footpad reaction and protection comparable to sham mice, while cells from immune nude (nu/nu) mice did not. This abilty was completely eliminated by the treatment of cells with anti-Thy1, anti-Lytl or anti-L3T4 antibody plus complement but not with anti-Lyt2 antibody plus complement. These results indicated that NTx mice can normally mount the immunity to L. monocytogenes by generating Lyt1⁺2^–, L3T4⁺ T cells. Immune competence of NTx mice and thymus dependency of various immune responses are discussed.     

Spatio-temporal relation between cadherin switching and cytogenesis of hormone-producing cells in the developing rat adenohypophysis

Cadherins are a family of transmembrane glycoproteins that mediate cell-to-cell adhesion in solid tissues and have been reported to regulate not only morphogenesis but also cell motility, proliferation, and function by activating intracellular signaling pathways. We recently found that primordial cells in the developing rat adenohypophysis co-expressed E- and N-cadherins, but endocrine cells lost E-cadherin to possess only N-cadherin at certain embryonic stages. In the present study, we aimed to elucidate the temporal relationships between cadherin expression and cell proliferation as well as between cadherin expression and the onset of hormone production in embryonic adenohypophyses. Adenohypophyses and their primordia from embryonic and postnatal rats were fixed in Bouin’s fluid and paraffin sections were routinely prepared. Multiple fluorescence immunohistochemistry was performed for combinations of E-cadherin, N-cadherin, proliferating cell nuclear antigen (a marker of proliferating cells), cyclin D1, and pituitary hormones. In primordia from embryonic days 13 through 16, proliferative activities were seen in cells that co-expressed E- and N-cadherin. Cells arrested proliferation coincidentally when they lost E-cadherin after embryonic day 16. Possession of E-cadherin was closely related with expression of cyclin D1 at this stage. Moreover, hormone production was observed from embryonic day 16 only in cells that lost E-cadherin. In the developing adenohypophysis, proliferation and differentiation of hormone-producing cells have been reported to be regulated by a variety of external humoral factors. Our results raise the possibility that changes in cadherins are closely involved in these processes.