Eugene H. Perkins

The effect of age on antigen retention in lymphoid follicles and in collagenous tissue of mice

Our major objectives were to determine (1) when mice develop the cellular mechanisms necessary to trap and retain immune complexes in lymphoid follicles, (2) whether the ability to trap and retain immune complexes in lymphoid and collagenous tissues was maintained in old animals, and (3) whether the pattern of antigen localization in lymphoid follicles was altered as a consequence of ageing. Mice were passively immunized with a standardized amount of specific antibody and then challenged in the foot pads with radioactive antigen. The results indicated that newborn, and 1- and 2-week-old BALB/c mice lacked the cells or cellular mechanism necessary for trapping, localizing, and retaining immune complexes in lymphoid follicles. By 3 weeks, however, this ability to trap, retain, and localize antigen became apparent. The ability to trap and retain immune complexes on tendons and in lymphoid tissues was maintained as long as 27-30 months. A comparison of the quantity of antigen retained per mg of tissue in young-adult (6 months of age) and in old (27-30 months of age) mice indicated that old mice retained slightly more antigen in lymphoid tissues and substantially more on tendons. Antigen retained in spleens of both young-adult and old mice was localized in follicles by 24 h. However, the antigen retained in lymph nodes of old mice remained in the subcapsular sinus and adjacent superficial cortex and was not localized in follicles even after a full week. The cellular mechanisms necessary to trap and retain immune complexes in lymphoid tissue appear to develop in BALB/c mice a few weeks after birth and persist throughout life. However, localization of antigen in the lymphoid follicles of lymph nodes diminishes in old mice. The antigen trapping capability of collagenous tissues was not only maintained in old mice but was substantially increased.

A multiple-parameter comparison of immunocompetence and tumor resistance in aged balb/c mice.

Several parameters of cell-mediated and humoral immunity were measured in young-adult and aged BALB/c mice and compared with resistance to the ascites form of intraperitoneal induced P815 mastocytoma. It was found that the age-related decline of in vitro phytohemagglutinin (PHA) responsiveness by spleen cells approximated the marked decrease of old mice to tumor-cell challenge and approached that characteristically seen in humoral immunity. Thus, decreased PHA responsiveness of splenic lymphocytes provided as sensitive an estimate of the age-related decline of immunocompetence in old mice as other classical parameters of cell-mediated immunity (e.g. graft-vs-host reaction or in vivo cellular proliferation of parental spleen cells in lethally-irradiated F1 recipients). Results could be interpreted to represent a decreased ability of noncycling T-cells to be released to a functional cycling state.

Antigen presentation by peritoneal macrophages from young adult and old mice

Abstract Macrophages perform vital inductive and regulatory functions in immune processes and host defense mechanisms. However, macrophage function during senescence has not been extensively studied. Although antibody response is dramatically reduced in old animals, antigen presentation has never been directly assessed. Therefore, the antigen-presenting capabilities of purified peritoneal macrophages from young adult and old mice were studied by quantitatively measuring their ability to induce antigen specific proliferation of lymph node T lymphocytes. Increasing numbers (10 2 to 10 5 ) of macrophages from nonimmunized young adult (3 to 6 months) or aged (27 to 36 months) animals were cultured in the presence of antigen with a constant number (2 × 10 5 ) of column-separated popliteal lymph node cells from young adult mice. The latter had been immunized with the dinitrophenyl conjugate of bovine γ-globulin in complete Freunds adjuvant by footpad injection. Macrophages from old animals were equal to macrophages from young adult animals in stimulating T-lymphocyte proliferation, and the kinetics of incorporation was identical with increasing numbers of macrophages from either young adult or old animals. However, greater numbers of resident or induced peritoneal macrophages were always harvested from old animals. Differences in macrophage activity as assessed by different functional parameters may be reconciled by implicating subpopulations of macrophages that perform separate functions e.g., Ia-positive antigen presenter and Ia-negative scavenger macrophages.

A positive correlation between declining immune competence and early mortality associated with diethylnitrosamine carcinogenesis in aging mice

The effect of age (2.5, 9.5, and 17 months) at time of treatment upon diethylnitrosamine (DEN) carcinogenesis and immune competence has been assessed in female BALB/c mice. Median times of death were 193, 168, and 125 days, respectively, after termination of DEN treatment. Immune competence as a measure by both cell-mediated and humoral immune parameters immediately after DEN treatment was not significantly different among treated and age-matched non-treated control animals. In contrast, a significant age-related decline in immune competence was seen in both DEN-treated and non-treated controls, thereby demonstrating a direct and positive correlation between the natural age-related decrease in immune competence and cancer-induced advanced mortality.

The late effects of selected immunosuppressants on immunocompetence, disease incidence, and mean life-span II. Cell-mediated immune activity

The effect of different immunosuppressive treatments during young adulthood or humoral immune competence late in life was determined. It was found that the marked reduction in humoral immune competence in aged mice is further compromised when severe insults are administered early in life. Thus, thymectomy, splenectomy, and sublethal X-irradiation produced lasting immunodepression as measured (1) in situ and (2) by the hemolysin, direct and indirect plaque forming cell responses of adoptively transferred spleen cells. In contrast, treatment with cyclophosphamide and cortisone acetate were without effect, indicating that drug-damaged cells of the immune system were replaced by competent cells during the course of time. Decrease in immune competence of aged thymectomized animals could not be correlated with a decrease in numbers of theta-bearing T or immunoglobulin receptor-bearing B lymphocytes. The significance of the observed unequal effects of these immunosuppressants on immune competence, as they relate to disease incidence and life expectancy, are dealt with in the third paper in this series.

The late effects of selected immunosuppressants on immunocompetence, disease incidence, and mean life-span. III. Disease incidence and life expectancy.

The effect of various immunosuppressive treatments on mean life-span and disease incidence have been studied. Significant life shortening was seen only in mice which recieved X-irradiation early in life and can be ascribed primarily to an increased incidence of certain malignancies. Marginal life shortening was seen in cyclophosphamide-treated animals, however, survival patterns between those and control animals did not differ until 30 months of age and the magnitude of life-shortening never approached that seen in X-irradiated animals. Thymectomy, splenectomy or cortisone treatment did not alter survival. All immunosuppressive treatments enhanced mortality due to non-neoplastic diseases, however, only a small percentage of animals die with these disease entities. With the exception of cortisone all immunosuppressive treatments increased the incidence of neoplastic disease. However, their effects on various neoplastic processes were variable and unpredictable. Four primary patterns in terms of relative immune competence, disease incidence and life expectancy were seen. Thus, immunodepression may of may not correlate with increased disease incidence, which in turn may or may not have a life-shortening effect. These findings are discussed in terms of the marked reduction of both humoral and cell-mediated immunity normally seen in aged mice and the significance of postulated immune surveillance mechanisms to survival.

The Engulfing Potential of Peritoneal Phagocytes of Conventional and Germfree Mice

It is not known what role naturally occurring microbial flora play in the development of the phagocytic capacity of peritoneal phagocytes. A quantitative comparative study of phagocytes from conventional and germfree animals is therefore desirable. With the development of a simple quantitative method, we have been able to measure the in vitro engulfing potential of peritoneal cells from different strains of mice raised in conventional or germfree environments. In the work to be presented we found no difference in the engulfing potential of peritoneal phagocytes from conventional and germfree mice. The importance of a defined population of cells and quantitative techniques are stressed, and the in vitro functional maturation of peritoneal phagocytes is reported.

The proliferative capacities of popliteal lymph node lymphocytes and of their functionally distinct T-cell subpopulations from young-adult and old mice

Young adult and old mice were immunized by footpad injection of dinitrophenyl-conjugated bovine gamma-globulin (DNP-BGG) in complete Freunds adjuvant. A comparison of lymph node weight and total number of nucleated cells per lymph node as a function of time after antigen injection demonstrated a significantly greater absolute increase in lymph node weight and peak number of nucleated cells per lymph node in young-adult than in old animals. However, as judged by this increase in total nucleated cells, other than being delayed in old mice, the magnitude of these in situ proliferative responses appeared comparable for young-adult and old mice. That is, the antigen-stimulated to non-stimulated cell ratio did not differ significantly between young-adult and old animals. This was because lymph nodes from old animals prior to antigen injection always weighed less and had fewer numbers of nucleated cells compared with young-adult animals. Therefore, the in vitro cellular proliferative response of three T-cell-enriched lymphocyte subpopulations from young-adult and old mice was further characterized. This was done by measuring [3H]thymidine incorporation following antigen- (DNP-BGG)- or mitogen-[phytohemagglutinin (PHA) or Concanavalin A (Con A)]-induced proliferation and assessing their quantitative and/or qualitative requirements for macrophages. In contrast to the markedly reduced proliferation of the two T-cell subpopulations from popliteal lymph nodes which respond to PHA and Con A in old animals primed 21-days earlier with DNP-BGG, antigen-induced in vitro cellular proliferation of the small T-cell subset in old mice specifically responsive to the immunizing antigen DNP-BGG always responded as well as, if not better than, cells from young-adult mice.

Genetic constraints in the induction of the immune response to ehrlich ascites tumor in mice

A single injection of irradiated Ehrlich ascites tumor (EAT) cells induces immunity in normal mice but fails to do so in T-cell-deficient-thymectomized, lethally irradiated, bone marrow-reconstituted (TIR) mice. TIR mice injected with normal syngeneic T cells develop an immune response to EAT when injected with irradiated EAT cells and reject a subsequent tumor cell challenge. In the present studies allogeneic T cells were unable to protect against EAT in TIR recipients even if harvested from donors tolerant to the recipients transplantation antigens and injected into the TIR mice tolerant to the transplantation antigens of the injected T cells. Tolerance was produced by establishing long-term radiation chimeras of the P → F1 type. Semiallogeneic T cells also failed to afford protection against EAT in TIR recipients. Whereas tolerance to other parental-strain transplantation antigens did not reverse the inability of parental T cells (cells from P → F1 chimeric donors) to protect against EAT in F1 TIR mice, it did enable F1 T cells to afford protection in P → F1 TIR mice.