Leonard L. Seelig

The temporal relationship among anti-parasite immune elements expressed during the early phase of infection of the rat with Trichinella spiralis

Abstract Immunological parameters were measured during the first 20 days of infection with Trichinella spiralis in the rat. Expulsion of adult worms was complete by day 15 postinfection. Eosinophil and neutrophil numbers rose in the blood of infected rats above preinfection levels on days 3 and 6, respectively, and remained high to day 20 postinfection. Release of cytokines by Trichinella-antigen-stimulated mesenteric lymph node cells was measured, and a significant elevation in interferon (IFN)-γ release was detected during the early stage of infection. Although initiated later, interleukin (IL)-10 release showed a pattern similar to IFN-γ. Biphasic release of IL-5 was seen with significant elevation above the preinfection level on day 3 and after day 6 postinfection to the end of the study. IL-4 and IL-2 showed biphasic secretion as well, with the level of IL-4 high in the early and middle part of infection, while the level of IL-2 was detectable only at days 2, 3 and 6 postinfection. Serum anti-parasite IgE rose above preinfection levels after day 6 postinfection. Anti-parasite Ig-positive mesenteric lymph node (MLN) cells were evident by day 3 postinfection for IgM, and day 9 postinfection for IgA and total IgG. The number of Ig-positive MLN cells for all antibody classes returned to preinfection levels by day 20 postinfection. Evaluation of the temporal interactions of the key anti-parasite immune components with which the host engages Trichinella shows a complex interplay between Th1 and Th2 helper subsets.

Distribution of lymphocyte subsets in rat milk from normal and Trichinella spiralis-infected rats

We have shown that T. spiralis-specific T lymphocytes can mediate maternal-to-neonatal immunity during lactation. This study addresses the change of lymphocyte populations in rat milk during normal and disease conditions. Two color flow cytometric analysis was performed for milk lymphocytes. T cells (OX19+) made up 45% of rat milk lymphocyte population. T helper cells (Th) composed 35% of total T cells while T cytotoxic/suppressor (Tcs) cells constituted 34%, giving a Th/Tcs ratio of 1.03. The corresponding ratio Th/Tcs in peripheral blood was 2.8. Approximately 21% of OX8+ cells in rat milk were OX19- natural killer (NK) cells. When using the monoclonal antibody 3.2.3 (NKR-P1), 43% of lymphocytes in control rat milk and 14% of blood lymphocytes were NK cells. This indicates a selective passage of these cells into milk. In T. spiralis-immunized rats, the percentage of total T cells was slightly decreased; however, Th and Tcs cells were consistent as compared to control milk. The percentage of NK cells (OX8+OX19-) in milk from T. spiralis-immunized rats was significantly higher than that from control milk (65% vs. 21%, respectively, P less than 0.01). This result was confirmed using the monoclonal antibody 3.2.3 which showed that milk from immunized rats contained 63% NK cells compared to 43% in normal milk (P less than 0.01). This study suggests that NK cells are selectively passaged into rat milk and T. spiralis infection induces an increase of NK cells in milk.

In vitro TNF, IL-6 and IL-2 production by rat milk cells following Trichinella spiralis infection

Trichinella spiralis-specific immunity can be transferred from immune mothers to suckling neonates via lactation, suggesting that milk from immune dams contains specific factors to T. spiralis. TNF and IL-6 are important cytokines in inflammatory processes, and IL-2 is essential in lymphocyte activation. Using cell line bioassays, we examined the capacity of rat milk mononuclear cells from immune and non-immune control dams to produce these cytokines following in vitro stimulation with mitogens or T. spiralis antigen. Milk cells were capable of producing IL-2, IL-6 and TNF upon Con A stimulation, and TNF and IL-6 upon LPS stimulation. The amount of these cytokines produced by mitogen-stimulated milk cells from T. spiralis-infected rats was similar to that produced by non-infected controls, although lower than that of corresponding blood mononuclear cells. Upon stimulation with T. spiralis antigen, milk cells from infected rats produced a significantly higher amount of TNF (158 +/- 39 U/10(6) cells) compared to non-infected controls (16 +/- 6 U/10(6) cells, P < 0.01), and also higher than that of the corresponding blood cells (60 +/- 10 U/10(6) cells, P < 0.01). Only small amounts of IL-6 and no IL-2 was secreted by milk cells from control or infected groups after stimulation with antigen. This study shows that rat milk cells are capable of synthesizing cytokines, and TNF produced by immune mothers may play a role in augmenting the neonate resistance to T. spiralis infection.

Immunohistochemical analysis of the stage-specific expression of Ia antigens in the rat mammary gland during pregnancy and lactation

The expression of Class II MHC antigens in the rat mammary gland was assessed by immunoperoxidase using monoclonal antibodies against rat homologues of the murine I-A and I-E antigens. Our results showed that, while I-A and I-E were inconspicuous in undifferentiated mammary epithelium in virgin rats, during early pregnancy, the ductal epithelium becomes intensely positive for both antigens. A monoclonal antibody specific for macrophages showed that they were abundant in the stroma but did not correspond to the epithelial staining. During late pregnancy and lactation, the ductal epithelium expressed varying levels of Ia, while the alveolar epithelial cells were consistently Ia-negative. Ia-positive mononuclear cells of round or dendritic morphology present in the alveolar areas were enumerated as cells per high power field (HPF). Maximal cell counts for I-A and I-E were seen in late pregnancy and early lactation, with a significant decline in mid-lactation. Counts were higher in intraepithelial locations than in the subjacent connective tissue at all stages. The possible role of these stage-specific alterations in regulating local immune responses and transfer of immune components into milk is discussed.

Immunohistochemical comparison of T cell and macrophage population in mammary tissue of control and Trichinella spiralis infected rats

We have shown that antigen-specific T lymphocytes can mediate maternal-to-neonatal immunity during lactation. Present studies address the dynamics of lymphocyte accumulation in the mammary gland during normal and disease stimulated conditions. Monoclonal antibodies specific for total T cells, suppressor/cytotoxic and helper subsets, and macrophages were used in conjunction with immunohistochemistry to identify and count the individual cell types. In unstimulated mammary tissue, following a rise in T cells to maximal numbers in late pregnancy, the total number of T cells/high power field (HPF) was significantly diminished in early lactation and continued to decline to the late lactation period. Both the numbers of T cells/HPF located in the mammary alveolar epithelium and surrounding connective tissue were significantly reduced in early lactation as compared to late pregnancy. This indicates the possible passage of cells into the milk during lactation. Prior infection of the mother with Trichinella spiralis and a secondary challenge 48 h. before sacrifice caused a significant reduction in the number of T cells in the mammary tissue in early lactation as compared with unstimulated controls, indicating the possibility of an even greater outflow of T cells into milk. In controls, the T-suppressor/cytotoxic subtype showed a reduction in early lactation versus late pregnancy but showed no shifts in total cells/HPF during infection. The T-helper subtype in controls remained unchanged from late pregnancy to early lactation with a considerable decline in late lactation. However, the T-helper cells were significantly decreased in T. spiralis-treated animals as compared with noninfected controls in early lactation.(ABSTRACT TRUNCATED AT 250 WORDS)