Lymphocyte Trapping
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Immunology 1976 30 749 Lymphocyte trapping DIFFERENTIAL EFFECTS OF ATS AND IRRADIATION ON TRAPPING IN LYMPH NODES AND SPLEEN MARION M. ZATZ Departments of Microbiology and Surgery, Yale University School of Medicine, New Haven, Connecticut, U.S.A. Received 20 August 1975; acceptedfor publication 13 November 1975 Summary. Increased sequestration (trapping) of lymphocytes in lymphoid organs (Zatz and Lance, lymphocytes occurs in lymphoid organs following 1971; Ford, 1972; Hall and Morris, 1965; Rowley, antigenic challenge. The effects of irradiation and Gowans, Atkins, Ford and Smith, 1972). This anti-thymocyte serum (ATS) upon lymphocyte sequestration of cells has been termed lymphocyte trapping in lymph nodes and spleen were studied. 'trapping' (Zatz and Lance, 1971). Both the above agents diminished or abrogated It seems likely that lymphocyte trapping indirectly trapping in the draining lymph nodes; these same plays an important role in vivo in the regulation of a agents resulted in enhanced trapping in the spleen. broad spectrum of immune responses, via control of Suppression of lymph node trapping could be lymphocyte traffic (Zatz and Lance, 1971; Ford, achieved with a low dose of ATS and with 850 R, 1972; Hall and Morris, 1965; Rowley et al., 1972; but not 200 R. Emeson and Thursh, 1971; Dresser, Taub and These results are interpreted as showing that Krantz, 1970; Zatz and Lance, 1971; Zatz, White lymphocyte trapping in lymph nodes is initiated and Goldstein, 1973; Gershon and Fightlin, 1973; primarily by an ATS- and irradiation-sensitive Gillette and Bellanti, 1973; Asherson and Barnes, population, whereas in the spleen a cell population 1973; Frost and Lance, 1973; Zatz and Gershon, resistant to the above agents activates the lymphocyte 1974). However, relatively little is known about the trap. These data demonstrate that the cell popula- mechanisms and cell subpopulations which are tions and/or the mechanisms involved in lymphocyte involved in production of the lymphocyte trap. trapping in lymph nodes and spleens are distinct. While the majority of cells that are trapped lack antigenic specificity (Zatz and Lance, 1971; Rowley et al., 1972), evidence suggests that the trigger for INTRODUCTION initiation oftrapping involves activation ofT cells by antigen (Taub and Gershon, 1972; Zatz and Ger- In vivo administration of antigen results, within shon, 1974) and that the ensuing sequestration of hours, in a transient sequestration of circulating large numbers of circulating lymphocytes serves to Correspondence: Dr Marion M. Zatz, Immunology concentrate antigen-reactive cells at the major site of Branch, National Cancer Institute, Bethesda, Maryland antigen depot. Previous reports by Frost and Lance 20014, U.S.A. (1973, 1974) have suggested that the macrophage is 749 750 Marion M. Zatz the cell responsible for trapping and that T cells are groups of mice. Twenty-four hours following injec- unimportant for eliciting this phenomenon. In con- tion of labelled lymphocytes, the recipients were trast, other studies (Zatz and Gershon, 1974, 1975) killed, and the percentage of injected radioactivity clearly show that T-cell activation is required for localizing in the left and right popliteal lymph nodes production and regulation of the lymphocyte trap. (following f.p. antigen injection) and in the spleen In order to try to clarify the nature of the cell (following i.v. antigen injection) was determined. populations involved in lymphocyte trapping, the In studies of spleen trapping the results are ex- effects of anti-thymocyte serum and irradiation upon pressed for each immunosuppressed or normal the antigen-induced sequestration of lymphocytes in group as a percentage ratio of " Cr-labelled cell spleens and lymph nodes were investigated. localization in the spleens of SRBC-injected/saline- injected mice. In the studies of lymph node trapping, the results are expressed as a ratio of " Cr cell MATERIALS AND METHODS localization in the left (draining ipsilateral)/right (contralateral) popliteal nodes. Since pretreatment of Animals mice with ATS generally diminishes the baseline Experiments were performed with BDF1 mice of localization of 5'Cr-labelled lymphocytes in control either sex, obtained from Jackson Laboratories, Bar animals, the data were also analysed for a specific Harbor, Maine. Following arrival, mice were rested increase in 5'Cr cell localization by the formula: for at least 1 week in the animal quarters prior to use. (percentage localization in spleen of SRBC-injected Mice were used at 6-8 weeks of age. mice) -(per cent localization in spleen of saline- injected mice). For studies of lymphocyte trapping in Cell suspensions lymph nodes, a specific increase was calculated by Lymphocyte suspensions were prepared from the the difference in per cent "'Cr cell localization in the peripheral and mesenteric lymph nodes of normal left-right popliteal nodes. donors, and labelled in vitro with "Cr as previously described (Zatz and Lance, 1971). Statistics The significance of the differences between groups Antigens was determined using Student's t-test. P values of SRBC (Gibco, Grand Island, New York) were washed < 0-05 are considered significant. three times in saline and diluted to a 25 per cent final suspension (v/v). 0 2 ml of antigen was injected i.v., RESULTS and 0 05 ml was injected f.p. Effects of ATS upon lymphocyte trapping in spleen Immunosuppressive agents and lymph nodes ATS (Microbiological Associates, Bethesda, Mary- land) was injected s.c. in the mid-dorsal region. This Distinct effects of ATS upon SRBC-induced trap- ATS preparation kills 30-40 per cent of spleen cells, ping in spleen and draining nodes were observed 60-80 per cent of lymph node cells, and greater than (Tables 1 and 2). Whereas prior treatment with ATS 90 per cent of thymocytes in a "1Cr release cytotoxic totally abrogated lymphocyte trapping in the left assay. Whole body irradiation was delivered from a popliteal nodes, trapping in spleen was augmented. Siemens 250 kV machine at a dose rate of 85 R/min, This enhancement of splenic trapping in ATS- and a distance of 70 cm. Under these conditions, treated mice was significant (P < 0 001). Similar 850 R is a lethal dose. results were obtained with a low (0-05 ml) dose of ATS. Experimental protocol Since residual ATS in the circulation of recipients Mice were pretreated with ATS on day -3, or with can deplete the lymph node-seeking population of irradiation on day -1. Each group of immuno- injected lymphocytes, it was necessary to determine suppressed animals was given antigen or saline at whether ATS-induced abrogation of lymph node - 24 h in the left footpad (f.p.) or at - 6 h i.v. At 0 h, trapping was attributable to loss of the lymph node 5 x 106 5"Cr-labelled BDF1 lymph node cells, ob- seeking population, rather than to an influence of tained from normal donors, were injected i.v. into ATS directly on trapping in lymph nodes. Therefore, Lymphocyte trapping 751 Table 1. Effect of ATS treatment on lymphocyte trapping in draining nodes Recipient treatment NRS (0 40 ml) ATS (0 40 ml) ATS (0-05 ml) Saline SRBC Saline SRBC SRBC Left popliteal node* 0-28+0-03 1-10+0-07 0-13+0-01 0-16+0-04 0-17+0-02 Right popliteal node* 0-26+0-02 0-36+0 03 0 11+0 01 0-14+0 04 0-23+0-04 Left/right 1-08 3-55 1-18 1-14 0 74 Left-right 0-02 0 74 0-02 0-02 -0-06 Pt n.s. < 0 001 n.s. n.s. n.s. * Values given are the mean + s.e. percentage localization of labelled cells in the left and right popliteal nodes. Results are based on four to six experiments containing three or four mice per group. t Significance of difference between localization in left and right nodes; n.s. = not significant. Table 2. Effect of ATS treatment on lymphocyte trapping in spleen Recipient treatment NRS ATS (0 04 ml) ATS (0 05 ml) Saline* 21-0+ 0 5 10-7+ 0 5 10 9+ 0 4 SRBC* 26-2+0-6 20-5+ 1-5 18-4+0-4 Percentage SRBC/saline 125-0 192-0 169-0 SRBC-saline 5 2 9-8 7-5 Pt < 0-001 < 0-001 < 0-001 * Values given are mean+ s.e. of percentage localization of labelled cells in spleen. Results are based on four experiments containing four mice per group. t Significance of difference between localization in spleens of SRBC- and saline-injected mice. 51Cr lymph node cells from ATS-treated donors trapping response (Table 4). 200 R was without (already depleted of their recirculating cell popula- effect upon lymph node trapping, but consistently tion) were compared to 51Cr lymph node cells from caused a significant (P < 0-05) enhancement of trap- NRS-treated donors in their ability to be trapped in ping in spleen. Thus both irradiation and ATS pre- NRS- or ATS-treated recipients. The results (data treatment selectively eliminated lymphocyte trapping not shown) reveal that, regardless of whether 51Cr in draining lymph nodes. lymphocytes are obtained from NRS- or ATS- treated donors, SRBC-induced trapping was abro- DISCUSSION gated in lymph nodes of ATS-treated recipients, but enhanced in spleens of the same mice. Earlier studies have demonstrated that lymphocyte trapping in both lymph nodes and spleen is, at least Effects of irradiation upon lymphocyte trapping in partially, a result of a T-cell response to antigen spleens and lymph nodes (Zatz and Gershon, 1974), although conflicting results have suggested that trapping is solely macro- The radiation sensitivity of lymphocyte trapping was phage dependent (Frost and Lance, 1974). The investigated. High (850 R) dose irradiation eliminated present studies reveal that lymphocyte trapping in trapping in draining lymph nodes (Table 3), al- spleen and lymph nodes differs markedly in its though the same treatment failed to alter the splenic sensitivity to modification by ATS and irradiation, 752 Marion M.