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. Zatz

Table 3. Effect of irradiation on trapping in lymph nodes

Recipient treatment (R) 0 200 850 Saline Left* 0-26+ 0-06 0 35+ 0-02 0 39+ 0-06 Right* 0-26+0 003 0-36+ 003 0 33+ 003 Left/right 1.0 0 97 1-18 Left-right 0 0 01 0-06 Pt n.s. n.s. n.s. SRBC Left* 0 77+ 0 03 0-81 + 0 05 0-48+ 0-06 Right* 0-22+ 0-02 0-28+ 0-02 0 40+ 003 Left/right 3 50 2-89 120 Left-right 0-55 0 53 0-08 Pt <00001 < 0001 n.s.

* Values given are percentage localization of labelled cells in left and right popliteal nodes. Results are based on two ex- periments containing four mice per group. t Significance of difference between localization in left and right nodes; n.s. = not significant.

Table 4. Effect of irradiation on trapping in spleen

Recipient treatment (R) 0 200 850 Saline* 15-7+0-6 15 4+0-5 14 2+1 0 SRBC* 19 5+0 8 21-1+1 0 18-6+1-1 Per cent SRBC/saline 124 2 137 0 131-0 SRBC- saline 3-8 5-7 4-4 Pt <0-001 <0-001 <0-01

* Values given are mean + s.e. for percentage localization of labelled cells in spleen. Results are based on three experiments containing three to six mice per group. t Significance of difference between localization in spleens of SRBC- and saline-injected mice. suggesting that different populations may be trig- In the spleen, an ATS and irradiation-resistant gering and/or regulating this response to antigens in splenic cell population is responsible for the expres- these two organs, possibly via different trapping sion of the lymphocyte trap. It was probably the mechanisms. Similar results have been obtained existence of this resistant population in spleen, and following treatment with cortisone (Zatz, 1975). the failure to totally abolish trapping in lymph nodes The magnitude of the lymphocyte trap in lymph following irradiation, that led Frost and Lance nodes appears to be critically dependent upon the (1974) to conclude that trapping is a T cell-indepen- presence of an ATS and irradiation sensitive dent phenomenon. The present experiments indicate, lymphocyte. Since even small doses of ATS can however, that, in addition to the ATS and irradia- diminish the lymph node trap, the compromised tion-resistant population (which might still represent population is most likely that of the recirculating a subset of T cells), there is a sensitive set of cells T cell. with the ability to limit the magnitude of the splenic Lymphocyte trapping 753 trap. Thus in ATS- and 200 R-treated mice, the acknowledged. This work was supported by a grant magnitude of trapping is actually enhanced. The from the U.S.P.H.S., number CA-14216. sensitivity of these cells to small doses of ATS sug- gests that they too belong to the recirculating T-cell pool. The cells participating in the trap in both REFERENCES lymph node and spleen are not dependent upon a continuing thymic presence, since adult thymectomy ASHERSON G.L. & BARNES R.M.R. (1973) The role of im- alone or in combination with ATS does not further munological inflammation in the movement of lympho- cytes to immunized lymph nodes. Immunology, 24, 885. influence the data (unpublished observations). CANTOR H. & ASOFSKY R. (1972) Synergy among lymphoid The existence of a splenic T cell in spleen, but not cells mediating the graft versus host response. III. J. exp. in lymph node, capable of suppressing the magni- Med. 135, 764. tude of the lymphocyte trap would help to explain DRESSER D.W., TAUB R.N. & KRANTZ A. (1970) The effect other observations (Zatz and Gershon, 1975) of of localized injection of adjuvant material on the draining lymph node. II. Circulating lymphocytes. Immunology, regulation of lymphocyte trapping. Those studies 13, 553. show that antigen can induce a net decrease in EMESON E.E. & THURSH D.R. (1971) Immunologically lymphocyte traffic to spleen, i.e. a 'negative trap', specific retention of long lived lymphoid cells in antigenic- and that thymocyte subpopulations can enhance or ally stimulated lymph nodes. J. Immunol. 106, 635. the size of a the FORD W.L. (1972) The recruitment of recirculating lympho- suppress splenic trap induced during cytes in the antigenically stimulated spleen. Clin. exp. course of a graft vs host response. Immunol. 12, 243. Since the mode of lymphocyte traffic through FROST P. & LANCE E.M. (1973) Immunopotentiation. 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(1968) Migration of reconcile claims of macrophage dependence (Frost lymphocytes and thymocytes in the rat. I. J. exp. Med. 127, and Lance, 1974), and of T-cell dependence (Zatz 155. HALL J.G. & MORRIS B. (1965) The immediate effect of and Gershon, 1974, 1975; Taub and Gershon, 1972) antigens on the cell output of a lymph node. Brit. J. exp. of lymphocyte trapping, and also to suggest a cellu- Path. 45, 450. lar basis for recent findings of both a negative trap ROWLEY D.A., GOWANS J.L., ATKINS R.D., FORD W.L. & and bidirectional enhancement and suppression of a SMITH M.E. (1972) The specific selection of recirculating positive trap in spleen (Zatz and Gershon, 1974), as lymphocytes by antigen in normal and preimmunized rats. J. exp. Med. 136, 499. follows: Trapping in lymph nodes is initiated by an TAUB R.N. & GERSHON R.K. (1972) The effect of localized ATS-sensitive T! cell, which directly activates the injection ofadjuvant material on the draining lymph node. trapping mechanism; an ATS- and irradiation- III. Thymus dependence. J. Immunol. 108, 377. resistant T cell or macrophage plays only a minor ZATZ M.M. (1975) Effects of cortisone on lymphocyte hom- ing. Israeli J. Med. Sci. (Special issue on the lymphocyte role, if any, in this process. In spleen, an ATS- and and its membrane) (In press.) irradiation-resistant T cell activates the trapping ZATZ M.M. & GERSHON R.K. (1974) Thymus dependence of mechanism, possibly via a macrophage intermediate. lymphocyte trapping. J. Immunol. 112, 101. However, the magnitude and duration of this trap is ZATZ M.M. & GERSHON R.K. (1975) Regulation of lympho- subject to regulation by T cells. cyte trapping: the negative trap. J. Immunol. 115, 450. ZATZ M.M. & LANCE E.M. (1971) The distribution of 51Cr- labeled lymphocytes into antigen stimulated mice. J. exp. Med. 134, 224. ACKNOWLEDGMENTS ZATZ M.M., WHITE A. & GOLDSTEIN A.L. (1973) Alterations in lymphocyte populations during tumorigenesis. I. Lymphocyte trapping. J. Immunol. 111, 706. The excellent assistance of Susan M. Baker in the per- formance of these experiments is very gratefully