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 New Haven, Connecticut, U.S.A.
School
of Medicine,
Received 20 August 1975; acceptedfor publication 13 November 1975
Summary. Increased sequestration (trapping) of lymphocytes occurs in lymphoid organs following antigenic challenge. The effects of irradiation and anti-thymocyte serum (ATS) upon lymphocyte trapping in lymph nodes and spleen were studied. Both the above agents diminished or abrogated trapping in the draining lymph nodes; these same agents resulted in enhanced trapping in the spleen. Suppression of lymph node trapping could be achieved with a low dose of ATS and with 850 R, but not 200 R. These results are interpreted as showing that lymphocyte trapping in lymph nodes is initiated primarily by an ATS- and irradiation-sensitive population, whereas in the spleen a cell population resistant to the above agents activates the lymphocyte trap. These data demonstrate that the cell populations and/or the mechanisms involved in lymphocyte trapping in lymph nodes and spleens are distinct.
INTRODUCTION In vivo administration of antigen results, within hours, in a transient sequestration of circulating Correspondence: Dr Marion M. Zatz, Immunology Branch, National Cancer Institute, Bethesda, Maryland 20014, U.S.A.
749
lymphocytes in lymphoid organs (Zatz and Lance, 1971; Ford, 1972; Hall and Morris, 1965; Rowley, Gowans, Atkins, Ford and Smith, 1972). This sequestration of cells has been termed lymphocyte 'trapping' (Zatz and Lance, 1971). It seems likely that lymphocyte trapping indirectly plays an important role in vivo in the regulation of a broad spectrum of immune responses, via control of lymphocyte traffic (Zatz and Lance, 1971; Ford, 1972; Hall and Morris, 1965; Rowley et al., 1972; Emeson and Thursh, 1971; Dresser, Taub and Krantz, 1970; Zatz and Lance, 1971; Zatz, White and Goldstein, 1973; Gershon and Fightlin, 1973; Gillette and Bellanti, 1973; Asherson and Barnes, 1973; Frost and Lance, 1973; Zatz and Gershon, 1974). However, relatively little is known about the mechanisms and cell subpopulations which are involved in production of the lymphocyte trap. 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 initiation of trapping involves activation of T cells by antigen (Taub and Gershon, 1972; Zatz and Gershon, 1974) and that the ensuing sequestration of large numbers of circulating lymphocytes serves to concentrate antigen-reactive cells at the major site of antigen depot. Previous reports by Frost and Lance (1973, 1974) have suggested that the macrophage is
Marion M. Zatz
750
the cell responsible for trapping and that T cells are unimportant for eliciting this phenomenon. In contrast, other studies (Zatz and Gershon, 1974, 1975) clearly show that T-cell activation is required for production and regulation of the lymphocyte trap. In order to try to clarify the nature of the cell populations involved in lymphocyte trapping, the effects of anti-thymocyte serum and irradiation upon the antigen-induced sequestration of lymphocytes in spleens and lymph nodes were investigated.
MATERIALS AND METHODS Animals Experiments were performed with BDF1 mice of either sex, obtained from Jackson Laboratories, Bar Harbor, Maine. Following arrival, mice were rested for at least 1 week in the animal quarters prior to use. Mice were used at 6-8 weeks of age.
Cell suspensions Lymphocyte suspensions were prepared from the peripheral and mesenteric lymph nodes of normal donors, and labelled in vitro with "Cr as previously described (Zatz and Lance, 1971). Antigens SRBC (Gibco, Grand Island, New York) were washed three times in saline and diluted to a 25 per cent final suspension (v/v). 0 2 ml of antigen was injected i.v., and 0 05 ml was injected f.p.
Immunosuppressive agents ATS (Microbiological Associates, Bethesda, Maryland) was injected s.c. in the mid-dorsal region. This ATS preparation kills 30-40 per cent of spleen cells, 60-80 per cent of lymph node cells, and greater than 90 per cent of thymocytes in a "1Cr release cytotoxic assay. Whole body irradiation was delivered from a Siemens 250 kV machine at a dose rate of 85 R/min, and a distance of 70 cm. Under these conditions, 850 R is a lethal dose.
Experimental protocol Mice were pretreated with ATS on day -3, or with irradiation on day -1. Each group of immunosuppressed animals was given antigen or saline at - 24 h in the left footpad (f.p.) or at - 6 h i.v. At 0 h, 5 x 106 5"Cr-labelled BDF1 lymph node cells, obtained from normal donors, were injected i.v. into
groups of mice. Twenty-four hours following injection of labelled lymphocytes, the recipients were killed, and the percentage of injected radioactivity localizing in the left and right popliteal lymph nodes (following f.p. antigen injection) and in the spleen (following i.v. antigen injection) was determined. In studies of spleen trapping the results are expressed for each immunosuppressed or normal group as a percentage ratio of " Cr-labelled cell localization in the spleens of SRBC-injected/salineinjected mice. In the studies of lymph node trapping, the results are expressed as a ratio of " Cr cell localization in the left (draining ipsilateral)/right (contralateral) popliteal nodes. Since pretreatment of mice with ATS generally diminishes the baseline localization of 5'Cr-labelled lymphocytes in control animals, the data were also analysed for a specific increase in 5'Cr cell localization by the formula: (percentage localization in spleen of SRBC-injected mice) -(per cent localization in spleen of salineinjected mice). For studies of lymphocyte trapping in lymph nodes, a specific increase was calculated by the difference in per cent "'Cr cell localization in the left-right popliteal nodes. Statistics The significance of the differences between groups was determined using Student's t-test. P values of < 0-05 are considered significant.
RESULTS Effects of ATS upon lymphocyte trapping in spleen and lymph nodes
Distinct effects of ATS upon SRBC-induced trapping in spleen and draining nodes were observed (Tables 1 and 2). Whereas prior treatment with ATS totally abrogated lymphocyte trapping in the left popliteal nodes, trapping in spleen was augmented. This enhancement of splenic trapping in ATStreated mice was significant (P < 0 001). Similar results were obtained with a low (0-05 ml) dose of ATS. Since residual ATS in the circulation of recipients can deplete the lymph node-seeking population of injected lymphocytes, it was necessary to determine whether ATS-induced abrogation of lymph node trapping was attributable to loss of the lymph node seeking population, rather than to an influence of ATS directly on trapping in lymph nodes. Therefore,
751
Lymphocyte trapping Table 1. Effect of ATS treatment on lymphocyte trapping in draining nodes
Recipient treatment ATS (0 40 ml)
NRS (0 40 ml)
Left popliteal node* Right popliteal node* Left/right Left-right
Pt
ATS (0-05 ml)
Saline
SRBC
Saline
SRBC
SRBC
0-28+0-03 0-26+0-02 1-08 0-02
1-10+0-07 0-36+0 03 3-55 0 74 < 0 001
0-13+0-01 0 11+0 01 1-18 0-02
0-16+0-04 0-14+0 04 1-14 0-02
0-17+0-02 0-23+0-04 0 74 -0-06
n.s.
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
Saline* SRBC* Percentage SRBC/saline SRBC-saline
Pt
21-0+ 0 5 26-2+0-6 125-0 52 < 0-001
ATS (0 04 ml) ATS (0 05 ml) 10-7+ 0 5 20-5+ 1-5 192-0 9-8 < 0-001
10 9+ 0 4 18-4+0-4 169-0 7-5 < 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 (already depleted of their recirculating cell population) were compared to 51Cr lymph node cells from NRS-treated donors in their ability to be trapped in NRS- or ATS-treated recipients. The results (data not shown) reveal that, regardless of whether 51Cr
lymphocytes are obtained from NRS- or ATStreated donors, SRBC-induced trapping was abrogated in lymph nodes of ATS-treated recipients, but enhanced in spleens of the same mice. Effects of irradiation upon lymphocyte trapping in spleens and lymph nodes The radiation sensitivity of lymphocyte trapping was investigated. High (850 R) dose irradiation eliminated trapping in draining lymph nodes (Table 3), although the same treatment failed to alter the splenic
trapping response (Table 4). 200 R was without effect upon lymph node trapping, but consistently caused a significant (P < 0-05) enhancement of trapping in spleen. Thus both irradiation and ATS pretreatment selectively eliminated lymphocyte trapping in draining lymph nodes. DISCUSSION Earlier studies have demonstrated that lymphocyte trapping in both lymph nodes and spleen is, at least partially, a result of a T-cell response to antigen (Zatz and Gershon, 1974), although conflicting results have suggested that trapping is solely macrophage dependent (Frost and Lance, 1974). The present studies reveal that lymphocyte trapping in spleen and lymph nodes differs markedly in its sensitivity to modification by ATS and irradiation,
Marion M. Zatz
752
Table 3. Effect of irradiation on trapping in lymph nodes Recipient treatment (R)
Saline Left*
Right* Left/right Left-right
Pt
0
200
850
0-26+ 0-06 0-26+0 003 1.0 0
0 35+ 0-02 0-36+ 003 0 97 0 01
0 39+ 0-06 0 33+ 003 1-18 0-06
n.s.
n.s.
n.s.
0 77+ 0 03 0-22+ 0-02 3 50 0-55
0-81 + 0 05 0-28+ 0-02 2-89 0 53 < 0001
0-48+ 0-06 0 40+ 003 120 0-08
SRBC
Left* Right* Left/right Left-right
Pt
< 00001
n.s.
* Values given are percentage localization of labelled cells in left and right popliteal nodes. Results are based on two experiments 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)
Saline* SRBC* Per cent SRBC/saline SRBC- saline
Pt
0
200
850
15-7+0-6 19 5+0 8 124 2 3-8
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 New Haven, Connecticut, U.S.A.
School
of Medicine,
Received 20 August 1975; acceptedfor publication 13 November 1975
Summary. Increased sequestration (trapping) of lymphocytes occurs in lymphoid organs following antigenic challenge. The effects of irradiation and anti-thymocyte serum (ATS) upon lymphocyte trapping in lymph nodes and spleen were studied. Both the above agents diminished or abrogated trapping in the draining lymph nodes; these same agents resulted in enhanced trapping in the spleen. Suppression of lymph node trapping could be achieved with a low dose of ATS and with 850 R, but not 200 R. These results are interpreted as showing that lymphocyte trapping in lymph nodes is initiated primarily by an ATS- and irradiation-sensitive population, whereas in the spleen a cell population resistant to the above agents activates the lymphocyte trap. These data demonstrate that the cell populations and/or the mechanisms involved in lymphocyte trapping in lymph nodes and spleens are distinct.
INTRODUCTION In vivo administration of antigen results, within hours, in a transient sequestration of circulating Correspondence: Dr Marion M. Zatz, Immunology Branch, National Cancer Institute, Bethesda, Maryland 20014, U.S.A.
749
lymphocytes in lymphoid organs (Zatz and Lance, 1971; Ford, 1972; Hall and Morris, 1965; Rowley, Gowans, Atkins, Ford and Smith, 1972). This sequestration of cells has been termed lymphocyte 'trapping' (Zatz and Lance, 1971). It seems likely that lymphocyte trapping indirectly plays an important role in vivo in the regulation of a broad spectrum of immune responses, via control of lymphocyte traffic (Zatz and Lance, 1971; Ford, 1972; Hall and Morris, 1965; Rowley et al., 1972; Emeson and Thursh, 1971; Dresser, Taub and Krantz, 1970; Zatz and Lance, 1971; Zatz, White and Goldstein, 1973; Gershon and Fightlin, 1973; Gillette and Bellanti, 1973; Asherson and Barnes, 1973; Frost and Lance, 1973; Zatz and Gershon, 1974). However, relatively little is known about the mechanisms and cell subpopulations which are involved in production of the lymphocyte trap. 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 initiation of trapping involves activation of T cells by antigen (Taub and Gershon, 1972; Zatz and Gershon, 1974) and that the ensuing sequestration of large numbers of circulating lymphocytes serves to concentrate antigen-reactive cells at the major site of antigen depot. Previous reports by Frost and Lance (1973, 1974) have suggested that the macrophage is
Marion M. Zatz
750
the cell responsible for trapping and that T cells are unimportant for eliciting this phenomenon. In contrast, other studies (Zatz and Gershon, 1974, 1975) clearly show that T-cell activation is required for production and regulation of the lymphocyte trap. In order to try to clarify the nature of the cell populations involved in lymphocyte trapping, the effects of anti-thymocyte serum and irradiation upon the antigen-induced sequestration of lymphocytes in spleens and lymph nodes were investigated.
MATERIALS AND METHODS Animals Experiments were performed with BDF1 mice of either sex, obtained from Jackson Laboratories, Bar Harbor, Maine. Following arrival, mice were rested for at least 1 week in the animal quarters prior to use. Mice were used at 6-8 weeks of age.
Cell suspensions Lymphocyte suspensions were prepared from the peripheral and mesenteric lymph nodes of normal donors, and labelled in vitro with "Cr as previously described (Zatz and Lance, 1971). Antigens SRBC (Gibco, Grand Island, New York) were washed three times in saline and diluted to a 25 per cent final suspension (v/v). 0 2 ml of antigen was injected i.v., and 0 05 ml was injected f.p.
Immunosuppressive agents ATS (Microbiological Associates, Bethesda, Maryland) was injected s.c. in the mid-dorsal region. This ATS preparation kills 30-40 per cent of spleen cells, 60-80 per cent of lymph node cells, and greater than 90 per cent of thymocytes in a "1Cr release cytotoxic assay. Whole body irradiation was delivered from a Siemens 250 kV machine at a dose rate of 85 R/min, and a distance of 70 cm. Under these conditions, 850 R is a lethal dose.
Experimental protocol Mice were pretreated with ATS on day -3, or with irradiation on day -1. Each group of immunosuppressed animals was given antigen or saline at - 24 h in the left footpad (f.p.) or at - 6 h i.v. At 0 h, 5 x 106 5"Cr-labelled BDF1 lymph node cells, obtained from normal donors, were injected i.v. into
groups of mice. Twenty-four hours following injection of labelled lymphocytes, the recipients were killed, and the percentage of injected radioactivity localizing in the left and right popliteal lymph nodes (following f.p. antigen injection) and in the spleen (following i.v. antigen injection) was determined. In studies of spleen trapping the results are expressed for each immunosuppressed or normal group as a percentage ratio of " Cr-labelled cell localization in the spleens of SRBC-injected/salineinjected mice. In the studies of lymph node trapping, the results are expressed as a ratio of " Cr cell localization in the left (draining ipsilateral)/right (contralateral) popliteal nodes. Since pretreatment of mice with ATS generally diminishes the baseline localization of 5'Cr-labelled lymphocytes in control animals, the data were also analysed for a specific increase in 5'Cr cell localization by the formula: (percentage localization in spleen of SRBC-injected mice) -(per cent localization in spleen of salineinjected mice). For studies of lymphocyte trapping in lymph nodes, a specific increase was calculated by the difference in per cent "'Cr cell localization in the left-right popliteal nodes. Statistics The significance of the differences between groups was determined using Student's t-test. P values of < 0-05 are considered significant.
RESULTS Effects of ATS upon lymphocyte trapping in spleen and lymph nodes
Distinct effects of ATS upon SRBC-induced trapping in spleen and draining nodes were observed (Tables 1 and 2). Whereas prior treatment with ATS totally abrogated lymphocyte trapping in the left popliteal nodes, trapping in spleen was augmented. This enhancement of splenic trapping in ATStreated mice was significant (P < 0 001). Similar results were obtained with a low (0-05 ml) dose of ATS. Since residual ATS in the circulation of recipients can deplete the lymph node-seeking population of injected lymphocytes, it was necessary to determine whether ATS-induced abrogation of lymph node trapping was attributable to loss of the lymph node seeking population, rather than to an influence of ATS directly on trapping in lymph nodes. Therefore,
751
Lymphocyte trapping Table 1. Effect of ATS treatment on lymphocyte trapping in draining nodes
Recipient treatment ATS (0 40 ml)
NRS (0 40 ml)
Left popliteal node* Right popliteal node* Left/right Left-right
Pt
ATS (0-05 ml)
Saline
SRBC
Saline
SRBC
SRBC
0-28+0-03 0-26+0-02 1-08 0-02
1-10+0-07 0-36+0 03 3-55 0 74 < 0 001
0-13+0-01 0 11+0 01 1-18 0-02
0-16+0-04 0-14+0 04 1-14 0-02
0-17+0-02 0-23+0-04 0 74 -0-06
n.s.
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
Saline* SRBC* Percentage SRBC/saline SRBC-saline
Pt
21-0+ 0 5 26-2+0-6 125-0 52 < 0-001
ATS (0 04 ml) ATS (0 05 ml) 10-7+ 0 5 20-5+ 1-5 192-0 9-8 < 0-001
10 9+ 0 4 18-4+0-4 169-0 7-5 < 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 (already depleted of their recirculating cell population) were compared to 51Cr lymph node cells from NRS-treated donors in their ability to be trapped in NRS- or ATS-treated recipients. The results (data not shown) reveal that, regardless of whether 51Cr
lymphocytes are obtained from NRS- or ATStreated donors, SRBC-induced trapping was abrogated in lymph nodes of ATS-treated recipients, but enhanced in spleens of the same mice. Effects of irradiation upon lymphocyte trapping in spleens and lymph nodes The radiation sensitivity of lymphocyte trapping was investigated. High (850 R) dose irradiation eliminated trapping in draining lymph nodes (Table 3), although the same treatment failed to alter the splenic
trapping response (Table 4). 200 R was without effect upon lymph node trapping, but consistently caused a significant (P < 0-05) enhancement of trapping in spleen. Thus both irradiation and ATS pretreatment selectively eliminated lymphocyte trapping in draining lymph nodes. DISCUSSION Earlier studies have demonstrated that lymphocyte trapping in both lymph nodes and spleen is, at least partially, a result of a T-cell response to antigen (Zatz and Gershon, 1974), although conflicting results have suggested that trapping is solely macrophage dependent (Frost and Lance, 1974). The present studies reveal that lymphocyte trapping in spleen and lymph nodes differs markedly in its sensitivity to modification by ATS and irradiation,
Marion M. Zatz
752
Table 3. Effect of irradiation on trapping in lymph nodes Recipient treatment (R)
Saline Left*
Right* Left/right Left-right
Pt
0
200
850
0-26+ 0-06 0-26+0 003 1.0 0
0 35+ 0-02 0-36+ 003 0 97 0 01
0 39+ 0-06 0 33+ 003 1-18 0-06
n.s.
n.s.
n.s.
0 77+ 0 03 0-22+ 0-02 3 50 0-55
0-81 + 0 05 0-28+ 0-02 2-89 0 53 < 0001
0-48+ 0-06 0 40+ 003 120 0-08
SRBC
Left* Right* Left/right Left-right
Pt
< 00001
n.s.
* Values given are percentage localization of labelled cells in left and right popliteal nodes. Results are based on two experiments 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)
Saline* SRBC* Per cent SRBC/saline SRBC- saline
Pt
0
200
850
15-7+0-6 19 5+0 8 124 2 3-8
