Migration of human lymphocytes. II. Variation of lymphocyte distribution.

Variations in the distribution of 51Cr-labelled human lymphocytes from several donors and from one donor on several occasions have been determined after their injection into carbon-treated mice and reasons for the variation discussed. Natural alteration of the lymphocyte surface (leukaemic lymphocytes) or induced change (trypsin-treatment) reduced lymph node localisation as did treatment with formalin, sodium azide and antilymphocyte globulin. We have concluded that the presence of radioactivity in the lymph nodes is due to the presence of viable, metabolically active lymphocytes with normal surfaces capable of interacting with the endothelium of the post-capillary venules.     

Lymphocyte traffic through chronic inflammatory lesions: differential migration versus differential retention.

Afferent lymphatics draining granulomas and efferent lymphatics from normal lymph nodes were cannulated in sheep. Cells collected from these lymphatics were radiolabelled in vitro with 111In (afferent lymph cells) and 51Cr (efferent lymphocytes) and both labelled cells were returned to the animal simultaneously by i.v. injection. The reappearance of these labelled cells in lymph, and the amount of 111In and 51Cr in normal or antigenically stimulated lymph nodes, cutaneous inflammatory sites (FCA-granulomas, NLT- and BCG-induced lesions) and blood was determined 24 hr later. As previously reported, labelled afferent cells preferentially migrated from the blood back through the granuloma into afferent lymph, and efferent lymphocytes back into efferent lymph. Forty per cent as many 111In- as 51Cr-labelled cells ;appeared in efferent lymph. This was caused by the greater migration of 51Cr-labelled cells appeared in efferent lymph. This was caused by the greater migration of 51Cr- than 111In-labelled cells out of the blood into the node. Neither cell type was selectively retained in the node, and 28% of the labelled cells that entered the node migrated on into efferent lymph in 24 hr. Similarly, there was no selective retention of either cell type in the granuloma, and equal amounts of 111In the 51Cr appeared in afferent lymph. The ratio 111In/51Cr in the blood suggested that in the lymph node the two labelled cell populations were extracted equally, while in the granuloma selectively at the level of the vascular endothelium resulted in the preferential extraction of 111In-labelled (afferent lymph) cells.     

Evidence for a loss of recirculation capacity of T-cells after antigenic stimulation

The distribution of i.v. injected ⁵¹Cr-labelled normal or antigen-activated thymic or thymus-derived cells (T-cells) was studied in isologous recipients.

Activated lymphocytes were obtained from the spleens and lymph nodes of lethally X-irradiated allogeneic mice infused 5 days earlier with lymphoid cells. Practically all of the cells of these organs were found to be of donor type origin and they exhibited an increased mean cell volume. Both activated and non-activated T-cells exhibited a high accumulation to spleen and liver within 40 hr after injection into isologous recipients. The non-activated cells also exhibited a high tendency to seek to lymph nodes whereas this capacity was largely absent in the activated cell populations. Since the lymph node seeking capacity of lymphocytes is considered to be a specific trait for the recirculating pool of lymphocytes, it is possible that T-cells may temporarily lose their recirculating capacity when activated by an antigen.

     

The effect of localized injection of adjuvant material on the draining lymph node: II. Circulating lymphocytes

The stimulation of a draining lymph node by the injection of a substance possessing adjuvanticity leads to a marked increase in the influx of lymphocytes into the node. The influx of cells has been followed in experimental mice injected intravenously with ⁵¹Cr-labelled normal mesenteric lymph node cells. The increase in size of the draining node is not due to an increased blood volume.     

Contact sensitivity in the mouse: IX. The role of immunological and non-immunological inflammation in the movement of lymphocytes to immunized lymph nodes

Normal mice were immunized with picryl chloride. ⁵¹Cr-labelled normal lymph node cells were injected 1–10 days afterwards and the net arrival of radioactivity at the draining lymph nodes estimated. The net arrival at normal unimmunized lymph nodes was 5.3 per cent of the injected dose. This rose to 13.4 per cent 1 day after immunization and then gradually declined.

This increased arrival caused by picryl chloride was reduced in mice rendered unresponsive by the repeated injection of picryl sulphonic acid or given a single injection of picryl sulphonic acid 1–6 days before immunization. Blast cells labelled with ¹²⁵IUDR from lymph nodes immunized with an unrelated agent also showed an increased arrival at draining lymph nodes. This increased arrival was reduced in unresponsive mice painted with picryl chloride.

A single intravenous injection of picryl sulphonic acid increased the arrival of normal lymph node cells at lymph nodes 1 day later and this effect was also diminished in mice rendered unresponsive by repeated injections of picryl sulphonic acid.

The arrival of normal lymph node cells at the spleen was reduced by painting with picryl chloride or by a single injection of picryl sulphonic acid given a few days beforehand.

Painting the ears of unimmunized mice with picryl chloride increased the arrival of normal lymph node cells and blast cells at the ears. However, the arrival was unaffected by pretreatment with picryl sulphonic acid.

It was concluded that the increased arrival of cells in lymph nodes caused by picryl chloride and picryl sulphonic acid had both an immunological and a chemical inflammatory component. In contrast the arrival at painted ears lacked an immunological component. This provided evidence for antibody, or antigen-sensitive cells in apparently unimmunized mice which rapidly release pharmacologically active agents on exposure to antigen.

     

Radiosensitivity of T and B lymphocytes. I. Effect of irradiation on cell migration

To study the effect of irradiation on cell migration, populations of recirculating lymphocytes consisting predominantly of either T or B lymphocytes were labeled with ⁵¹Cr, exposed to various doses of irradiation in vitro and transferred intravenously to syngeneic mice. Cell recipients were killed at intervals to determine levels of radioactivity in various organs. On the basis of radioactive counts, small T lymphocytes homed normally to the lymphoid tissues within 4 h of injection (primary migration) in spite of prior exposure to heavy irradiation, e.g. 5000 r. Primary migration, especially to the lymph nodes, was greatly reduced, however, when irradiated cells were cultured in vitro for 1–7 h before injection. This suggested that cell damage following irradation was not manifested immediately, but developed slowly over several hours. This was supported by observations that, with cells injected immediately after irradiation, even doses as low as 50 r caused a marked reduction in secondary migration, i.e. migration occurring between 4 and 24 h after cell injection. Activated T cells proved to be more radioresistant than small T cells in that secondary migration of activated cells was not abolished, although reduced, by high doses of irradiation. B cells were found to be extremely radiosensitive. Thus, in contrast to T cells, primary migration of B cells was greatly reduced by heavy irradiation. Secondary migration of B cells, although limited, was abolished by 100 r. When lymphocytes were irradiated and placed in tissue culture for 4 days, virtually no B cells survived exposure to 300 r. This dose reduced viability of T cells by 90 %. Five to 10 % of T cells, however, remained viable after exposure to doses as high as 1000 r. The results, as a whole, suggested that both T and B small lymphocytes die in interphase soon after irradiation, death occurring very rapidly with B cells, but less rapidly with T cells. Interphase death of activated T cells seemed to occur comparatively slowly.     

Age-related changes in cell localization and proliferation in lymph nodes and spleen after antigenic stimulation.

Antigen-dependent localization of 51Cr-labelled lymphocytes, and the subsequent uptake of IUdR into lymphoid organs has been studied as a function of age. Measures of cell localization indicated that while old age can alter the patterns of entry of lymphocytes into lymph nodes and spleen, these changes are variable and probably not sufficient alone to explain decreased primary antibody responses in old animals. Proliferation of cells, however, was consistently affected in both organs and this phenomenon is discussed in terms of abnormal T-cell function.     

Radioresistant T lymphocytes in mice: distribution in organs, Thy-1 antigen content and helper activity.

Radioresistant T lymphocytes (RRTL) were derived from spleens of CBA mice after gamma-irradiation (2000 rad). RRTL comprise 2.2-3.5% of the total T-cell population. Adult thymectomy and treatment with ATS or cortisol do not affect the yield of RRTL. The Thy-1 antigen content on RRTL surfaces exceeds that on thymocytes and splenic T lymphocytes. 51Cr-labelled RRTL fail to home into lymph nodes: a considerable number of labelled RRTL persist in the blood. In an adoptive transfer system RRTL display poor helper activity. The adoptive response of normal B cells plus RRTL was reduced when RRTL donors were preimmunized with the same antigen. It is not clear if the unusual properties of RRTL were induced by irradiation or if they pre-existed.     

Restrictions to the use of 111indium-oxine as a radiolabel in lymphocyte migration studies

Guinea pig T lymphocytes were labelled with various concentrations of ¹¹¹In-oxine. The capacity of these cells to migrate into a skin inflammatory site was compared with that of ⁵¹Cr-labelled cells.The results show that even with low dosages of ¹¹¹In-oxine which did not impair lymph node localization (1?10 μCi/10⁸ cells/ml), migration into an inflammatory site was markedly reduced. Moreover, using this isotope, a significant contribution to the radioactivity recovered from an inflammatory site is made by the local accumulation of non-cell-bound label. These results stress the limited use of ¹¹¹In-oxine as a radiolabel in lymphocyte migration studies.     

Studies of allograft immunity in mice: I. Induction, development and in vitro assay of cellular immunity

Cellular immunity induced by tumour allografts in inbred mice was studied with the help of an in vitro assay system measuring the cytotoxic effect of sensitized lymphocytes on ⁵¹Cr-labelled target cells. It is shown that lymphoid cells from spleen, lymph nodes and blood of the allograft recipients reach a peak of cytotoxic activity on days 10–11 after immunization. Incubated with labelled target cells at a ratio of 100:1, the sensitized lymphocytes caused the specific release of up to 70 per cent of the radioactivity within 1 hour. A second population of target cells added to the same cell suspension was destroyed at a slightly accelerated rate, suggesting stimulation of the effector cells by the first interaction. The cytotoxic activity of circulating lymphocytes was found to reach a plateau between days 20 and 60 after immunization, while the activity of spleen cells dropped to low levels in the same time period. The specificity of target cell destruction by sensitized lymphocytes is demonstrated by the lack of lytic activity for syngeneic target cells, and by the selective destruction of target cells carrying a tumour specific antigen. Tumour cells, lymphocytes and embryonic fibroblasts of the donor strain are shown to differ considerably in their sensitivity to lysis by immune lymphocytes.     

Route of lymphocyte migration in pigs. I. Lymphocyte circulation in gut-associated lymphoid tissue.

Evidence is presented which indicates that recirculating lymphocytes originating from the intestine in pigs are returned to the blood circulation at the level of the mesenteric lymph nodes (MLN) and not via efferent intestinal lymph. This was demonstrated by three observations: (1) removal of all MLN resulted in a thirty-fold increase in lymphocyte numbers in efferent lymph of pigs, but not in rats; (2) there are about twenty-five times more lymphocytes in afferent intestinal lymph than efferent intestinal lymph in normal pigs; (3) 51Cr-labelled lymphocytes injected into afferent lymphatics are mostly recovered in the node tissue or efferent lymph of sheep, and very few in the venous drainage, whereas in pigs relatively few labelled cells are recovered in the node or in efferent lymph.     

Adoptive immunity to Mycobacterium bovis strain bacillus Calmette-Guérin by long-term cultured T-cell line in nude mice.

Tuberculin-active peptide-reactive T-cell lines were established from the popliteal lymph node of BALB/cA mice immunized with heat-killed Mycobacterium tuberculosis to investigate the cellular mechanisms in the protective immunity of tuberculosis. These T-cell lines, consisting mainly of L3T4 surface antigen-positive cells, were transferred intravenously into nude mice infected with M. bovis strain bacillus Calmette-Guérin (BCG). Four or 6 weeks after transfer, footpad swelling and hepatic granuloma formation were observed and viable counts in the liver were decreased. Reduction of viable counts in the liver was obviously preceded by the hepatic granuloma formation. An effect of Lyt-2+ T cells which might be included in the inoculum could be ruled out by the experiment using T-cell lines pretreated with anti-Lyt-2 monoclonal antibody and complement. These results indicated that T cells required for protective immunity in these experiments belong to the L3T4-positive TDTH subset. However, their protective activity was inferior to that of freshly prepared immune lymph node T cells. From the observation of migration patterns of 51Cr-labelled T cells in BCG-infected nude mice, a relatively high proportion of freshly isolated T cells, but only a small part of T-cell line, deposited in the spleen of infected nude mice. This difference in migration pattern of freshly isolated and cultured T cells could be one of the reasons for the less in vivo anti-bacterial activity of the latter.     

Contact sensitivity in the mouse. X. Non-specific cytotoxicity of T blasts in the draining lymph nodes

CBA mice were immunized by painting the skin with the contact sensitizing agent `oxazolone''. The draining lymph nodes were taken at 4 days and mixed with <sup>51</sup>Cr-labelled mastocytoma tumour cells. Cytotoxic killing was assessed by the release of <sup>51</sup>Cr. Little killing occurred in the absence of phytohaemagglutinin. However, immunized but not normal lymph node cells killed the mastocytoma cells providing phytohaemagglutinin (PHA) was present during the killing reaction. Analysis by the `one hit'' hypothesis suggested that up to 14 per cent of the lymphocytes in immunized lymph nodes were effector or killer cells.The cytotoxicity was non-specific. The evidence for this was that DBA/2 lymph nodes immunized with oxazolone killed DBA/2 mastocytoma cells. The killing reaction did not require macrophages but was due to large cells. These were shown to be θ-positive in carefully controlled experiments. The non-specific cytotoxicity was greatest 4 days after immunization with oxazolone. Reimmunization of the mice on day 10 did not cause a classical secondary response, as assessed by non-specific cytotoxic killing. Instead the time course resembled the primary while the magnitude of killing was slightly reduced.There were several similarities between non-specific cytotoxic cells and the large pyroninophilic cells and the cells which move to sites of inflammation which are found in lymph nodes immunized with contact sensitizing agents. These similarities included θ-positivity, large size and peak occurrence 4 days after immunization. It is postulated that non-specific cytotoxicity and movement to sites of inflammation are due to blasts or their immediate descendants which originated from T lymphocytes.     

The effect of histocompatibility antigens on lymphocyte migration in the mouse

Migration patterns of ⁵¹Cr-labelled murine lymph node cells were studied in syngeneic, allogeneic, semi-allogeneic and congenic strain combinations. In certain allogeneic and semi-allogeneic combinations with strong H-2 histoincompatibility, reduced migration of a donor population representing recirculating lymphocytes into recipient lymph nodes was observed. This phenomen could be reproduced in congenic strains differing only at the H-2 locus, and was not seen in congenic strains differing at the Ly-A, Ly-B, RZ, M, or θ loci.

The kinetics of reduced lymphocyte homing to allogeneic lymph nodes and the role of host and donor recognition were investigated. These studies are discussed in relation to possible mechanisms of action, and relevance to factors regulating lymphocyte recirculation.

     

Studies on the lymphocytes of sheep. I. Recirculation of lymphocytes through peripheral lymph nodes and tissues

Recirculating lymphocytes were collected from the efferent ducts of peripheral somatic nodes and incubated in vitro with ⁵¹Cr. After washing, the labeled cells were returned to the sheep by i.v. injection. The partitioning of the labeled cells between blood and efferent (intermediate) lymph, or between efferent and afferent (peripheral) lymph was monitored for the next 24 h by assaying the radioactivity in appropriate samples of cells. The labeled lymphocytes took several hours to migrate through the nodes so that they equilibrated between blood and lymph in about 10 h. This pattern was not altered grossly during the inductive phase of an immune response. The small numbers of lymphocytes which migrated through peripheral tissues entered the lymph at least as quickly as those that migrated through lymph nodes.     

Recruitment of effector lymphocytes by initiator lymphocytes. In vivo migration of in vitro sensitized initiator T lymphocytes.

We previously found that mouse T lymphocytes sensitized in vitro against allo- or syngeneic fibroblasts, upon injection into syngeneic recipients, do not themselves differentiate into effector cells, but recruit effector T lymphocytes within the draining lymph nodes. As a result of sensitization, these initiator lymphocytes acquire a trypsin-sensitive membrane property which is necessary for recruitment. We now report studies on the in vivo migratory behavior of initiator lymphocytes following sensitization. We injected 51Cr-labeled initiator lymphocytes into recipient footpads and found significantly increased migration of sensitized cells to the draining popliteal lymph node (PLN) during the first day. By amputation of the foot at various times, we showed that migration during the first 12-24 hours was critical for subsequent recruitment. Trypsin treatment of initiator lymphocytes abolished this accelerated migration. Lymphocytes triggered nonspecifically by Con A migrated to the PLN like antigen-sensitized cells. We also compared the migration of injected lymphocytes from the footpad to the PLN in graft-versus-host and host-versus-graft reactions, and found these reactions to differ both from each other and from recruitment in terms of lymphocyte migration. These findings are discussed in terms of the physiology of the cell-mediated immune response and the notion of peripheral sensitization.     

Allogeneic lymphocyte cytotoxicity in rats: the effects of various pharmacological agents.

In a number of strain combinations among inbred rats, intravenously injected 51Cr-labelled lymphocytes are rapidly destroyed by unsensitized allogeneic hosts. This phenomenon has previously been referred to by a number of terms, the most explicit and least confusing of which is allogeneic lymphocyte cytotoxicity (ALC). It is characterized by reduced lymph node radioactivity, together with increased kidney and/or urine radioactivity 24 hr after injection, in allogeneic hosts as compared with syngeneic recipients of the same cell suspension. ALC has a number of features in common with other natural resistance systems. Nevertheless, the administration of hydrocortisone or silica in doses comparable to those causing diminished NK activity in rats was without effect on ALC; likewise cyclophosphamide at a dose capable of significantly impairing NK activity in rats had, at most, a minor effect on ALC. Under the conditions of administration cyclosporin A was without effect. The interferon inducer polyinosinic polycytidilic acid (poly I:C) brought about a significant augmentation of ALC.     

Influence of reserpine on in vivo localization of injected lymph node cells in the mouse.

The effects of reserpine, and other agents that affect the storage and availability of 5-hydroxytryptamine (5HT), on the localization of injected 51Cr-labelled syngeneic lymph node cells have been investigated. A high dose (5 mg/kg) of reserpine to the recipients reduced localization in the lymph nodes and prevented the usual accumulation of lymphocytes in lymph nodes draining the site of an antigen (sheep erythrocytes: SE) injection. These effects were partially reversible by the monoamine oxidase inhibitor nialamide. This dose of reserpine produced deep sedation throughout the period of the experiment. Lower doses, up to 2.5 mg/kg, produced little sedation and had no effect on the localization of lymphocytes. Other workers had previously reported reduced localization of cells in delayed-type hypersensitivity (DTH) lesions after treatment of the recipients with 5 mg/kg reserpine, and had interpreted this in terms of a role of 5HT in promoting vascular permeability and egress of blood cells. The effect of lower doses of reserpine was not reported. We suggest that the effects on cell localization in both sets of experiments may have been secondary to the general state of sedation and not attributable to a direct local influence of 5HT. Other effects of reserpine included prolonged retention of lymphocytes in lungs and blood, and a reduction of cellularity and DNA synthesis in the thymus, spleen and lymph nodes.     

Measurement of lymphocyte traffic with indium-111.

This study was designed to assess the use of 111indium as a radioactive marker for the investigation of lymphocyte recirculation in the sheep. Lymphocytes were collected from sheep with indwelling catheters in the efferent lymphatic ducts of peripheral lymph nodes and labelled with 111In-oxine or Na2 51CrO at doses of 10 microCi and 50 microCi/10(8) cells respectively. After intravenous injection the lymphocyte specific activity (c.p.m./10(7) cells) in blood and lymph was measured for several days. The maximum specific activity in efferent lymph was twelve-fold greater with 111In than with 51Cr-labelled cells. The kinetics of lymphocyte traffic as measured in double labelling experiments was very similar. The modal transit time was 21.6 hr with each isotope. The recovery of 111In-labelled cells was not significantly different from cells labelled with 51Cr. In vivo viability of the labelled cells was further supported by the normal proliferative response observed with 111In-labelled lymphocytes compared to unlabelled cells in the normal lymphocyte transfer reaction. In conclusion, 111In-oxine is an excellent radioactive label for lymphocytes in the sheep. Because of its high counting efficiency and cell labelling characteristics one can label as few as 10 million lymphocytes, or a subpopulation of cells, and assess their recirculation.     

Recruitment of effector lymphocytes by initiator lymphocytes. In vivo migration of in vitro sensitized initiator T lymphocytes

We previously found that mouse T lymphocytes sensitized in vitro against allo- or syngeneic fibroblasts, upon injection into syngeneic recipients, do not themselves differentiate into effector cells, but recruit effector T lymphocytes within the draining lymph nodes. Asaresult of sensitization, these initiator lymphocytes acquire a trypsin-sensitive membrane property which is necessary for recruitment. We now report studies on the in vivo migratory behavior of initiator lymphocytes following sensitization. We injected ⁵¹Cr-labeled initiator lymphocytes into recipient footpads and found significantly increased migration of sensitized cells to the draining popliteal lymph node (PLN) during the first day. By amputation ofthe foot at various times, we showed that migration during the first 12—24 hours was critical for subsequent recruitment. Trypsin treatment of initiator lymphocytes abolished this accelerated migration. Lymphocytes triggered nonspecifically by Con A migrated to the PLN like antigen-sensitized cells. We also compared the migration of injected lymphocytes from the footpad to the PLN in graftversus-host and host-versus-graft reactions, and found these reactions to differ both from each other and from recruitment in terms of lymphocyte migration. These findings are discussed in terms of the physiology of the cell-mediated immune response and the notion of peripheral sensitization.