Kinetics and morphology of chemically induced popliteal lymph node reactions compared with antigen-, mitogen-, and graft-versus-host-reaction-induced responses

Changes in the popliteal lymph node (PLN) in mice evoked by a local graft-versus-host (GVH) reaction and by a single injection of various agents into the hind footpad were compared. The drug diphenylhydantoin induced similar weight changes in time as the GVH reaction. More vigorous and protracted reactions were induced by the drug nitrofurantoin and the contact sensitizer dinitrochlorobenzene, whereas the antigens lipopolysaccharide and sheep erythrocytes caused very moderate and short-lasting weight changes. Alterations of lymph node architecture upon injection of diphenylhydantoin resembled those observed during the GVH response. Some quantitative and qualitative differences were noted for nitrofurantoin, but clearly deviant morphological alterations were seen in response to lipopolysaccharide and sheep erythrocytes. The PLN reaction to dinitrochlorobenzene had features of both the GVH reaction and the antigen-induced responses. These findings support the concept that some drugs and chemicals may induce or exacerbate lymphoproliferative disorders by GVH-like mechanisms.     

Immunity to D-penicillamine: genetic, cellular, and chemical requirements for induction of popliteal lymph node enlargement in the mouse

To elucidate the pathogenesis of immunological diseases induced by the drug D-Penicillamine (D-Pen) the requirements for sensitization to this drug were investigated. Mice were subcutaneously (s.c.) injected into one hind footpad with a solution of D-Pen without adjuvant, and reactivity to D-Pen was determined in the popliteal lymph node assay (PLNA) by weight increase of the draining PLN, the incorporation of 3H-thymidine, and trapping of 51Cr-labeled syngeneic lymphocytes in the draining PLN. The peak of the primary PLN response was obtained between day 7 and 10 after injecting 1 mg of D-Pen per mouse. Likewise, PLN enlargement could be induced by injecting 18 hr nonadherent spleen cells s.c. that had been pretreated overnight with D-Pen in vitro. D-Pen-induced PLN enlargement was primarily caused by cell proliferation within the lymph node, and only a minor portion was due to trapping of circulating lymphocytes. The majority of the cells in the enlarged PLN were B cells; T cells, however, were required for generation of PLN enlargement. For induction of PLN reactivity to D-Pen, the stereoisomer L-Pen, and the dimer D-Pen disulfide, it was mandatory that the respective molecules were administered in ionized form. PLN reactivity to D-Pen is controlled by at least two loci, one mapping to the I region, possibly A beta A alpha, the other(s) to the non-H-2 background. As far as studied, high responsiveness was inherited dominantly. The PLN reaction proved to be antigen-specific, since D-Pen-primed mice exhibited an enhanced reaction when challenged with a suboptimal dose of D-Pen, but not when challenged with an unrelated drug, diphenylhydantoin (DPH). The possible relationship between immunity to D-Pen and autoimmunity induced by this drug is discussed.     

Allogeneic cell interactions in the rat: I. Changes in the rate of cell proliferation during local graft-versus-host and host-versus-graft reactions

Cell proliferation was investigated during local host-versus-graft (HVG) and graft-versus-host (GVH) reactions in rats by means of the popliteal lymph node weight assay. Dose-response regression lines were obtained at early, peak, and late response times for both reactions. Changes in the rate of cell proliferation were demonstrated by changes in the slopes of the dose-response regression lines and were confirmed statistically. In the local HVG reaction the rate of cell proliferation was constant from early (Day +2) to peak (Day +4) response times but possibly was reduced later (Day +8), when the response was dose dependent only at low doses. In the local GVH reaction the rate of cell proliferation increased markedly between early (Day +4) and peak (Day +8) response times and then remained constant. Examination of time-response curves following the injection of a fixed cell dose confirmed these findings. In the HVG reaction lymph node weight increased linearly with respect to time whereas in the GVH reaction the increase in lymph node weight was linear initially but became exponential before peak response. These results are consistent with the concept that, whereas HVG reactions involve proliferation of one principal population of cells (host cells), GVH reactions involve proliferation of two principal populations of cells (host and donor cells). The change in the rate of proliferation occurring between early and peak response in the GVH reaction probably reflects an initial proliferation of donor cells being joined by proliferation of host cells.     

Apparent T cell function of bone marrow cells from mice experiencing a graft-versus-host reaction.

The graft-versus-host (GVH) reaction, induced in adult F₁ mice by the injection of parental strain lymphoid cells (GVH mice), suppressed the in vitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) of spleen cells obtained from the GVH mice (GVH-SC). In vitro restoration of the PFC response of GVH-SC was carried out employing a modified Marbrook culture chamber consisting of an inner culture compartment (IC) separated from an outer culture compartment (OC) by a cell-impermeable membrane. Thymus cells (TC) and lymph node cells (LNC) but not bone marrow cells (BMC) from normal mice placed in the IC restored the PFC response of GVH-SC cultured with SRBC in the OC. The restoring ability of TC and LNC was markedly reduced following treatment with anti-theta serum plus complement. BMC taken from GVH mice 3 or more days post-GVH induction (GVHBMC) and placed in the IC restored the PFC response of GVH-SC as well as TC and LNC. Treatment of GVH-BMC with anti-theta serum plus complement did not affect their restoring ability; furthermore, the number of theta-bearing cells in the bone marrow did not increase as a consequence of the GVH reaction. Two possible explanations are proposed for the T-like function of GVH-BMC.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

Graft versus host-induced immunosuppression: mechanism of depressed T-cell helper function in vitro.

The cellular basis of graft versus host (GVH)-induced immunosuppression was investigated. Results showed that thymus, lymph node, and splenic T cells from normal mice and thymus and lymph node T cells from GVH mice, when cultured on one side of a cell impermeable membrane, restored the plaque-forming cell (PFC) response to sheep erythrocytes of GVH-immunosuppressed spleen cells (GVH-SC) cultured on the other side of the membrane. The restoring ability of T cells present in GVH-SC was inhibited by splenic accessory (A) cells. A direct relationship was shown between the proportion of splenic A cells and the degree of suppression of the PFC response during the first 10 days of the GVH reaction. Normal or GVH A cells reconstituted the PFC response of normal cells and GVH-SC depleted of their A-cell fraction. An optimum ratio of A: nonadherent (NA) cells (1: 10) was required for maximum reconstitution. Larger proportions of A cells inhibited the PFC response. The results suggest that GVH-induced immunosuppression is due, at least in its initial phase, to a depressed T-cell helper function caused by a marked increase of A cells in the spleen.     

Prostaglandin synthesis by lymphoid tissue of mice experiencing a graft-versus-host reaction: relationship to immunosuppression

Studies were carried out on the induction of PGE synthesis during the GVH reaction and its role in GVH-induced immunosuppression. The results demonstrated that spleen, lymph node cells and, to a much lesser degree, thymus cells obtained from adult C57BL/6 × AF₁ mice treated with 50–75 × 10⁶ C57BL/6 lymphoid cells were stimulated to produce PGE during the course of the GVH reaction. The spleen and lymph node PGE production peaked at Day 9 post-GVH induction (30- and 15-fold higher than normal, respectively). Thereafter, it declined to near normal levels by Days 25–30 post-GVH induction. Passage of GVH spleen cells through a rayon column removed macrophages but not mitogen-responsive T and B cells and also removed nearly all of the PGE-producing cells, except during the later course of the GVH reaction. Removal of PGE-producing cells from GVH-immunosuppressed spleen cells significantly reconstituted the mitogen response to PHA and LPS. Treatment of mice experiencing a GVH reaction with indomethacin delayed the onset of suppression of the plaque-forming cell response to sheep erythrocytes. These results suggest that early GVH-induced immunosuppression which may represent an amplified normal regulatory mechanism is mediated by increased macrophage production of PGE which suppresses both B- and T-cell functions, whereas at later stages other immunosuppressive mechanisms become operational.     

Recruitment of effector lymphocytes by initiator lymphocytes. Recruited lymphocytes are immunospecific and include graft-versus-host-reactive lymphocytes.

We have shown previously that initiator T lymphocytes (ITL), sensitized in vitro against fibroblast antigens, recruit effector T cells in vivo. After injection into hind footpads of syngeneic recipients, sensitized ITL migrated to the draining popliteal lymph nodes (PLN) and activated a trapping mechanism by which circulating lymphocytes were recruited in the PLN. This paper reports experiments designed to test the immunospecificity of these recruited T lymphocytes (RTL). We found that immunospecific RTL were depleted from other lymphoid organs during recruitment in the PLN. However, immunospecific ITL were not depleted from spleens during PLN recruitment. Thus ITL and RTL are functionally distinguishable. We show that specific GVH reactive lymphocytes were also lost from spleens and distal lymph nodes during trapping of RTL in the PLN. Thus, the trapping phase of the recruitment response is immunospecific, as are the sensitization and effector phases. The trapped RTL are antigen-specific, and include the pool of GVH-reactive-lymphocytes committed to the same alloantigen. Thus, it appears that GVH-reactive cells respond to syngeneic ITL sensitized against allogeneic fibroblasts.     

Allogeneic cell interactions in the rat: II. The relationship between DNA synthesis and cell proliferation during local graft-versus-host and host-versus-graft reactions

DNA synthesis and cell proliferation were measured, using [¹²⁵I]iododeoxyuridine (¹²⁵IUDR) incorporation and lymph node weight, respectively, in the popliteal nodes of rats undergoing local graft-versus-host (GVH) and host-versus-graft (HVG) reactions in response to a wide range of cell doses. The relationship between ¹²⁵IUDR incorporation and lymph node weight was investigated at various times during the course of these reactions. Good correlation was demonstrated on linear-linear, log-log, and log-linear plots at early, peak, and late response times in both reactions. These results confirm the usefulness of ¹²⁵IUDR incorporation as a measure of the local GVH reaction at peak response and show that its use extends to the local HVG reaction and to the measurement of both these reactions at early and late response times. There were no statistically significant quantitative differences in the correlations obtained with linear-linear, log-log, and log-linear plots at any time in either reaction, but comparison of the distributions of the residuals about the regression lines indicated that at peak and late response times in the GVH reaction the log-linear plot gave a qualitatively better fit of the results to the regression line. This could imply that at peak and late response times in the local GVH reaction more DNA synthesis is occurring than is required for cell proliferation alone. The theoretical implications of these findings are discussed.     

Stimulation of humoral immune responses to a thymic-independent antigen in mice immunosuppressed by a graft-versus-host reaction.

The immunosuppressive effect of the graft-versus-host (GVH) reaction was studied in CBA × A F₁ (CAF₁) mice which had been rendered immunologically unresponsive by the injection of parental A-strain lymphoid cells (GVH mice). When challenged with a single injection of either sheep red blood cells or Escherichia coli lipopolysaccharide (LPS), GVH mice failed to produce a significant number of plaque-forming cells (PFC) or a significant level of antibody against either the thymic-dependent or the thymic-independent antigen. Multiple challenges with SRBC also failed to stimulate a significant humoral immune response to the thymic-dependent antigen. Multiple challenges with LPS, however, resulted in the production of a significant number of LPS-specific PFC and a high titer of anti-LPS hemagglutinating antibodies. These results suggest that GVH-induced suppression of humoral immune responses is directed partly at B-cell activity and partly at the activity of helper T cells.     

Immunosuppression of normal lymphoid cells by serum from mice undergoing chronic graft-vs-host disease.

Spleen cells from F1 mice undergoing chronic graft-vs-host (GVH) reaction, induced by injection of parental cells, were shown to be immunosuppressed since their in vitro responses to the mitogens concanavalin A (Con A) and bacterial lipopolysaccharide (LPS) were substantially lower than control animals. Serum, from mice undergoing GVH, when cultured in vitro with normal spleen cells was immunosuppressive. The proliferation response to Con A and allogeneic cells of normal syngeneic, allogeneic, and parental spleen cells was 90% suppressed when serum from mice undergoing chronic GVH was added in comparison to the addition of serum from untreated F1 mice. Similarly, the in vitro antibody response to a T-dependent antigen was impaired; however, the antibody response to a T-independent antigen was not impaired. These results indicate that T cell functions are more sensitive than are B cell functions to immunosuppressive factors in the serum of mice undergoing GVH.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.     

Soluble factors and the immune response: in vitro studies of the immunosuppression induced by the graft-versus-host reaction

In vitro experiments were carried out to investigate the cause(s) of the immunosuppression induced by the graft-versus-host (GVH) reaction in F₁ hybrid mice injected with parental strain lymphoid cells. A modified Marbrook culture chamber, made up of two cell compartments separated by a cell impermeable membrane, was used in these studies. Spleen cells from either normal animals (NSC) or from animals experiencing a GVH reaction (GVH-SC) were cultured with sheep red blood cells (SRBC) and the direct plaque-forming cell (PFC) response to SRBC was measured. It was found that normal thymus, lymph node and spleen cells, separated from the GVH-SC by a cell impermeable membrane, restored partially or totally the immune response of the suppressed cells, while bone marrow cells did not. It was also found that GVH-SC inhibited the PFC response to SRBC of NSC when mixed in culture at a ratio of 1:5. Conversely the inhibitory effect of GVH-SC on the immune response of NSC was abrogated when the two cell populations were separated by a cell impermeable membrane. These observations demonstrate that GVH-induced immunosuppression is caused, at least in part, by the deficiency of a T-cell derived factor which is a necessary component of the normal immune response. It is suggested that the suppressive effect of GVH-SC on the immune response of NSC is mediated by a non-T cell which regulates the release and/or production of the T-cell derived factor.     

Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1-Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Lymphoid tissue responses to perfluorocarbon emulsion in mice

The effects of either intraperitoneal or intravenous injection of low doses (5 or 10 ml/kg) of the proprietary emulsified perfluorocarbon-based blood substitute, Fluosol-DA 20%, on mouse lymphoid tissue and antibody production against sheep erythrocytes (SRBC) have been investigated. Mean liver weight was significantly increased and gut mesenteric lymph node (MLN) weights decreased in all animals injected with Fluosol-DA, irrespective of route of administration. In contrast, spleen weight decreased following intravenous injection of emulsion at 5 ml/kg. The mean plasma haemagglutination response to SRBC was significantly (P less than 0.01) increased in animals injected intraperitoneally with Fluosol at both doses but was similar to control in all other cases. These results show that lymphoid tissue responses to Fluosol-DA in mice are variable and that antibody production against intraperitoneally-injected SRBC is enhanced by prior injection of emulsion into the peritoneal cavity.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. II. Significance of MHC antigens in anti-Mls tolerance

Specificity of anti-Mlsa tolerance induced in BALB/c (H-2d, Mlsb) neonates was investigated by a popliteal lymph node (PLN)-swelling assay for the local graft-versus-host (GVH) reaction by injecting tolerant thymus cells into the footpads of several types of F1 hybrid mice. When thymus cells were obtained from 1-week-old normal BALB/c, they evoked enlargement of PLNs of (BALB/c X DBA/2)F1 (H-2d, Mlsb/a) [CDF1] recipients and of other hybrid recipients, heterozygous in Mlsa,c,d alleles, irrespective of the major histocompatibility complex (MHC) haplotypes. The same thymus cells did not cause the response in MHC-heterozygous F1 hybrids when the hybrids were homozygous in Mlsb, identical with BALB/c mice. Therefore, the PLN response to Mls antigens, known to be closely associated with MHC-class II antigens, was not directed to the class II antigens themselves. This enabled us to examine the effects of MHC on tolerance induction to the Mls antigens. When BALB/c neonates were injected with CDF1 bone marrow cells, complete tolerance to Mlsa-H-2d antigens of CDF1 cells was induced in the thymus, while responsiveness to Mlsa antigens in the context of H-2k and H-2b antigens, was not affected. This indicates MHC-restriction of neonatal tolerance to Mls antigens. Furthermore, when Mls and H-2-heterozygous (BALB/c X AKR)F1 (H-2d/k, Mlsb/a) bone marrow cells served as the tolerogen, thymus cells of BALB/c neonates were also tolerized to Mlsa-H-2k antigens as well as to Mlsa-H-2d antigens, which suggests the involvement of MHC, probably class II antigens of tolerance-inducing cells.     

Recruitment of effector lymphocytes by initiator lymphocytes. Circulating lymphocytes are trapped in the reacting lymph node.

In previous studies, we have shown that initiator T lymphocytes (ITL) sensitized in vitro recruit effector T lymphocytes in vivo. When ITL were sensitized against fibroblast antigens in vitro and injected into footpads of syngeneic recipients, they induced enlargement of the draining popliteal lymph node (PLN) and the development there of specific effector lymphocytes of recipient origin. To study the basis of this lymph node response in recruitment, we injected 51Cr-labeled spleen cells i.v. into recipients of sensitized ITL and found that the labeled circulating lymphocytes were trapped in the reacting PLN. The trapping depended on surface properties of the labeled circulating lymphocytes, as revealed by various enzymatic treatments. The trapping process was radiosensitive, both on the part of the trapped lymphocytes and the lymph node-trapping mechanism. Thus, sensitized ITL injected into the hind footpads migrate to the PLN and induce the trapping of circulating recruitable lymphocytes, which either differentiate into or regulate the differentiation of effector T lymphocytes.     

Adult thymectomy promotes the manifestation of autoreactive lymphocytes.

Spleen cells from adult thymectomized mice (ATX) were assayed in a syngeneic graft vs host (GVH) model based upon enlargement of the draining popliteal lymph node following syngeneic cell inoculation into the hind footpad. Spleen cells from ATX mice have been found to induce a significantly higher increase in the weight of the regional lymph node than that induced by the injection of normal spleen cells. Irradiated spleen cells from ATX donors did not cause a similar increase, suggesting either that proliferation of the transferred cells was required at some stage of the reaction or that autoreactive cells are radiosensitive. Autoreactive cells were found in the spleen of mice 2 to 3 months after the thymectomy but were never found in the lymph nodes of such animals or in the thymus of intact mice. They are not phagocytic adherent cells and are not retained on nylon wool columns, which suggests that they belong to the T-cell lineage. Autoreactivity is lost when spleen cells from ATX donors are depleted of autologous rosette-forming cells (A-RFC) by centrifugation on a Ficoll-Hypaque gradient after rosette formation. Autoreactive spleen lymphocytes might belong to the population of A-RFC previously characterized as a population of immature T cells.     

Characteristics of the immunosuppressive activity of lymphoid organ cells in the process of sensitization with ram erythrocytes and exposure to antilymphocyte serum

In the adoptive transfer of cells obtained from the thymus, lymph nodes and the spleen to intact syngeneic animals the suppression of immune response was induced by lymph node cells. If the donors were previously sensitized, the cells of the thymus and lymph nodes showed suppressive activity in the adoptive transfer test. A single injection of antilymphocytic serum to the donors of lymphoid cells, previously sensitized with sheep red blood cells, enhanced the immunosuppressing action of thymocytes and lymph node cells.