The distribution of 51Cr-labeled lymphocytes into antigen-stimulated mice. Lymphocyte trapping.

The localization of syngeneic (51)Cr-labeled lymph node cells was investigated in CBA/J mice previously challenged with sheep erythrocytes, Salmonella H antigen, keyhole limpet hemocyanin, C57BL/6J skin, or rat skin. The effect of time, dose, and route of antigen administration on lymphocyte migration was studied in both primary and secondary responses. When the distribution pattern of lymphocytes was examined after 20-24 hr, it was found that increased localization of labeled cells occurred in spleen after intravenous or intraperitoneal antigen injection, and in draining lymph nodes after subcutaneous antigen injection or skin grafting. Increased localization (trapping) of lymphocytes was antigen dose dependent and could be demonstrated when 1-6 hr had elapsed between intravenous antigen administration, or when 24 hr had elapsed between subcutaneous antigen administration and intravenous cell infusion. Trapping was transient, lasting approximately 24 hr. Maximal trapping of lymphocytes in the draining nodes occurred 9 days after skin grafting in the first-set allograft response, and 3 days after grafting in the second-set allograft and first-set xenograft responses. The cell type trapped, the specificity and mechanism of action of the trap, and the role of lymphocyte trapping in the initiation of immune responses are discussed.

Immunoreactive thymosin alpha 1 is associated with murine T-cell lymphomas.

Growth of murine spontaneous and transplanted AKR T-cell lymphomas results in marked elevations of serum immunoreactive thymosin alpha 1. Thymosin alpha 1 is one of the peptide hormones believed to be secreted primarily by the thymic epithelium. This elevation, however, is not mediated by the thymus but rather, seems to be directly associated with the tumor cells. Growth of a B-cell lymphoma does not generate elevated immunoreactive thymosin alpha 1 in the serum, thus, a thymosin alpha 1-like peptide is selectively associated with these T-cell lymphomas. The possible relationship between expression of T-leukemia viruses and alpha 1 expression is discussed.

Lymphocyte trapping. Differential effects of ATS and irradiation on trapping in lymph nodes and spleen.

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.

Effects of BCG on lymphocyte trapping.

The effects of Bacillus-Calmette Guerin (BCG) administration upon lymphocyte traffic was studied in BALB/cJ mice. Intravenous or subcutaneous injection of BCG resulted in increased localization (trapping) of 51Cr-labeled syngeneic lymphocytes in the spleen or draining nodes. BCG-induced splenic trapping was biphasic, occurring 1 to 2 days after BCG injection, and reappearing 3 weeks later. Trapping in draining lymph nodes persisted and increased during the 24 days of study. These results are in contrast to those obtained with SRBC, where trapping was evident only within the first few days of antigen injection. Additional studies revealed that prior injection of BCG influencdd the ensuing magnitude of SRBC-induced trapping. Intravenous administration of BCG suppressed SRBC-induced splenic trapping; both intravenous and subcutaneous injection of BCG enhanced trapping to SRBC in the draining lymph nodes. These studies suggest that one mechanism for potentiation of immune responses by BCG may operate via BCG's effects upon regulation of lymphocyte traffic to the lymphoid tissues.

F1 resistance to AKR lymphoma cells in vivo and in vitro.

AKR X DBA/2 (AKD2F1) mice resist growth of injected spontaneous AKR lymphoma cells. In vitro, normal F1 responder cells generate Thy-1 positive effector cells specifically cytotoxic for the sensitizing antigens of parental AKR normal or lymphoma cells. The possibility is considered that the homozygous normal and neoplastic AKR cells express antigenic determinants, analogous to hemopoietic histocompatibility (Hh) antigens, which are recognized by the F1 hybrid, resulting in in vivo tumor resistance and in vitro cell-mediated cytotoxicity.

Enhancement of murine thymocyte cytotoxic T cell responses by thymosin.

Incubation of murine thymocytes with thymosin Fraction 5 (F5) results in a twofold enhancement of the cytotoxic T lymphocyte response (CTL). The assay exhibits requirements for optimal concentrations of thymosin (100 micrograms/ml) and optimal responder/stimulator ratios. Enhancement of CTL activity can be demonstrated in several responder/stimulator strain combinations. The data indicate that thymosin F5 acts via the responder thymocyte population rather than the stimulator cells, since comparable effects were obtained using nude spleen stimulator cells devoid of mature T cells. This system provides a useful bioassay for identifying the component peptides of thymosin F5 which promote thymocyte differentiation and/or maturation and for elucidating the mechanisms of action of the biologically active thymosin peptides.

Regulation of lymphocyte trapping: the negative trap.

Suppression of 51Cr-labeled lymphocyte homing to the spleen occurs after administration of insoluble protein antigen and as a late sequela to the graft-vs-host response (GVHr). This decrease in splenic localization of labeled cells, termed "negative trapping," has elements of immunologic specificity in that tolerance induction or presensitization abrogates the negative trap. Increasing the dose of antigen accelerates the appearance of the negative trap, which is, however, evanescent, lasting from 24 to 48 hr. Synergistic and antergistic regulation of the GVHr-induced lymphocyte trap is produced by populations of 850 R-irradiated thymocytes and by F1 cortisone-resistant thymocytes. The time at which these subpopulations cause suppression or amplification of the lymphocyte trap correlates with the activity of the GVHr-producing inoculum. These findings suggest that regulation of lymphocyte traffic may provide a mechanism for control of immune responses in vivo.

Thymosins, lymphokines, and the immunology of aging.

Recent data point to a significant role for thymosins, lymphokines, and other soluble mediators in the senescence of the immune response that occurs with aging. In recent years, considerable progress has been made in the isolation and physiochemical characterization of several of these soluble mediators. We are now beginning to define the mechanisms by which these molecules regulate and mediate immune responses. In this paper we review the properties of the best characterized thymic hormones and lymphokines and focus on the role of the endocrine thymus in modulating immune responses. Of particular interest is the recent observation that thymosin fraction 5 can enhance production of interleukin-2 (IL-2) and colony-stimulating factor (CSF), and that IL-2 production, but not CSF production, is selectively diminished in aging mice. Several of the products of the immune system also can act as neuroactive immunotransmitters and modulate a number of neuroendocrine responses. Current studies point to an important role for these molecules in modulating neuroendocrine function, suggesting a broader role for the endocrine thymus in the aging process.

Mechanism of action of thymosin. I. Thymosin fraction 5 increases lymphokine production by mature murine T cells responding in a mixed lymphocyte reaction.

The effect of thymosin on the murine thymocyte mixed lymphocyte response was studied. Thymosin fraction 5 (TF5) caused a two- to threefold enhancement of the proliferative response and production of IL 2 when murine thymocytes were cultured with alloantigenic stimulator cells. Production of a second lymphokine, CSF, was increased up to sevenfold. The target cell for thymosin was a mature T cell, because the PNA- subpopulation of thymocytes, as well as peripheral lymph node lymphocytes, responded to culture with TF5 and alloantigen by enhanced proliferation and lymphokine production. The active component of TF5 appears to be one or more as yet unidentified peptides, because neither of the well-characterized TF5 component peptides, alpha 1 or beta 4, were active. After incubation with TF5 in primary culture, cells remaining after 10 to 14 days were increased both in number and in secondary response to alloantigen, as measured by lymphokine production. These results suggest that TF5 contains one or more biologically active components which can modulate mature T cell activity and lymphokine production, and which provide the basis for understanding some of the previously reported diverse effects of thymosin.

The biologic activity of affinity cross-linked oligomers of rabbit immunogloblin G as tested by antigody-dependent cell-mediated cytolysis (ADCC).

The abilities of normal spleen cells and macrophage-like and lymphocyte-like tumor cells to serve as effectors in antibody-dependent cell-mediated cytolytic (ADCC) reactions were examined. It was observed that those cells which contained Fc receptors on their surfaces were effective mediators of ADCC whereas those not bearing Fc receptors were inactive. Oligomers of IgG were able to inhibit the ADCC reaction and, with the macrophage line, inhibition and binding closely paralleled each other. With the lymphocyte lines, a large degree of inhibition of ADCC occurred when only small amounts of oligomers were bound; with normal spleen cells inhibition occurred in the same range of oligomer concentrations required to inhibit the tumor-mediated ADCC reactions. The data suggest that the interaction between antibody-coated erythrocytes and effector cells is unexpectedly weak.

Purification and biological activity of thymosin, a hormone of the thymus gland.

The purification and chemical properties of thymosin, obtained from bovine thymus tissue, are described. The biological activity of the thymic hormone has been assessed by a newly developed rosette assay, which permits measurement of thymus-dependent lymphoid cells. Thymosin activity is associated with a physico-chemically homogeneous protein of molecular weight 12,600. The hormonal activity is evident in in vitro incubation assay, after injection into adult thymectomized mice, and in prolonging survival of neonatally thymectomized mice and the reconstitution of their response to a skin allograft.

Suppressor cell responses in patients with rheumatoid arthritis: the effect of thymosin.

Peripheral blood lymphocytes (PBL) from patients with rheumatoid arthritis (RA) and age and sex-matched normal controls were compared in a Concanavalin A (Con A)-induced suppressor cell assay. Suppression induced by pre-incubation with Con A was significantly greater in PBL from RA patients than in PBL from normal controls. Preincubation with thymosin fraction 5, in the absence of Con A, also induced greater suppressor cell activity in PBL from normal controls. Preincubation with Con A and thymosin, simultaneously, induced suppression similar to that seen with Con A alone. These results suggest the presence of an immunoregulatory defect in RA, characterized by an excess of both Con A and thymosin-inducible suppressor cells, that may play a role in disease pathogenesis. The implications of these observations for immunotherapy of rheumatoid arthritis with thymosin are discussed.

Separation and characterization of two component tumor lines within the AKR lymphoma, AKTB-1, by fluorescence-activated cell sorting and flow microfluorometry analysis. I. the coexistence of sIg+ and sIg- sublines.

The AKTB-1 tumor line, previously described as having both T and B cell characteristics, was separated by electronic cell sorting (ECS) on a fluorescence-activated cell sorter (FACS) on the basis of surface immunoglobulin (sIg). This resulted in 2 distinct sIg+ and sIg- subpopulations, which were transferred in graded doses (10(1) to 10(3)) into groups of normal AKR animals. The resulting splenic tumor cells were analyzed at several time points by flow microfluorometry for surface characteristics (sIg, Thy 1, Lyt 2, and Ly9) and by erythrocyte-antibody rosetting for FcR. Two distinct sublines were identifiable--one of which was sIg+, FcR-, Thy 1.1+, Lyt 2.1+, PNA+, Ly9+ and the other of which was sIg+, FcR+, Thy 1.1-, Lyt 2.1-, PNA-, Ly 9+. When these 2 sublines, generated by ECS fractionation and high dilution transfer, were serially transferred at high doses (10(5)), they maintained their unique characteristics as distinct sublines, now designated AKTB-1t and AKTB-1b.

Synthesis and characterization of membrane immunoglobulin, Ia, and H-2 molecules of thy 1+ AKR/J lymphomas.

Three AKR lymphomas displaying B cell and T cell characteristics have been described. Because of the proclivity of normal AKR/J mice to develop T cell lymphomas, and the rarity of lymphomas with dual characteristics, the B cell markers of these tumors were studied more intensively. Fluorescence data with class-specific anti-immunoglobulin reagents demonstrated that the tumor cells stained only with class-specific anti-IgM reagents. Because of the possibility that the surface Ig was passively acquired and of reports that certain anti-mu-chain sera react with "IgT", chemical characterization of the immunoglobulin molecules was performed. Using 3H-leucine internal labeling, we showed that all three tumor lines synthesized the immunoglobulin found on their surface, and that the immunoglobulin had the chemical and immunologic characteristics most typical of monomeric surface IgM, and was composed of mu-chains and light chains. The Ia antigens found on these cells were also examined. These antigens were also synthesized by the cells and were present in the same molecular form and in the same approximate quantity as Ia antigens on normal spleen cells.

Multiple occurrence of spontaneous AKR/J lymphomas with T and B cell characteristics.

Three Thy 1.1-positive and surface IgM-positive (Thy 1+, SIg+) AKR/J lymphoma lines are described. These doubly marked tumors arose spontaneously in the peripheral lymphoid organs of 14- to 16-month-old AKR/J mice that either had spontaneous thymus atrophy or had been thymectomized at 1 month of age. All lines bore surface Thy 1.1, Ly,Ig(micron-chain) and Fc receptor (FcR), detectable by immunofluorescence. Immune response region (Iak) antigen was present on the two lines tested. Persistence of Thy 1.1 antigen and SIg after long-term tissue culture provided evidence that these markers were not passively acquired. One of these tumor lines, AkTB-1 always grows in lymph nodes as Thy 1.1-positive,SIg-negative tumors cells, whereas tumor cells growing in the spleen are initially Thy 1.1 positive, SIg negative, but they rapidly acquire SIg, FcR, and 1a between 18 and 21 days of passage.

Comparison of the effects of thymosin and other thymic factors on modulation of interleukin-2 production.

Thymosin fraction 5 (TF5) enhances interleukin-2 (IL-2) production by human peripheral blood mononuclear leukocytes (PBL) when cocultured in the presence of phytohemagglutinin (PHA). This important biological activity of TF5 is not due to the presence of two previously well-characterized component peptides, thymosin alpha 1 and thymosin beta 4, but rather to a new, still to be identified component of TF5. Comparison of TF5 with other well-defined thymic preparations and peptides indicates that this biological activity is unique to TF5 and a closely related extract prepared from porcine thymus tissue. Using a standard human PBL cell population obtained by leukopheresis and cryopreservation, it should now be possible to establish a reproducible bioassay for the isolation and characterization of one or more components of TF5 with the property of enhancing PHA-induced IL-2 production by human PBL.

Mechanism of action of thymosin. II. Effects of aspirin and thymosin on enhancement of IL-2 production.

Recent studies have shown that thymosin fraction 5 (TF5) enhances production of interleukin-2 (IL-2) by phytohemagglutinin (PHA)-stimulated normal human peripheral blood mononuclear leukocytes (PBL). In this study we sought to determine whether this effect of TF5 might be mediated via the cyclooxygenase or lipoxygenase pathways. Our studies demonstrate that aspirin, an inhibitor of the cyclooxygenase pathway, given in vivo, or added to cultures in vitro, results in two-fold increased IL-2 production by PHA-stimulated PBL. This increase is comparable to that seen when PBL are cultured in vitro with TF5. When aspirin and TF5 are added simultaneously to PBL in the presence of PHA, an additive response is seen. An inhibitor of the lipoxygenase pathway, 15-hydroxyeicosatetraenoic acid, did not significantly change IL-2 production by PBL or influence the enhancement by TF5. Augmentation of IL-2 production by aspirin and/or TF5 was prevented by monocyte depletion of the PBL population. These results are interpreted as demonstrating (a) that TF5 and aspirin augment, by distinct mechanisms, IL-2 production by normal human PBL, (b) that the effects of both of these agents are mediated directly or indirectly via a monocyte population and (c) that aspirin, in addition to its analgesic and anti-inflammatory properties, may act as a modulator of immunological responsiveness, either alone or in combination with other biological response modifiers such as thymosin.

Thymosin increases production of T-cell growth factor by normal human peripheral blood lymphocytes.

The in vitro incubation of phytohemagglutinin-stimulated peripheral blood lymphocytes with thymosin results in a marked and reproducible increase in production of T-cell growth factor, which is dose dependent and most pronounced in the first 24 hr of culture. Incubation of lymphocytes with thymosin alone failed to induce any production of T-cell growth factor. The biological activity of thymosin fraction 5 cannot be attributed to the activity of thymosin alpha 1, one of the well-characterized peptide components of fraction 5. These data provide the basis for (i) a potential mechanism for the in vivo immunorestorative effects of thymosin in primary and secondary immunodeficiencies and (ii) identification of an additional, but as yet undefined, immunoregulatory component of thymosin fraction 5.