Regulation of lymphocyte motility by macrophages: characterization of a lymphocyte migration inhibitory factor derived from a macrophage-like cell line

An inhibitory factor on lymphocyte migration was detected using a capillary random migration assay in the culture supernatant of peritoneal exudate macrophages cultured at concentrations greater than 8 x 10(6) cells/ml. After examining different macrophage-like cell lines, J774A.1 cells were found to produce this inhibitory factor, which was termed lymphocyte migration inhibitory factor (LMIF). The inhibitory effect of LMIF on the migration of spleen lymphocytes, thymocytes, and bone marrow cells was determined. The migration of thymocytes was more sensitive to LMIF than was the migration of spleen lymphocytes and bone marrow cells. Interestingly, when the effect of LMIF was tested on the migration of spleen T cells and B cells, T cells were more sensitive than B cells. When the thymocytes were separated by peanut agglutinin into mature and immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes to the effect of LMIF, suggesting that the greatest effect of LMIF was on the migration of mature T cells. Partial purification of LMIF by ion-exchange and gel-filtration chromatography revealed that it is approximately 14,000 in molecular weight and could exist in either monomeric or dimeric forms. The possible role of this factor in an immune response is discussed.     

Functional differentiation of thymocytes induced by macrophages: cellular, humoral and genetic aspects

The cellular, humoral and genetical mechanisms of induction of T-effectors of the graft vs. host reaction (GHR) were studied in a double-cell culture of phagocytizing mononuclears with thymocytes. Experiments were carried out on mice of inbred and recombinant strains. The GHR intensity was estimated by the increase in the number of cells in the popliteal lymph node, regional with reference to the introduction of parental thymocytes into the F1 hybrid. For the induction of the GHR T-effectors from the immature population of thymocytes to be realized, a direct physical contact and identity by the H-2K locus of the major histocompatibility complex between the cooperating cells in culture are indispensable. An antiserum containing antibodies against the H-2K locus products prevents the induction. At the same time antibodies against antigens controlled by loci of I-region or the H-2D locus do not affect the accumulation of T-effectors. Contact interaction of phagocytizing mononuclears with thymocytes results in accumulation of a 65,000 D humoral factor in the culture medium. Incubation of the intact thymocytes with this factor ensures functional transformation of immature thymocytes to corresponding effector cells. For the humoral induction of T-effectors to be successfully realized, identity by the T-2K locus between the factor producents and intact thymocytes is indispensable, as well as in the conditions of direct intercellular interaction. It is suggested that H-2K specificity is incorporated into the factor structure.     

Thymocyte-stimulating factor from peripheral blood cells.

Human peripheral blood leukocytes (HPBL) produce a thymocyte-stimulating factor (TSF-HPBL) that enhances the phytohemagglutinin (PHA) and concanavalin A (Con A) responsiveness of murine thymocytes. This activity is considerably specific for thymocytes. TSF-HPBL is not mitogenic by itself. Experiments with cell cultures pretreated with carbonyl iron particles showed that phagocytic cells are not involved in the production of mouse and rat TSF but are involved in the production of TSF-HPBL. The dose-response profile to PHA of murine thymocytes cultured in the presence of TSF-containing supernatants is similar to that of mature, immunocompetent spleen cells. TSF-HPBL, however, does not enhance the PHA responsiveness of murine thymocytes at low (<0.25 μg/microwell) concentrations of mitogen. TSF enhances the PHA and Con A responsiveness of the high-density subpopulations of thymocytes isolated on a Ficoll-Hypaque gradient. In general, the enhancing effect of TSF-HPBL on these subpopulations of thymocytes is smaller than that exerted by TSF. While supernatants containing TSF confer to thymocytes the ability to participate in a mixed lymphocyte reaction, this effect is not exerted by supernatants containing TSF-HPBL. A factor enhancing the PHA and Con A responsiveness of murine thymocytes is also produced by murine peripheral blood leukocytes (TSF-MPBL). This factor, similarly to TSF-HPBL, is produced by phagocytic cells and does not confer to murine thymocytes the ability to participate in a mixed lymphocyte reaction. Human T-cell lines do not enhance the PHA or Con A responsiveness of murine thymocytes. TSF-HPBL has a molecular weight of about 30,000 daltons, as measured by Sephadex filtration. Its half-time of inactivation as 56 °C is 162 ± 8 min.     

Amplification of the proliferative response to alloantigen by a factor present in an extract of syngeneic thymic lymphoid cells: demonstration of optimal conditions and target cell for factor activity.

A factor extracted from syngeneic thymic lymphoid cells (thymocytes) is shown to amplify the proliferative (MLC) response of syngeneic lymphoid cells to alloantigen in vitro. The optimal conditions for an effect of the thymus factor are quantitatively defined by kinetic and dose-response studies. Other variables that could potentially influence the activity of the thymus factor, such as the presence of 2-mercaptoethanol and the source of alloantigen, are identified. Factor activity can be recovered from semi-allogeneic thymocytes, as well as syngeneic thymocytes. The factor appears to predominantly effect the proliferative response of T cells localized in peripheral lymphoid organs. As such, this factor appears to be distinct from the variety of previously described factors derived from thymic reticuloepithelial elements that are thought to primarily induce the differentiation of T cell precursors found predominantly in bone marrow. Several possible mechanisms of action of this thymocyte-derived factor are considered.     

Enhancement of DNA synthesis and cAMP content of mouse thymocytes by mediator(s) derived from adherent cells.

Supernatants of adherent mouse peritoneal exudate cells or human mononuclear cells were used as the source of lymphocyte activation factor (LAF). LAF was found to potentiate the effect of mitogens such as PHA and Con A on DNA synthesis by mouse thymocytes. However, LAF also was capable of reducing vigorous thymosyte reactions to Con A. Thus, LAF usually enhanced the effect of PHA on DNA synthesis by BALB/c thymocytes to a relatively greater degree than that of Con A. This change in the ratio of Con A to PHA response of thymocytes suggests that LAF can serve as a regulator of thymocyte DNA synthesis. Moreover, in the presence of LAF, allogeneic thymocytes developed the ability to have bidirectional mixed thymocyte reactions. Exposure to LAF not only improved the ability of parental thymocytes to act as responder cells, but, in addition, led to increased stimulatory activity of F1 thymocytes, presumably by promoting the differentiation of stimulator cells. These indications that LAF affected differentiation were investigated further by studying its effect on the cAMP content of thymocytes. LAF stimulated significant immediate but transient elevations of intracellular cAMP and adenylate cyclase activity in thymocyte membranes. In contrast, the mitogens themselves failed to elevate or to influence the effect of LAF on the content of intracellular cAMP of thymocytes. Furthermore, the potentiating effect of LAF on mitogen-induced thymocyte DNA synthesis at times was enhanced by exogenous cGMP, carbachol, or imidazole. These findings suggest that LAF, through its stimulation of cAMP levels in thymocytes may in turn promote thymocytes to differentiate sufficiently to become competent to proliferative in response to mitogens.     

Distinct patterns of lymphokine requirement for the proliferation of various subpopulations of activated thymocytes in a single cell assay

The frequency and capacity for clonal expansion of several murine thymocyte subpopulations responsive to various IL (fetal day 15, and adult CD4-8-, CD4+8- and CD4-8+) were investigated using a single-cell limiting-dilution cell culture system without filler cells. This assay requires the presence of PMA and ionomycin. The main conclusions of these studies are the following: 1) IL-4 is a better growth factor than IL-2 for immature thymocytes (fetal day 15 or adult CD4-8-). 2) IL-2 is a better growth factor than IL-4 for mature phenotype thymocytes (CD4+8- and CD4-8+). 3) IL-4 is a relatively poor growth factor for adult CD4-CD8- thymocytes and CD4+CD8- thymocytes, while it induced strong responses in fetal day 15 and CD4-8+ thymocytes. 4) IL-6 enhanced the response of CD4+8- thymocytes to either IL-2 or IL-4. 5) Cortisone-resistant thymocytes grown initially with IL-4 and then switched to IL-2 showed a significant decrease in cloning efficiency. No inhibitory effect was observed when cells were cultured first with IL-2 and then switched to IL-4. 6) Finally, supernatant from Con-A stimulated rat spleen cells induced maximal growth of all adult thymocyte populations tested, suggesting that unidentified thymocyte growth factor(s) remain to be characterized. These results indicate that the maturational stage of thymocytes determines their requirements for activation and proliferation.     

Effect of thymocyte-stimulating factor-containing supernatants on the surface antigens of murine thymocytes.

Incubation of murine thymocytes in thymocyte-stimulating factor (TSF)-containing supernatants causes a four- to fivefold increase in the expression of the H-2^k and H-2^d antigens and a similar decrease in the expression of the TL antigen (in TL⁺ strains) on the surface of these cells. Experiments with antisera directed toward the private H-2K and H-2D antigens showed that TSF-containing supernatants cause approximately the same increase in the expression of the H-2K and H-2D antigens of thymocytes of the d and b haplotypes. With thymocytes of the k haplotype, only an increase in the expression of the H-2D antigens takes place, while no significant increase was found for the H-2K antigens. TSF-containing supernatants cause no significant change in the expression of the following antigens on the surface of thymocytes: Thy-1.1, Thy-1.2, Ly-1.2, Ly-2.2, Ly-6.2, Th-B, Ia-1,2,3,7, and G_IX. A factor similar to murine TSF, produced by human peripheral blood leukocytes, does not affect appreciably the expression of the H-2 antigens on the surface of murine thymocytes. The factor(s) causing the increased expression of the H-2 antigens on the surface of murine thymocytes appears to be produced by T lymphocytes. The factor(s) is eluted from a Sephadex G-100 column in at least two broad peaks with molecular weights of 300,000 and 90,000-25,000. Most of the activity enhancing the expression of the H-2 antigens is lost at pH 2, while most of it is maintained at pH 11.5 and at 56 °C. On the basis of these properties, it is concluded that the factor under study is probably different from the factor enhancing the phytohemagglutinin responsiveness of thymocytes.     

Influence of age on the ability of thymic adherent cells to produce factors in vitro which modulate immune responses of thymocytes

Thymic adherent cells from BALB/c mice ranging in age from 1 day to 20 months were cultured in vitro for 1 month. The supernatants, collected at weekly intervals, were assessed for their ability to augment the antigen/mitogen responses of thymocytes from 2- or 4-week-old BALB/c mice and spleen cells from 3-month-old nude BALB/c mice. The results indicate that (a) the ability of thymic adherent cells to produce an augmenting factor(s) declines sharply between 2.5 and 5 months of age; (b) thymic adherent cells of 1-day-old mice synthesize an inhibitory factor(s) in addition to the augmenting factor, while those of young adult mice synthesize only the augmenting factor, and those of 20-month-old mice synthesize primarily the inhibitory factor; (c) supernatants containing the augmenting factor can be neutralized by mixing them with supernatants containing the inhibitory factor; (d) thymocytes which are responsive to the augmenting factor are immature as judged by their sensitivity to dexamethasone and by their ability to bind macrophages; and (e) spleen cells of normal and nude mice are not responsive to either the augmenting or the inhibitory factor of the supernatant.     

Calcitonin gene-related peptide and its receptor in the thymus

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat -CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8–37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.     

Structural and functional characteristics of the CD3 (T3) molecular complex on human thymocytes

The CD3 (T3) molecular complex is noncovalently associated with the antigen receptor molecule on T cells. The mitogenic properties of anti-CD3 antibodies have suggested that this complex may be the transducer of the antigenic signal to the intracellular environment. In the present investigation, we studied some of the structural and functional characteristics of the CD3 complex on human thymocytes. In 11 specimens tested, we found that anti-CD3 antibodies react with 50 to 76% of the thymocytes. Two-color immunofluorescence analysis revealed that the majority (greater than 50%) of thymocytes express both CD3 and CD1 on their surfaces. The latter is a marker of immature thymocytes. However, a distinct subpopulation comprising 13 to 19% of the total cells displays only CD3, while an approximately equal percentage of cells expresses only CD1. The mitogenic potential of anti-CD3 antibodies on peripheral T cells is dependent on the presence of monocytes. Anti-CD3 antibodies by themselves cannot activate thymocytes, indicating that functionally active monocytes are absent from the thymocyte population. Even the addition of peripheral monocytes does not allow a response of thymocytes to anti-CD3 antibodies. However, when the anti-CD3 antibody 64.1 is added in the presence of exogenous rIL 2, a strong antibody and lymphokine dose-dependent response ensues. Only CD1- CD3+ thymocytes are stimulated by the addition of antibody and IL 2. The mere expression of CD3 on the CD1+ CD3+ subpopulation of thymocytes apparently is not sufficient to render the cells responsive to the signals of anti-CD3 and IL 2.     

Selective stimulation of human thymocyte subpopulations by recombinant IL-4 and IL-3

Human thymocytes and thymocyte subsets were examined for their proliferative response to recombinant interleukin-4 (IL-4) and interleukin-3 (IL-3) in serum-free cultures. IL-4 induced marked proliferation of thymocytes after PHA and TPA stimulation, in contrast to the marginal response of T cells from adult peripheral blood. However, depletion of thymocytes bearing the CD3 antigen diminished the IL-4-induced proliferation of thymocytes, indicating that the response of thymocytes to IL-4 is mainly mediated by the CD3-positive cells. Phenotypic changes after culture with IL-4 showed an increase in the percentage of total thymocytes expressing mature T cell antigens (CD3, CD5, and TCR-1) and a decrease in CD1-positive cells. In addition there was an increase in the percentage of CD4+8- cells in both nylon wool-separated thymocytes and CD3-depleted cells with the disappearance of most of the CD4+8+ cells. However, an increase in the percentage of CD4-8- cells was also observed. The IL-4-responding cells do, however, express the mature T cell antigen, CD5, in high density. The effect of IL-3 on the proliferation of human thymocytes was very low and detected only when the thymocytes were cultured in serum-free medium. Depletion of CD3-positive cells did not diminish the IL-3-mediated proliferation of thymocytes, indicating that IL-3-responsive thymocytes are more immature than the subset of thymocytes which responds to IL-4. These results suggest that IL-4 and IL-3 play different roles in the development of human T cells.     

Differences in the pattern of proliferative response with age in thymocytes undergoing spontaneous and induced proliferation

The proliferative capacity of thymocytes from C3H/HeJ mice decrease as the animals attain maturity. The proliferative response of thymocytes from 24- to 28-week-old mice to stimulation with concanavalin A (Con A) is only 20% of that observed at 4 weeks of age. The decreased proliferative capacity of thymocytes in response to Con A stimulation observed between 4 and 24 weeks of age closely correlates to the drop in thymic weight and cellularity observed during this period. In contrast, the spontaneous proliferative capacity of thymocytes, as well as proliferation of thymocytes in response to stimulation with phorbol myristate acetate (PMA) and ionomycin, drops only slightly during this period, as proliferation under these condition in thymocytes from 24- to 28-week-old mice is approximately 65-70% of that observed in 4-week-old animals. We have previously shown that cytoplasmic extracts from proliferating lymphoid cells contain a factor, termed the activator of DNA replication (ADR), which is capable of inducing DNA synthesis in isolated, quiescent nuclei. We show in this study that the decreased proliferative capacity of thymocytes during whole organism maturation and thymic involution is associated with decreased endogenous levels of ADR, while nuclear sensitivity of thymocyte to ADR was retained during these process. The diminution of ADR activity during thymic involution was quantitatively greater than the loss in proliferative capacity.     

Disparate functional properties of two interleukin 1-responsive Ly-1+2- T cell clones: distinction of T cell growth factor and T cell-replacing factor activities

We describe the properties of two Ly-1+2- T cell clones (Ly-1.14 and Ly-1.21), which are maintained in long-term culture in the absence of other cell types. The clones require media containing a source of interleukin 1 as well as interleukin 2. They retain physiologic responses to interleukin 1, which is required for optimal production of T cell lymphokines by these clones in response to concanavalin A (Con A). The two Ly-1+2- T cell clones differ in their production of lymphokines after stimulation by Con A. The supernatant of clone Ly-1.21 promotes the proliferation of T cells maintained in long-term culture, induces antibody synthesis in cultures of B cells and antigen, and induces the differentiation of cytolytic cells in cultures of thymocytes and antigen; these assays define the properties of T cell growth factor (TCGF), T cell-replacing factor for B cells (TRF-B), and T cell-replacing factor for cytolytic cells (TRF-C), respectively. In contrast, the supernatant of clone Ly-1.14 contains only TCGF activity and does not promote antibody synthesis by B cells or differentiation of cytolytic cells from thymocytes. The results indicates that TCGF and TRF activities reside on independent, although perhaps related, molecules.     

A spleen-derived maturational factor allows immature thymocytes, prepared as cells bearing low amounts of surface sialic acid, to become cytotoxic T cells

A population of immature mouse thymocytes bears low levels of surface sialic acid and can be separated from the more mature high sialic acid-bearing thymocytes by selective agglutination with the sialic acid-specific lectin, lobster agglutinin 1. These immature thymocytes do not proliferate in response to concanavalin A (Con A). They do not produce interleukin 2 (IL-2), do not provide T cell help to B cells for an in vitro antibody response, and as shown here, do not become cytotoxic T lymphocytes when polyclonally stimulated with Con A + IL-2. We describe here a spleen-derived maturational factor which stimulates these immature thymocytes, in the presence of Con A and IL-2, to become cytotoxic T lymphocytes. The maturational factor is a protein secreted by Con A-stimulated mouse or rat spleen cells; it is apparently neither interleukin 1, IL-2, interleukin 3, gamma-interferon, nor combinations of these cytokines, because these materials do not replace the maturational factor. The active material in Con A-stimulated mouse spleen cell supernatant was recovered from a G-75 column in the 33,000-48,000 m.w. range. These experiments suggest that within the lobster agglutinin 1-negative thymocyte population there are cells which can mature under the influence of a spleen-derived factor. It is possible that these cells represent the small subpopulation of immature cells destined to become immunocompetent peripheral T cells. On the other hand, the factor may be rescuing cells destined to die in the thymus.