Lymphokines secreted from sodium periodate-treated lymphocytes

This study was designed to determine if sodium metaperiodate (NaIO₄)-treated lymphocytes secrete lymphokines and if these lymphokines are similar to those obtained from mitogen- or antigen-stimulated lymphocytes. A brief exposure of CBA spleen cells to NaIO₄ induced the secretion of significant amounts of migration inhibitory factor (MIF). This MIF had a molecular weight range between 30,000 and 58,000, and was stable when heated at 56 °C for 30 min, but unstable at 80 °C. These characteristics are similar to those previously reported for mitogen- and antigen-induced MIF. In addition, NaIO₄ induced the secretion of lymphotoxin (LT) from CBA and Balb/c spleen cells, as well as from guinea pig lymph node cells. NaIO₄ was compared to the other inducers in regard to the quantity of LT secreted. Supernatant derived from NaIO₄-treated mouse spleen cells contained less LT than supernatants derived from concanavalin A- or phytohemagglutinin-treated cells, but contained more activity than those supernatants derived from lipopolysaccharide-treated cells. CBA spleen cells secreted significantly more LT than Balb/c spleen cells after NaIO₄ stimulation. NaIO₄-stimulated CBA spleen cells secreted LT in cultures with or without serum, but stimulated Balb/c spleen cells secreted LT only in serum-containing cultures. The advantages of NaIO₄ as an inducer of lymphokines, as opposed to other mitogens or antigens, is the brief exposure of this agent to the cells after which the NaIO₄ is removed, and the lymphokines can be obtained free from the inducer.     

Production of macrophage migration inhibitory factor and lymphotoxin by leukocytes from normal and Wiskott-Aldrich syndrome patients

The production of macrophage migration inhibitory factor (MIF) and lymphotoxin (LT) by cultured leukocytes from patients with Wiskott-Aldrich syndrome (WAS) and normal controls was studied. The presence of these lymphokines in leukocyte culture supernatants usually correlated directly with the dose of stimulant used. Doses of nonspecific mitogens and specific antigens, which produced maximal in vitro lymphocyte transformation, stimulated maximal production of these mediators. When the incorporation of tritiated thymidine by stimulated leukocyte cultures from patients with Wiskott-Aldrich syndrome (WAS) was deficient, they usually produced less MIF and lymphotoxin than normal. However, when their in vitro lymphoproliferative responses were normal, the lymphotoxin activity in supernatants of WAS leukocyte cultures was normal.     

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells

 Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D₃ eliminated natural suppressor activity. In mice that were first treated with vitamin D₃ and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D₃/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D₃/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D₃ to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor. Received: 15 December 1995 / Accepted: 22 March 1996     

Eimeria tenella and E. acervulina: lymphokines secreted by an avian T cell lymphoma or by sporozoite-stimulated immune T lymphocytes protect chickens against avian coccidiosis

The role of avian lymphokines as nonspecific immunomodulators of host immunity against the intracellular parasite Eimeria was investigated. Prophylactic treatment of normal chickens with crude cell-free supernatants obtained from JMV-1 culture, concanavalin A (Con A)-stimulated normal spleen cells, or sporozoite-stimulated immune T cells prior to inoculation with E. tenella or E. acervulina conferred significant protection. These crude cell-free culture supernatants also inhibited intracellular development of eimerian parasites in vitro. Avian macrophages pretreated with these supernatant preparations showed inhibitory activity against Eimeria. This inhibitory activity could not be ascribed to anti-Eimeria antibody, complement, or cell-free Marek's disease virus and was therefore considered to be due to immunomodulating lymphokines present in the culture supernatants. These results suggest that JMV-1-transformed T lymphoblastoid cells, immune T lymphocytes, and Con A-stimulated normal spleen cells secrete lymphokines that can enhance host immunity in a nonspecific manner and implicate cell-mediated immunity as a major mechanism of the protective host immune response against eimerian infections.     

Immunosuppressive factors from human breast carcinoma cell lines that affect initiation of lymphocyte proliferation

Summary Supernatants from two human breast carcinoma cell lines, 734B and 231, have been shown to inhibit lymphocyte activation by mitogens and antigens. This inhibition appears to be specific for lymphocytes or recently stimulated cells, while having no effect on the growth of established cell lines. Studies of the mechanism of inhibition revealed that the factors inhibit lymphocyte activation and that the factors must be present at the initiation of lymphocyte stimulation for inhibition to occur. The supernatants do not inhibit lymphocyte activation by blocking binding of PHA to lymphocytes. Preliminary purification steps have shown that the inhibitory factors present in the tissue culture supernatants are precipitated at 50% ammonium sulfate saturation and their molecular weights are greater than 100 000. The inhibitory capacity of the 734B supernatants was destroyed by heating at 70° C, while the factors present in the 231 supernatants were only partially destroyed by heating to 90° C. The possible mechanism of action of the inhibitory substances released by tumors and their relevance to tumor growth are important to understanding of immune responses to neoplasia.     

Experimental allergic encephalomyelitis in inbred guinea pigs: correlation of decrease in early T cells with clinical signs in suppressed and unsuppressed animals.

Lymphoid cells from MHA hamsters, Hartley guinea pigs, DA and Fischer rats, and BDF₁ mice can be stimulated to incorporate tritiated thymidine by the mitogens concanavalin A and phytohemmagglutinin P. Addition of soybean trypsin inhibitor to cultures of hamster thymocytes or spleen cells had no effect on stimulation. Similar concentrations inhibited stimulation of a subpopulation of hamster lymph node cells. The inhibitable population was concentrated in the peripheral lymph nodes. Concentrations of soybean trypsin inhibitor up to 1 mg/ml did not inhibit mitogen stimulation of guinea pig lymph node cells and partially inhibited the response of spleen cells. The inhibition of splenocyte stimulation was consistently 30–40% which may indicate a specific subpopulation of cells is inhibited. Mitogen stimulation of rat splenocytes was completely inhibited by addition of soybean trypsin inhibitor. On the other hand stimulation of lymph node cells was relatively resistant to inhibition by the protease inhibitor. Mitogen stimulation of BDF₁ splenocytes and lymph node cell cultures was inhibited 60–80% by the addition of soybean trypsin inhibitor.     

Fractionation of lymphocyte subpopulations which regulate mixed lymphocyte reactions.

Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.     

Fibroblast-type reticular stromal cells regulate the lymph node vasculature

The lymph node vasculature is essential to immune function, but mechanisms regulating lymph node vascular maintenance and growth are not well understood. Vascular endothelial growth factor (VEGF) is an important mediator of lymph node endothelial cell proliferation in stimulated lymph nodes. It is expressed basally in lymph nodes and up-regulated upon lymph node stimulation, but the identity of VEGF-expressing cells in lymph nodes is not known. We show that, at homeostasis, fibroblast-type reticular stromal cells (FRC) in the T zone and medullary cords are the principal VEGF-expressing cells in lymph nodes and that VEGF plays a role in maintaining endothelial cell proliferation, although peripheral node addressin (PNAd)(+) endothelial cells are less sensitive than PNAd(-) endothelial cells to VEGF blockade. Lymphotoxin beta receptor (LTbetaR) blockade reduces homeostatic VEGF levels and endothelial cell proliferation, and LTbetaR stimulation of murine fibroblast-type cells up-regulates VEGF expression, suggesting that LTbetaR signals on FRC regulate lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation. At the initiation of immune responses, FRC remain the principal VEGF mRNA-expressing cells in lymph nodes, suggesting that FRC may play an important role in regulating vascular growth in stimulated nodes. In stimulated nodes, VEGF regulates the proliferation and expansion of both PNAd(+) and PNAd(-) endothelial cells. Taken together, these data suggest a role for FRC as paracrine regulators of lymph node endothelial cells and suggest that modulation of FRC VEGF expression may be a means to regulate lymph node vascularity and, potentially, immune function.     

Activation of alveolar macrophages after lower respiratory tract infection.

Alveolar macrophage function has been studied in relation to bacterial infection of the lower respiratory tract. First, LRT macrophages were examined after exposure of rabbits to Listeria monocytogenes aerosols. Macrophages obtained from the LRT of animals 10 to 48 days after infection were activated, as evidenced by greater adherence to culture dishes and increased ability to ingest and kill both the original infecting organism and unrelated organisms, when compared to normal alveolar macrophages. Next, the in vitro effects on normal alveolar macrophages of incubation supernatants of control and antigen-stimulated lymphocytes (LRT and lymph node) from animals infected with L. monocytogenes or Streptococcus pneumoniae were evaluated. As manifested by increased adherence and phagocytosis, and an enhanced nonspecific bactericidal activity, alveolar macrophages were activated by the antigen-stimulated supernatants. These stimulated lymphocyte supernatants contain lymphokines (MIF), but the exact nature of the alveolar macrophage activating factor(s) remains to be determined. These observations, together with recent evidence that alveolar macrophages respond to lymphokines (MIF), suggest that the effector mechanism for cell-mediated immunity in the LRT is intact.     

Stochastic regulation of cell migration from the efferent lymph to oxazolone-stimulated skin

The systemic immune response is a dynamic process involving the trafficking of lymphocytes from the Ag-stimulated lymph node to the peripheral tissue. Studies in sheep have demonstrated several phases of cell output in the efferent lymph after Ag stimulation. When skin contact sensitizers are used as Ag, the efferent lymph cell output peaks approximately 96 h after Ag stimulation and is temporally associated with the recruitment of cells into the skin. To investigate the relative contribution of this high-output phase of efferent lymphocytes to lymphocytic inflammation in the skin, we used a common contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) to stimulate the skin and draining prescapular lymph node of adult sheep. The efferent lymph ducts draining the Ag-stimulated and contralateral control lymph nodes were cannulated throughout the experimental period. The lymphocytes leaving the lymph nodes during the 72-h period before maximum infiltration were differentially labeled with fluorescent tracers, reinjected into the arterial circulation, and tracked to the site of Ag stimulation. Quantitative tissue cytometry of the skin at the conclusion of the injection period (96 h after Ag stimulation) demonstrated more migratory cells derived from the Ag-stimulated lymph node than the contralateral control (median 18.5 vs 15.5 per field; p < 0.05). However, when corrected for total cell output of the lymph node, the Ag-stimulated migratory cells were 3.8-fold more prevalent in the skin than the contralateral control cells. These results suggest that the in situ immune response generally mirrors the frequency of recruitable lymphocytes in the peripheral blood.     

Natural cytotoxicity of lymphocytes from lymph nodes draining breast carcinoma and its augmentation by interferon and OK432

Summary Lymphocytes isolated from axillary lymph nodes draining breast carcinoma were tested for natural killer (NK) activity against K562 in a 4-h ⁵¹Cr-release assay, and the in vitro effects of interferon (IFN) and OK432 (a streptococcal preparation) on their cytotoxicity were examined in comparison with NK activity of autologous peripheral blood lymphocytes (PBL). The levels of NK activity were lower in lymph node lymphocytes (LNL) than in PBL of the same patients. Significant levels of LNL-mediated lysis were recorded in 14 of 42 (33%) lymph node samples and in nine of 14 (64%) patients. Purification of large granular lymphocytes (LGL) from lymph node cells by discontinuous Percoll density gradient centrifugation resulted in an induction or enhancement of cytotoxic activity, with no reactivity in LGL-depleted, small T-lymphocyte populations. Positive reactions were observed with 10 of 13 (77%) LGL samples. The low reactivity of LNL was not attributable to coexistent suppressor cells for NK function, since lymph node cells failed to suppress NK activity of normal PBL. Partially purified human IFN and OK432 augmented NK activity of patients' PBL in approximately 70% and 90% of the cases, respectively, while LNL-mediated lysis was augmented in only 7% and 36% of the lymph node samples by IFN and OK432, respectively. These results indicate that K562-reactive NK cells and/or their precursors may frequently be present at subthreshold levels in the lymph nodes draining breast carcinoma, and that the augmentation of LNL-mediated cytotoxicity by OK432 might provide a local potentiation of natural immune function at the host-tumor interface rather than IFN.     

Pulmonary metastases in guinea pigs as a consequence of dermal implantation of line-10 tumor cells

Summary Metastases to the lungs of guinea pigs occurred at high frequency as a consequence of intradermal implantation of tumor cells derived from the syngeneic hepatocellular carcinoma line-10. Surgery had a major influence on the proportion of guinea pigs found to have pulmonary metastases at necropsy. Without surgery all guinea pigs died with extensive lymph node metastases; macroscopic pulmonary metastases were present in a minority of the animals. Animals treated by excision of dermal tumors survived longer than untreated animals, and macroscopic pulmonary metastases were present in the majority of the animals. Animals treated by excision of dermal tumor and regional lymph nodes were rendered tumor-free. The data suggest that lymph node metastases were the most likely source of the tumor cells that spread to the lungs in animals from whom the dermal tumor transplant had been removed.     

T-cell mediated suppression in the MRL mouse

MRL/lpr mice possess an autosomal recessive gene, lpr, which is associated with lymphoproliferation and acceleration of autoimmune disease. Lymphoproliferation has been ascribed to a single gene defect predominantly affecting the T-lymphocyte component of the immune system. MRL/++ mice do not possess the lpr autosomal recessive mutation and do not develop early lymphadenopathy. T-lymphocyte functional activity was studied in these mice using the polyclonal T-cell mitogens PHA and Con A. Our results indicated a significant suppression of the spleen and lymph node response of MRL/lpr mice to these polyclonal mitogens as compared to the MRL/++ response noted as early as 6 weeks of age. In addition, there was a progressive decline in the MRL/lpr spleen and lymph node cell mitogenic responses with increasing age. Spleen and lymph node cells from 20-week MRL/lpr mice were also relatively unresponsive in the mixed lymphocyte culture reaction as compared to cells from MRL/ ++ or BALB/c mice. The in vitro proliferative response of the MRL mice was further examined with respect to possible accessory cell modulation by both macrophages and T cells. It was found that in 20-week MRL/lpr lymph nodes a significant degree of suppression of lymphocyte proliferation could be mediated by the MRL/lpr T cell. Increased lymphocyte proliferation to a mitogenic signal could only be demonstrated in those MRL/lpr mice 3 weeks of age.     

Differentiation of CD8+ T cells from tumor-invaded and tumor-free lymph nodes of melanoma patients: role of common gamma-chain cytokines

Differentiation of CD8(+) T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy of antitumor response developing naturally or induced through immunotherapy. To address this issue, CD8(+) T lymphocytes from tumor-invaded (n = 142) and tumor-free (n = 42) lymph nodes removed from the same nodal basin of melanoma patients were analyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8(+) T cells from all tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainly by CCR7(+) CD45RA(+/-) cytotoxic factor(-) cells. The remaining three clusters contained only samples from tumor-invaded lymph nodes and showed a progressive shift of the CD8(+) T cell population toward CCR7(-) CD45RA(-/+) perforin(+) granzyme B(+) differentiation stages. Distinct CD8(+) T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, were identified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 days of CCR7(+) perforin(-) CD8(+) T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly in CCR7(+)CD45RA(-) cells, proliferation coupled to differentiation to the CCR7(-) perforin(+) stage and acquisition of melanoma Ag-specific effector functions. Taken together, these results indicate that CD8(+) T cells differentiated toward CCR7(-) cytotoxic factor(+) stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients and suggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8(+) T cells.     

Polyclonal activation of primed rat B cells

In recent years, murine and human virgin B lymphocytes have been used to examine the steps necessary for polyclonal activation. In these models mitogens are used in conjunction with lymphokines to determine which signals are responsible for regulating B-cell triggering, proliferation, and differentiation. While progress has been made in understanding these events as they occur in virgin B cells, very little evidence exists to suggest whether these models of activation also apply to the memory B-cell population. In this report we have described an antigen-specific, secondary in vitro immune response using cells isolated from lymph nodes draining the site of antigen injection. Unfractionated cells, B cells, and size-fractionated cells from dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH)-primed rats were challenged in vitro with DNP-KLH, lipopolysaccharide plus dextran sulfate (LPS/DxS), and T-cell factors. We have consistently found, under all these conditions, that antigen challenge of primed cells results in the production of DNP-specific IgG antibody while stimulation with LPS/DxS plus T-cell factors results only in the polyclonal activation of virgin B cells; no antigen-specific IgG secretion is seen. This suggests that acquisition of memory status is associated with a loss in responsiveness to LPS/DxS-induced differentiation.     

Limb lymph node response to bone fracture

In previous clinical studies, dilation of afferent lymphatics and enlargement of inguinal lymph nodes (LN) were observed in lymphoscintigrams from patients with persistent posttraumatic edema of lower extremities after fractures and trauma of soft tissues. In this study, changes in rat popliteal and iliac lymph nodes draining lymph from the site of tibial fracture and adjacent soft tissue injury were investigated. The observed parameters were lymph node weight, cell number, phenotype frequency, cell cytokine expression, and reactivity to mitogens. The key observations included: a) increase in the weight and total cell number of the lymph nodes; b) increased autotransformation rate and responsiveness of lymph node cells to mitogen; c) decreased frequency of ED1 macrophages and activated OX8 cytotoxic cells in flow cytometry analysis; d) high expression of OX6 class II-positive, OX7 (stem cells), OX62 (migrating dendritic cells), ED1 (macrophages), and OX12 (B cells) on immunohistochemical sections of LNs with some few HIS48 (granulocytes); e) high expression of NOS3 and TGF beta by lymph node lymphocytes and endothelial cells. In summary, local lymph nodes reacted to internal wounds, such as bone fracture and injury to adjacent tissues, through mobilization of cells from the blood circulation, along with activation of cellular subsets. The molecular mechanism that provides the signal for this reaction remains unknown. The absence of major changes in the frequency of lymph node cell subpopulations indicates that lymph nodes are constitutively prepared for influx of antigens from damaged tissues and react only with increase in cell number and cell activation. The nature of the reaction, including lack of immunization against autoantigens, remains unclear. Further elucidation will require studies on the mechanism of cross-tolerance to self-antigens during wound healing.     

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.