Functional activation of immune lymphocytes by antigenic stimulation in cell mediated immunity: I. Requirement for macrophages in antigen-induced MIF production by guinea pig immune lymphocytes in vitro

The role of macrophages in the process of antigen-induced production of mediators in cellular immune response was studied, using the antigen-induced production of migration inhibitory factor (MIF) as a measure of the activation of immune lymphocytes.The production of MIF by guinea pig immune lymph node cells in response to the stimulation with PPD was abolished when the lymph node cells were depleted of adherent cell population by passing the cells through a Tetron fiber column and incubating the effluent cells in plastic dishes. These purified immune lymphocytes did not respond to particle-bound PPD, either. However, the response was obviously restored by the addition of a small number of the purified peritoneal adherent cells (macrophages) which had been pulse-treated with PPD. The PPD-pulsed macrophages produced no MIF by themselves. Thus, the results clearly indicated the requirement for macrophages in the process of antigen-induced MIF production by immune lymphocytes. Destruction of PPD-pulsed macrophages by freezing and thawing or by homogenization abrogated their ability to stimulate immune lymphocytes. Attempts to restore the response of the purified immune lymphocytes to PPD by adding 2-mercaptoethanol or the culture supernatant of macrophages to the medium have so far been unsuccessful.     

Staphylococcal enterotoxin B induced release of macrophage migration inhibition factor from normal lymphocytes

Staphylococcal enterotoxin B (SEB), a potent lymphocyte mitogen, inhibits migration of peritoneal exudate cells from most guinea pigs but does not inhibit migration of purified macrophages. Experiments were designed to test the ability of highly purified SEB to induce normal lymphocytes to release migration inhibition factor (MIF). Supernatants of lymph node lymphocytes cultured with SEB inhibited the migration of purified macrophages, indicating the release of a migration inhibition factor. Mitomycin-C blocked the SEB-induced release of MIF. SEB-induced MIF localized in the albumin fraction on Sephadex G-200 chromatography. Antibody to SEB specifically blocked the inhibitory effect of SEB on migration of normal guinea pig peritoneal exudate cells.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

Identification of the principal cell type yielding immune RNA capable of transferring tumor-specific cellular cytotoxicity

A search was made for the lymphoid cell type(s) which are the source of immune RNA (I-RNA) capable of transferring tumor-specific cell-mediated cytotoxicity (CMC). Hartley guinea pigs were immunized with syngeneic murine fibrosarcomas (BP-10 or BP-11) induced by 3,4-benzo(a)pyrene in C3H/HeJ mice, and the I-RNA was extracted individually from their spleens, lymph nodes, and peritoneal exudate (PE) cells. All three I-RNA preparations were able to convert normal C3H/HeJ mouse lymphocytes to effector cells significantly cytolytic to the specific syngeneic mouse tumor in vitro. Furthermore, lymphocytes and macrophages were purified from the spleens, lymph nodes, and PE cells of tumor-immunized guinea pigs. I-RNA was extracted from these purified cell populations and also from the pooled guinea pig lymphoid tissues. Normal C3H/HeJ lymphocytes were incubated with each type of I-RNA and tested in vitro for CMC against the specific tumor cells. Significant CMC against BP-10 targets was observed with mouse lymphocytes incubated with I-RNA extracted from pooled lymphoid tissues of BP-10 tumor-immunized guinea pigs. There was a reduced but still significant CMC when mouse lymphocytes were incubated with I-RNA extracted from purified guinea pig lymphocytes, whereas there was a markedly increased CMC when the I-RNA was extracted from purified guinea pig macrophages. As indicated by sucrose density gradient analysis, the lesser effectiveness of lymphocyte I-RNA was not due to RNA degradation resulting from lymphocyte purification or I-RNA extraction. Treatment of all types of I-RNA with RNase abrogated the transfer of CMC, whereas treatment of I-RNA with DNase or pronase did not. RNA extracted from the lymphoid tissues of guinea pigs immunized with complete Freund's adjuvant without tumor was ineffective. Mouse lymphocytes incubated with BP-10 macrophage I-RNA destroyed BP-10 but not BP-11 tumor cells, whereas lymphocytes incubated with BP-11 macrophage I-RNA killed BP-11 but not BP-10 tumor cells, thus indicating tumor specificity of the immunity transferred by macrophage I-RNA. Our results suggest that macrophages are the principal source of I-RNA capable of transferring tumor-specific CMC.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. IV. Synergy of lymphocytes in the formation of clusters.

T-lymphocyte-enriched lymph node lymphocytes from guinea pigs immunized with Mycobacterium tuberculosis produce clusters with macrophages when cultivated on monolayers of syngeneic purified protein derivative of tuberculin (PPD)-pulsed peritoneal macrophages. The clusters consist of a macrophage with a central lymphocyte attached to it, and several peripheral lymphocytes attached to the central one. By mechanical manipulation immune lymphocytes incubated on monolayers of PPD-pulsed macrophages were separated into those which adhered firmly to the macrophages after 4 hr of culture and those which did not adhere. While neither of the two populations was able to produce significant numbers of clusters alone, they did so in combination. The number of macrophage-lymphocyte clusters which are produced in a culture depends not only on the absolute number of immune lymphocytes in the culture, but also on the concentration of lymphocytes per area of the macrophage monolayer, with high concentrations resulting in high numbers of clusters. Autoradiographic studies showed that the DNA-synthesizing lymphocytes physically associated with macrophages were located mainly inside the clusters in cultures with high concentrations of lymphocytes but mainly outside the clusters in cultures with low concentrations of lymphocytes.     

Guinea pig T-cell in vitro proliferative responses to tyrosine-azobenzenearsonate-pulsed and azobenzenearsonate-coupled macrophages have different specificities

The cell interactions involved in azobenzenearsonate-N-acetyl-tyrosine (ABA-tyr)-induced delayed hypersensitivity in the guinea pig were studied by in vitro blastogenesis. The ABA-sensitive lymphocyte was demonstrated to be a T lymphocyte and its presence in peritoneal exudate cells was shown to be much higher than spleen or lymph node populations. The secondary response of ABA-sensitized lymphocytes to ABA-tyr in culture is dependent on the presence of an accessory cell, with both splenic and peritoneal macrophages being equally effective. ABA coupled directly to macrophages as an immunogen induced strong responses to itself and not to ABA-tyr-pulsed macrophages or ABA-tyr in solution. The reverse was true in animals, immunized with ABA-tyr. ABA conjugated to thymocytes, L₂C leukemia cells, and guinea pig erythrocytes however, did not elicit significant responses. The results obtained in animals immunized with ABA- or ABA-tyr-modified cells was similar whether or not CFA was used. The difference in specificity shown between ABA-coupled and ABA-tyr-pulsed macrophages favors a single receptor hypothesis for T-cell recognition.     

The alveolar macrophage: Its capacity to act as an accessory cell in mitogen-stimulated proliferation of guinea pig lymphocytes

The capacity of the alveolar macrophage to act as an accessory cell in PHA-induced lymphocyte proliferation was investigated and compared with that of the peritoneal and peritoneal exudate macrophages in guinea pigs. When lymph node cells were co-cultured with autologous lung cells recovered by airway lavage, the proliferative response to PHA was greatly enhanced over that of lymph node cells alone. In the presence of peritoneal cells or peritoneal exudate (glycogen-induced) cells, the PHA response was intermediate between that of lymph node cells alone and lymph node cells cultured with lung cells. Experiments using purified macrophages (≥98%) as accessory cells demonstrated that the difference observed between lung and peritoneal accessory cells was due to differences in macrophage function. Furthermore, when lymph node cells were cultured in the upper chamber of a double-chambered Marbrook apparatus, PHA-induced proliferation was enhanced only when lung and not peritoneal macrophages were present in the lower chamber. Additional experiments showed that this difference (1) was not an artifact of the thymidine incorporation assay to measure proliferation; (2) was not affected by changing the macrophage-lymphocyte ratio; and (3) was not simply a trephocytic or growth promoting effect of macrophages which could be replaced by 2-mercaptoethanol.These findings show that macrophages from different sources differ in their abilities to act as accessory cells in PHA-induced lymphocyte proliferation. Alveolar macrophages appear to have an enhanced capacity compared to unstimulated and stimulated peritoneal macrophages in this function. At least part of this difference may be due to a difference in the elaboration of soluble factor (s) by macrophages.     

Macrophage-lymphocyte interaction: antigen-independent binding of guinea pig lymph node lymphocytes by macrophages.

The antigen independent binding of guinea pig lymph node lymphocytes by glass-adherent macrophages was investigated. Binding was found to be mediated by a trypsin digestible, divalent cation-dependent, temperature-sensitive macrophage receptor mechanism that was not competitively inhibited by excess immunoglobulin. Data are presented to indicate that in the absence of antigen, macrophages were capable of binding both thymus-derived and bone marrow-derived lymphocytes without apparent selectivity, and further, that the binding of neither cell was mediated by surface membrane-associated immunoglobulin.     

Biological Activities of Guinea Pig Mitogenic Factor from Immune Lymphocytes Stimulated with Antigen

Mitogenic factor was produced by sensitized guinea pig lymph node cells stimulated with a specific antigen. Both T lymphocytes and macrophages were required for the production of this factor. The culture supernatant of lymphocytes containing the mitogenic factor exhibited a strong helping effect on the proliferative response of T lymphocytes to phytohemagglutinin (PHA). Mitogenic factor and the factor with the helping activity coeluted in the molecular weight range of 25,000-35,000 daltons in gel filtration. Furthermore the fraction containing mitogenic factor was found to support the proliferation of lymphoblasts induced by PHA or antigen, suggesting that the mitogenic factor may be the guinea pig equivalent of T cell growth factor (TCGF) reported in the mouse, rat, and human. On the other hand, the T cell-activating monokine of the guinea pig, possessing the helping activity for the proliferative response of T lymphocytes to PHA, never exhibited TCGF-like activity.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

The expression of Ia antigens on immunocompetent cells in the guinea pig. III. Functional selection of Ia-negative T cells by in vitro culture.

In the present study we examined the expression of I-region-associated (Ia) antigens by guinea pig T lymphocytes stimulated in vitro with antigen-pulsed macrophages. Treatment of lymph node (LNL) or peritoneal exudate (PEL) T cells taken directly from immune animals with anti-Ia serum and complement (C) dramatically reduced their proliferative response to antigen-pulsed macrophages when determined on the 4th day of culture. In contrast, the response of immune T cells that had been selected by culture for a week with antigen-pulsed macrophages and restimulated in a second culture was not affected by anti-Ia and C treatment. This same result occurred with selected LNL or PEL that were initially treated before the selection culture with either normal serum or anti-Ia serum and C. LNL became resistant to anti-Ia serum and C treatment by 3 days of culture whereas antigen-specific PEL were still sensitive at that time. These results indicate that in an immune animal two antigen-specific T cell subpopulations are generated based on their sensitivity to anti-Ia serum and C treatment, but that only the resistant population is selected by in vitro culture. In addition, we demonstrated that the Ig-negative T cell population can only be activated by histocompatible antigen-pulsed macrophages.     

DFP-sensitive polypeptides of the guinea pig peritoneal macrophage

Guinea pig peritoneal exudate macrophages were reacted with [³H] diisopropyl fluorophosphate (10^?7 to 10^?4 M) and subjected to sodium dodecyl sulfate-polyacrylamide disc electrophoresis and fluorography. Macrophages reacted with 10^?5 M [³H]diisopropyl fluorophosphate contain eight major ³H-labelled polypeptides which have apparent molecular weights of 83,000, 75,000, 63,000, 48,000, 41,000, 30,000, 26,000, and 25,000. The sensitive polypeptides were not seen when guinea pig polymorphonuclear leukocytes, lymph node lymphocytes, erythrocytes, serum or plasma were reacted with [³H]diisopropyl fluorophosphate, demonstrating that these components are particular to the macrophage. The finding of a large number of diisopropyl fluorophosphate-sensitive proteins associated exclusively with the macrophage supports the concept that serine esterases play a unique role in macrophage physiology.     

Possible role of macrophage glycolipids as receptors for migration inhibitory factor (MIF).

Guinea pig peritoneal exudate cells incubated with water soluble glycolipids obtained from macrophages show an enhanced response to migration inhibitory factor. Incorporation of these glycolipids into liposomes greatly facilitates their interaction with indicator cells. Enhancement of peritoneal exudate cell responsiveness to migration inhibitory factor was specific for glycolipids from guinea pig macrophages. Glycolipids extracted from guinea pig brain and polymorphonuclear leukocytes as well as several bovine and porcine glycolipids had no effect. Specificity of enhancement was not due merely to a preferential association of macrophage glycolipids with indicator cells. The possible role of macrophage glycolipids as receptors for MIF is discussed.     

A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

Phytohemagglutinin-induced proliferation of guinea pig thymus-derived lymphocytes. I. Accessory cell dependence.

The accessory cell requirement for mitogen-induced T lymphocyte proliferation has been investigated by using a population of guinea pig lymph node lymphocytes enriched in T cells and markedly depleted of macrophages and B lymphocytes. We have found that effective phytohemagglutinin-induced proliferation of T cells is dependent on the participation of accessory cells. Augmentation of PHA responsiveness was noted when cultural conditions were manipulated to increase cell density, suggesting that physical proximity between T cell and accessory cell is required for efficient triggering. Both syngeneic and allogeneic macrophages, as well as syngeneic fibroblasts, serve as accessory cells in this response whereas polymorphonuclear leukocytes or thymocytes do not. Thus, although PHA-induced T lymphocyte proliferation requires accessory cells, the specificity of these cells is strikingly less stringent than for antigen-mediated triggering of immune guinea pig T cells, a response which is dependent upon participation of syngeneic macrophages.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

The expression of Ia antigens on immunocompetent cells in the guinea pig. II. Ia antigens on macrophages.

Only 15 to 25% of purified oil-induced guinea pig macrophages could be lysed by treatment with anti-Ia serum and C. Those cells remaining alive after treatment were not damaged and were metabolically active since they readily phagocytized latex beads. However, the "Ia-negative" macrophages were markedly deficient in their ability to present protein antigens to immune T lymphocytes and to function as stimulator cells when mixed with allogeneic T cells in the mixed leukocyte reaction. It thus appears that Ia antigens are expressed on a subpopulation of macrophages and that this subpopulation plays a critical role in the activation of T cell proliferation to soluble protein antigens and to alloantigens.     

CRRP: a guinea pig protein, identified by sequence homology to human CR1, which contains two short consensus repeat motifs and appears not to be transmembrane or secreted.

cDNA from the C4b-binding site of the human C3b/C4b receptor (CR1) was used to find homologous sequences in the guinea pig. This cDNA identified an 18S mRNA species in guinea pig spleen, but not liver. Probing of a guinea pig spleen cDNA library identified clones with identical 1.5-kb inserts, which also hybridized to mRNA in spleen, but not liver. Sequence analysis of the insert revealed a single long open-reading frame coding for a 20,000 Mr protein consisting of two short consensus repeat motifs homologous to human CR1, and unique sequence at the amino- and carboxy-terminals of the short consensus repeats. This sequence did not encode peptides with features of transmembrane domains or signal peptides. Antibody to this complement receptor-related protein-beta galactosidase fusion protein recognized a 20,000 Mr protein in SDS lysates of guinea pig spleen, lymph node, lymphocytes, neutrophils, and peritoneal macrophages. Immunoprecipitation of human serum by this antibody revealed an 180,000 Mr protein reacting both with the anti-guinea pig protein antibody and with anti-human CR1 antibody. Immunoprecipitation of guinea pig serum revealed no protein reacting with the anti-guinea pig protein antibody. Tissue staining of cultured peritoneal macrophages with this antibody showed intracellular staining, as opposed to membrane staining obtained with anti-guinea pig Ig antibody. The lack of membrane expression was confirmed by surface protein radiolabeling experiments and by fluorescent staining of surface proteins. Thus, we have identified a guinea pig protein with homology to human CR1, which may have an unusual property for this class of proteins in that it appears to be intracellular.     

Cell surface receptors for lymphokines. I. The possible role of glycolipids as receptors for macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF).

Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM₁, GM₂, and GM₃), di- (GD_1a), and trisialogangliosides (GT₁) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.