Role of macrophages as modulators but not as autonomous accessory cells in the proliferative response of immune T cells to soluble antigen

The role of murine macrophages (M phi) and that of splenic dendritic cells (DC) were investigated in the antigen-specific proliferative response of memory T cells of mice primed with key-hole limpet hemocyanin (KLH) 6 weeks or more before. Peritoneal M phi, whether expressing Ia antigens or not, did not function as autonomous accessory cells (A cells). A-cell activity of the spleen adherent cell population, which comprised M phi in the majority and DC in the minority, was abolished by eliminating DC with a DC-specific monoclonal antibody and complement, and regained by the addition of a small number of DC. Though M phi did not function as autonomous A cells, they augmented the proliferative response in the presence of a small number of DC. This occurred not only in the presence of free antigen, but also when DC and/or M phi were pulsed with antigen. A culture supernatant of M phi having interleukin-1 activity was effective in enhancing the proliferation of T cells which responded to antigen-pulsed DC. On the other hand, interleukin-2 did not replace DC even in the presence of antigen-pulsed Ia+ M phi. We also investigated recently primed T cells, but no evidence was obtained in favor of the competence of M phi as autonomous A cells.     

Enhancement of antigen-presenting function of dendritic cells with culture supernatants of mouse peritoneal macrophages stimulated with certain particulate substances

The production from murine resident peritoneal macrophages (M phi) of a soluble factor, which was capable of enhancing the antigen-presenting (AP) function of dendritic cells (DC), was examined. The supernatants of peritoneal M phi (M phi sup) were prepared by culturing peritoneal M phi with particles, i.e., zymosan A, latex, and sheep red blood cells (SRBC), or antigen-antibody (Ag-Ab) complexes such as keyhole limpet hemocyanin (KLH)-anti-KLH, ovalbumin (OVA)-anti-OVA, and SRBC-anti-SRBC complexes. When exposed to M phi sup during antigen pulsing DC induced a marked antigen-specific T cell proliferation, relative to DC treated with the supernatants from M phi cultured without stimuli (control sup). On the other hand, M phi sup-treated splenic M phi stimulated antigen-specific T cell activation to almost the same extent as did splenic M phi treated with control sup. These results indicated that peritoneal M phi elaborated a soluble factor which preferentially enhanced the AP capacity of DC when stimulated with particles or Ag-Ab complexes. Analytical gel filtration of M phi sup revealed that the factor had an apparent molecular weight of 27,000 daltons which was distinct from interleukin 1.     

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.     

Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma

By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.     

Role of macrophages as modulators but not as autonomous accessory cells in primary antibody response

The role of macrophages (M phi) in the in vitro primary antibody response of murine lymphocytes to sheep erythrocytes was investigated. Peritoneal M phi were activated to express Ia antigens either in vitro or in vivo. Nonactivated Ia- M phi were also examined. We observed that only Ia- M phi but also Ia+ M phi failed to trigger the antibody response, in contrast with splenic dendritic cells (DC) which served as potent and autonomous accessory cells, but that M phi modulated the level of response which was dependent primarily on the DC content of culture. The modulation appeared to incline to suppression rather than enhancement, when M phi were allowed to remain throughout the culture period for 4 days. A highly enhancing capacity of M phi, however, could be revealed by removing M phi 2 days after the initiation of culture, indicating that M phi exerted their suppressive effect more strongly in the late phase than in the early phase of in vitro antibody response. The modulatory activity seemed higher in Ia+ M phi than in Ia- M phi.     

Characterization of regulatory (interferon-alpha/beta) and accessory (LAF/IL 1) monokine activities from liver granuloma macrophages of Schistosoma mansoni-infected mice

Previously it was shown that macrophages (M phi) isolated from the vigorous (Vig) or modulated (Mod) liver granulomas (Gr) of Schistosoma mansoni-infected mice restored mitogen and parasite egg antigen-induced proliferative responses to accessory cell-depleted lymphocytes. Furthermore, supraoptimal concentrations of highly activated VigGrM phi suppressed lymphoproliferation to a greater extent than did the lesser activated ModGrM phi. In this study we investigated the role of soluble mediators in GrM phi accessory/regulatory activity. Indomethacin released VigGrM phi-mediated inhibition of mitogen but not antigen-induced lymphoproliferation. Extensively dialyzed serum-free GrM phi culture supernatant nonspecifically suppressed SEA- or KLH-induced blastogenesis. Culture supernatants also reduced vesicular stomatitis virus-induced plaque formation in supernatant-pretreated L-929 fibroblasts. The 20 to 45 Kd GrM phi-derived lymphoproliferation suppressive factor (SF) and the 20 to 50 Kd viral plaque-reducing factor (PRF) were stable at low pH, but became inactivated by heat and trypsin digestion. Although freshly isolated Vig or ModGrM phi contained preformed SF and PRF, in vitro production of the factors were depressed by protein synthesis inhibitors. Moreover, SF was active only when added to cultures before day 3 of the 6-day proliferation assay. Both SF and PRF were specifically retained on rabbit anti-murine IFN-alpha/beta immunoaffinity columns. Thus, the suppressive activity of Vig or ModGrM phi is in part mediated by a monokine that shares physical, biological, and antigenic characteristics with murine IFN-alpha/beta. In contrast to the suppression of antigen-driven proliferation, GrM phi culture supernatant costimulated PHA-induced mitogenesis. The 13 to 21 Kd GrM phi-derived lymphocyte-activating factor (LAF) was stable to heat, low pH, and trypsin digestion. Freshly isolated Vig or ModGrM phi contained preformed LAF, although its in vitro production was depressed by protein synthesis inhibitors. The physical and biological characteristics of GrM phi-derived LAF appear similar to IL 1. It is concluded that both Vig and ModGrM phi secrete regulatory/accessory monokines that may contribute to the initiation and maintenance of the focal inflammatory granulomatous response.     

Macrophage-derived soluble factors mediate suppression induced by 2,4-dinitrophenyl-conjugated mouse IgG in hybridoma cells

Our laboratory has established that 2,4-dinitrophenyl-conjugated mouse IgG (DNP-MGG) can specifically suppress proliferation, antibody synthesis, and secretion in vivo in two anti-DNP secreting cell lines: hybridoma 35-12 and myeloma MOPC-315. In the present study an in vitro system was used to further analyze the mechanism of suppression of hybridoma 35-12 cells (HC) by DNP-MGG. It was found that DNP-MGG-induced suppression of HC requires macrophages (M phi) and occurs only in eclipsed HC which are mainly small, nonsecreting cells. The M phi-mediated suppression is DNP specific, requires no M phi-HC cell contact, and does not involve killing of eclipsed HC. M phi culture supernatant alone cannot mediate suppression, but supernatants obtained by culturing M phi with either HC or supernatant from HC culture can mediate suppression of eclipsed HC in the presence of DNP-MGG. DNP-MGG is not required for the generation of effective M phi factors, but it is required for suppression of HC in the presence of M phi factors. Indomethacin cannot reverse M phi-mediated suppression, suggesting prostaglandins may not be the M phi factors. These data suggest that M phi-derived factors which are not prostaglandins in nature may play a role in B-cell regulation and in B-cell suppression induced by tolerogenic forms of antigen.     

Establishment of T-cell hybridoma constitutively producing macrophage-dependent T-cell replacing factor

A T-cell hybridoma was established by the fusion of concanavalin A-stimulated splenic T cells with BW 5147. The hybridoma cells secrete a factor constitutively to support antibody formation of spleen cells depleted of T cells against TNP-Ficoll but not against horse red blood cells. The activity was indicated not to be due to interleukin 2, B-cell growth factor I, B-cell growth factor II, or interferon. The factor-mediated antibody response to TNP-Ficoll required the presence of adherent cells. The adherent cell function could be replaced by the macrophage culture supernatant containing interleukin 1. B cells responding to TNP-Ficoll in the culture with hybridoma factor were indicated to be Lyb 5+ and to bear receptors for third component of complement.     

A significant role of the macrophage accumulation induced by MCF in the protection of mice against Listeria monocytogenes in vivo

Analysis was done on macrophage chemotactic factor (MCF) produced in the culture supernatant of spleen cells from mice immunized with Listeria monocytogenes. MCF was produced by Thy-1+, Lyt-1+ lymphocytes. MCF activity was resistant against pH 2 treatment and heating at 56 degrees C for 30 min, but was abolished by digestion with trypsin. G-100 gel filtration chromatography revealed that the approximate molecular weight of MCF was 15,000. MCF-rich fraction obtained by gel filtration chromatography showed neither MAF activity nor interferon activity. MCF activity in MCF-rich fraction was not affected by treatment with anti-rIFN-gamma antibody. An injection of MCF-rich fraction into the peritoneal cavity of mice induced a significant degree of accumulation of polymorphonuclear leukocytes (PMN) in a very short time after injection and macrophages thereafter. Resistance against listerial infection was augmented at the site where macrophage accumulation was provoked by the injection with MCF-rich fraction. It was shown that MCF plays an important role by itself in the protection against listerial infection by the accelerated accumulation of macrophages.     

The effect of bone marrow depletion on prostaglandin E-producing suppressor macrophages in mouse spleen

The i.p. injection of Corynebacterium parvum (CP) into CBA/J mice effected increases in macrophage colony-forming cells (M-CFC) when spleen cells were cultured with L cell culture filtrate as a source of colony-stimulating factor. Significant increases in phagocytic macrophages (M phi) with Fc receptors for IgG2a and IgG2b immune complexes were additionally noted among the spleen cells in these mice. These M phi effectively inhibited Con A-induced lymphocyte proliferation, probably reflecting a 10-fold increase above normal controls in prostaglandin E to 47 ng/3 X 10(6) spleen cells/ml. To determine whether the suppressor M phi are immediate derivatives of splenic M-CFC, we tried to induce suppressor M phi by the injection of CP into mice depleted of bone marrow M-CFC by the earlier administration of the bone-seeking isotope, 89Sr. This procedure reduced M-CFC in the bone marrow to less than 1% of normal for more than 30 days. Monocytes in the blood fell to 5% of normal by day 10 and were 30% on day 30. Levels of resident peritoneal M phi showed relatively little change in this period. By contrast, splenic M-CFC increased to 20-fold higher than the "cold" 88Sr controls. CP-induced suppressor M phi activity, however, was sharply reduced in 89Sr marrow-depleted mice on day 10, despite the striking increase in M-CFC. There was a threefold increase in the number of phagocytic M phi binding IgG2a immune complexes, with no significant increase in IgG2b binding M phi. The kinetics of recovery of suppressor M phi activity showed that on days 20, 30, and 50 after 89Sr injection the activities reached 20%, 30%, and 70% of the "cold" control, respectively, and correlated with the recovery of significant levels of M-CFC in the bone marrow. Taken together, these observations suggest that splenic M-CFC are not an immediate source of PGE-suppressor M phi in vivo. It appears more likely that the CP-inducible suppressor M phi, in particular, originate from radiosensitive bone marrow cells or require for differentiation a microenvironment provided by bone marrow cells. The data also suggest that the expression of the Fc gamma 2b receptor and of suppressor activity by CP-induced splenic M phi are related phenomena.     

Uptake of HIV and latex particles by fresh and cultured dendritic cells and monocytes

Blood dendritic cells (DC) efficiently carry HIV-1 and transmit infection to CD4+ T cells in the absence of productive infection of the APC. Fluorescent latex beads were used to define the endocytic pathways that may contribute to this non-infectious pathway of virus carriage. Beads between 14 nm and 2300 nm in diameter were taken up by uncultured blood DC, but uptake of beads larger than 280 nm was much reduced in the DC compared to monocytes. After culture, there was a reduction in bead carriage in DC compared to monocytes. In the DC, beads were found as small aggregates in class II containing compartments or as single beads just below the cell surface. Beads accumulated in monocytes as aggregates in class II negative compartments. Bead recycling occurred in DC, but not in the fresh or cultured monocytes. Electron microscopy of HIV-1-pulsed DC cultured with CD4+ T cells showed accumulation of apoptotic debris and virions within endosomes in the DC. The peripheral location and recycling of endocytosed material in DC provides a pathway for virion transfer from DC to T cells that does not occur in monocytes.     

T cell specificity in twice-irradiated F1----parent bone marrow chimeras: failure to detect a role for immigrant marrow-derived cells in imprinting intrathymic H-2 restriction

In an attempt to resolve the issue of whether H-2-restricted T cell specificity is controlled by thymic epithelial cells or by cells of the macrophage/dendritic cell (M phi/DC) lineages, long-term F1----parent chimeras were subjected to secondary irradiation and reconstitution with F1 marrow cells. The rationale was that if F1 M phi/DC enter the thymus only quite slowly after irradiation, as claimed by other investigators, leaving F1----parent chimeras for a period of several months before re-irradiation would ensure that the new wave of T cells generated in the thymus of the chimeras would have no difficulty in making contact with donor-derived F1 M phi/DC. According to the view that M phi/DC rather than epithelial cells control H-2 restriction, the T cells differentiating in these chimeras would be expected to show H-2 restriction to both parental strains. In practice, T cells from twice-irradiated (1000 + 800 rad) chimeras showed strong restriction to host (thymic) H-2 determinants, the degree of restriction to host determinants being as marked as with T cells from once-irradiated chimeras. This finding applied both to T proliferative responses to KLH assayed in vitro and to T helper function for sheep erythrocytes measured in vivo. Preliminary experiments established that the initial dose of irradiation used for preparing the chimeras (1000 rad) resulted in almost total replacement of intrathymic M phi/DC by donor-derived cells within 4 wk of irradiation; M phi/DC were typed by determining their capacity to stimulate mixed-lymphocyte reactions. Collectively, the data imply that, at least under the conditions used, H-2-restricted T cell specificity is controlled by epithelial cells rather than by M phi/DC.     

Role of macrophages as modulators but not as stimulators in primary mixed leukocyte reaction

The role of macrophages (M phi) and that of splenic dendritic cells (DC) in the allogeneic mixed leukocyte reaction (MLR) in the mouse have been investigated. In contrast with the high stimulatory capacity of DC, we obtained no evidence in favor of the competence of M phi, whether Ia + or Ia-, as an autonomous stimulator of MLR. However, M phi were found to modulate the level of MLR. Thus, M phi amplified the low level MLR to low dose DC and apparently suppressed the high level MLR to high dose DC. Ia + M phi seemed superior to Ia- M phi in the MLR-enhancing effect. M phi syngeneic to the responder and those to the stimulator suggest that M phi are modulators of immune responses triggered through the mediation of DC.     

Prostaglandin-mediated suppression of macrophage phagocytosis of Listeria monocytogenes

Suppression of macrophage phagocytosis of Listeria monocytogenes has been shown to be due to a low-molecular-weight component of spleen cell culture supernatant. The possibility that the factor could be a prostaglandin was investigated. When murine peritoneal macrophages were treated with prostaglandin E2 (PGE2), L. monocytogenes was phagocytized at a rate comparable to that phagocytized when treated with a low-molecular-weight fraction of concanavalin A-generated spleen cell culture supernatant. Suppressive activity of the spleen cell culture supernatant was abrogated when supernatant was prepared in the presence of indomethacin, a prostaglandin synthetase inhibitor. Prostaglandins were identified in supernatants with thin-layer and high-pressure liquid chromatography. These results suggest a role for prostaglandins, particularly PGE2, as a modulator of macrophage phagocytosis of L. monocytogenes.     

T cell hybridomas coexpressing Fc receptors (FcR) for different isotypes. II. IgA-induced formation of suppressive IgA binding factor(s) by a murine T hybridoma bearing Fc gamma R and Fc alpha R

T2D4 murine T hybridoma cells have previously been shown to express Fc receptors (FcR) for IgG (Fc gamma R) and for IgA (Fc alpha R) and to produce an IgG binding factor (IgGBF) that suppresses IgG and IgM responses. In the present work we report on the behavior of IgA bound to T2D4 cells and on the production of IgA binding factor (IgABF) and its ability to suppress IgA antibody production. A dose-dependent binding of MOPC315 IgA with anti-TNP activity by T2D4 cells was demonstrated by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) and fixation of iodinated DNP-BSA. IgA bound to the cells disappeared after a short-term culture of 3 hr at 37 degrees C, but not at 4 degrees C. Because this phenomenon was inhibited by 0.1% sodium azide and 100 microM dansylcadaverine, a transglutaminase inhibitor, Fc alpha R-IgA complexes seemed to be released by an active process involving receptor movement. In the culture supernatant of IgA-treated T2D4 cells, we detected a factor(s) that binds to IgA-Sepharose and competitively inhibits the binding of IgA to T2D4 cells. The factor (IgABF) failed to inhibit the rosette formation of Fc gamma R(+) cells with IgG-sensitized ORBC (EAox gamma), indicating that it binds specifically to IgA. IgABF was undetectable in the culture supernatants of untreated T2D4 cells of Fc alpha R(-) BW5147 T lymphoma cells used as parent cells for the establishment of the hybridoma. To study the effect of IgABF on antibody formation, culture filtrates of IgA-treated or untreated T2D4 cells were fractionated on IgA-Sepharose beads and were added to BALB/c spleen cells cultured with pokeweed mitogen. By use of a reverse plaque assay, it was shown that the IgA plaque-forming cell (PFC) response was suppressed by the acid eluate but not by the effluent of IgA-Sepharose beads incubated with the filtrates of IgA-treated T2D4 cell cultures. The suppression was IgA specific, because neither IgG nor IgM responses were suppressed by the eluate. As expected, there was no significant IgA suppressive activity in the acid eluates of the beads incubated with the culture filtrate of untreated T2D4 cells or IgA-treated BW5147 cells. IgA-specific suppressive activity proved to be due to IgA binding factor(s), because suppressive activity in the eluate was completely adsorbed by IgA-Sepharose but not by IgG- nor BSA-Sepharose.     

TGFbeta is responsible for skin tumour infiltration by macrophages enabling the tumours to escape immune destruction

Infiltration of skin tumours by macrophages is an important step in tumour progression, although the mechanisms of macrophage recruitment to the tumour mass and the subsequent effects on tumour growth are poorly understood. Transfecting a murine regressing skin tumour with the gene for transforming growth factor (TGF)beta enabled the tumours to grow progressively in vivo thus allowing us to study the role of this cytokine in tumour growth. Flow cytometry was used to show that TGFbeta-mediated tumour progression was accompanied by an increase in tumour-associated macrophages (TAM) and a decrease in tumour-infiltrating dendritic cells (DCs). TAM in TGFbeta-secreting tumours expressed lower levels of major histocompatibility complex II and CD86 compared to DC in control tumours and had a high phagocytic capacity as measured by uptake of latex beads in vivo. Indeed, TGFbeta was directly responsible not only for the enhanced macrophage phagocytosis but also altering the ratio of antigen-presenting cells to favour macrophages over DC. Our results demonstrate that TGFbeta recruitment and retention of macrophages at the tumour site enable effective tumour evasion of the host immune system and reinforces the need to target TGFbeta in human cancer immunotherapy trials.     

Dendritic cells but not macrophages are targets for immune regulation by natural killer cells

Both dendritic cells (DC) and macrophages (M phi) stimulate lymphocyte proliferation in secondary mixed-lymphocyte (ML) reactions, though DC are approximately fourfold more effective. Natural killer (NK) cells suppress secondary ML reactions when DC are used, but NK cells do not suppress when M phi are used in these reactions. The findings are consistent with the idea that DC, but not M phi, are potential targets in immune regulation mediated by NK cells.     

Antigen-specific augmentation of murine immediate hypersensitivity-like footpad reaction by a T-cell factor in the culture supernatant of immune spleen cells

An antigen-specific factor capable of augmenting delayed-type hypersensitivity (DH) in the culture supernatants from immune spleen cells and erythrocyte antigen has been found. These culture supernatants also augmented an immediate hypersensitivity-like reaction which appeared in advance of the classical DH reaction. In this paper, the basic characteristics and cells producing the augmentation factor (IAF) involved in immediate hypersensitivity-like reaction were investigated. Maximum activity of IAF was detected in a supernatant from 24-hr culture of immune spleen cells and antigen. In vitro antigen stimulation was essentially required for the production or release of IAF. IAF showed antigen-specificity. IAF was produced or released by T cells. In addition to these facts, the DH-augmentation factor proved to be a T-cell product.     

Cytostatic peptide isolation from culture media of mouse peritoneal exudate cells

It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of [3H]TdR into DNA of mouse spleen cells. The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time. The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells. The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min. Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction. This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP). The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory. It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0. The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents. Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.     

Macrophage factors which enhance the mixed leukocyte reaction initiated by dendritic cells

Stimulator cells for the allogeneic MLR are dendritic cells but not macrophages (M phi). M phi, however, enhance the MLR initiated by relatively low doses of dendritic cells. The present report demonstrates that the enhancement of the MLR is mediated by two factors produced by M phi. One is IL-1, inasmuch as it has a Mr approximately 15 kDa, and both partially purified IL-1 and rIL-1 also enhance the MLR. The other has been identified as granulocyte-macrophage (GM)-CSF. It had a Mr approximately 25 kDa, and is reproduced by rGM-CSF. Moreover, the MLR-enhancing activity of both the 25-kDa molecule and rGM-CSF have been neutralized by anti-GM-CSF antiserum. Both IL-1 and GM-CSF have autonomous enhancing activity, but they collaborate with each other in enhancing the MLR. Both factors act on DC to augment their stimulatory activity for allogeneic T lymphocytes.