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.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

A comparison of the stimulatory activities of lymphoid dendritic cells and macrophages in T proliferative responses to various antigens

The identities of murine accessory cells and the mechanism by which they process antigen and stimulate T cell proliferation have been examined with cell separation techniques and specific agents to block antigen catabolism. Using preparations of splenic dendritic cells (DC) and macrophages (M phi) with minimal cross-contamination, we found that only DC could induce syngeneic mixed leukocyte reaction (MLR), whereas both DC and M phi could initiate allogeneic MLR. This observation may have significant implications for syngeneic MLR as a manifestation of self Ia recognition, and for the cell type that defines self Ia during ontogeny. DC and M phi could present soluble antigens such as purified protein derivative of tuberculin (PPD) and Salmonella flagellin about equally well to antigen-specific T cell lines. M phi, however, were much more effective than the non-phagocytic DC at inducing T cell proliferation to whole Corynebacterium parvum organisms. These differences could not be attributed to differences in antigen uptake. The results suggest that the bacteria must be ingested and processed by phagocytes before T cell activation. Using the lysosomotropic agent chloroquine to inhibit antigen catabolism in accessory cells, we found that the presentation of large antigens by M phi and DC was abolished by chloroquine treatment, whereas T cell activation by antigens (such as PPD or integral membrane Ia for MLR) that apparently required no processing was relatively insensitive to chloroquine. Thus, in addition to differences between cells, discrete functions within each cell type can also be distinguished.     

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.     

Mouse T lymphocytes proliferative responses specific for human MHC products in mouse anti-human xenogeneic MLR

It has been reported that human T cells recognize the polymorphism of murine Ia antigens in the human anti-mouse xenogeneic mixed lymphocyte reactions (MLR). In this study, murine T cell recognition of human Class II antigens of the major histocompatibility complex (MHC) was analyzed in mouse anti-human xenogeneic MLR responses. The xenoreactive murine T cell proliferative response was blocked by adding anti-HLA-DR monoclonal antibody to the xenogeneic MLR culture. The specificity of xenoreactive murine T cells was examined with regard to the secondary and tertiary xenogeneic MLR system. The xenoreactive murine T cells were restimulated by distinct human stimulator cells that had no shared HLA antigens with the stimulator used in the primary MLR. The data presented here show that the murine xenoreactive T cells recognize the shared determinant(s) of HLA-DR antigen on non-T, non-B stimulator cells. The xenoreactive murine T cell proliferative responses were mediated by Thy-1+, Lyt-1+, and Lyt-2- cells. Furthermore, the xenoreactive T cell responses required Ia+ cells, and Ia antigen on accessory cells plays a crucial role in eliciting the xenoreactive responses against human stimulator cells, while Ia+ accessory cells in the responding cell population are not essential for the elicitation of allogeneic MLR responses, as reported previously.     

Antigen-presenting cells for Lyt-2+ cells. I. Stimulation of unprimed Lyt-2+ cells by H-2 different Thy-1-Ia- cells prepared from spleen and bone marrow

In agreement with previous studies on Ia- tumor cells, evidence is presented that primary MLR of purified Lyt-2+ T cells to class I alloantigens can be elicited by a minor population of Thy 1- Ia- cells present in normal spleen, bone marrow, and day-13 fetal liver; these cells are non-stimulatory for L3T4+ T cells. The data strengthen the view that primary responses of Lyt-2+ cells do not require the presence of Ia+ cells.     

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.     

Immunologic effects of whole-body ultraviolet (uv) irradiation. III Defective splenic adherent cell function in alloantigen-stimulated T-cell proliferation

The daily exposure of a mouse to ultraviolet (uv) radiation causes a selective depletion of Ia-bearing adherent cells in that animal's spleen. This depletion manifests itself in functional deficiencies in the presentation of protein antigens and haptens to T cells. The present studies demonstrate a defect in splenic adherent cells (SAC) from uv-irradiated mice resulting in defective alloantigen presentation. We show that unfractionated splenocytes and SAC from uv-irradiated mice show decreased stimulatory activity in allogeneic MLR. We then utilize this phenomenon induced by uv radiation to characterize the stimulator cell in the M locus (Mls) determinant-driven MLR. We show that the stimulator cell in Mls determinant-driven MLR is an adherent cell and demonstrate that this stimulator cell bears Ia determinants by showing that whole spleen cells and SAC from mice treated with uv radiation are inefficient stimulators of the Mls determinant-driven MLR. The importance of the Ia determinant on the stimulator cells in Mls determinant-driven MLR is corroborated by the demonstration that a monoclonal antibody directed at this determinant fully blocks the Mls determinant-driven MLR. The significance of these studies to the problem of alloreactions in vivo is discussed.     

Two-color flow cytometric analysis of the expression of MAC and MHC class II antigens on macrophages during tumor growth

Tumor-bearing host (TBH) macrophages (M phi) exhibit immune dysfunction that is concomitant with phenotypic changes. We examined M phi subpopulations by changes in the expression of surface antigens Mac-1, -2, -3, and Ia on normal and TBH peritoneal and splenic M phi. M phi were double-labeled and analyzed by flow cytometry to observe multiple expression of surface antigens. Tumor growth alters the multiple expression of these M phi markers. Peritoneal and splenic M phi had different Mac+ and Mac+Ia+ population percentages. In TBH, peritoneal M phi had decreased percentages of Mac-1+2+, Mac-1+3+, Mac-2+3+, and Mac+Ia+ M phi. This decrease correlated with functional changes in TBH M phi. In contrast, there was an increase in Mac-2-Ia- TBH peritoneal M phi. Previously undiscovered Mac-1+2-3- and Mac-1-2-3+ populations were found. In contrast to peritoneal M phi, there was an increase in the percentage of Mac-1+2+, Mac-1+3+, and Mac-2+3+ splenic TBH M phi but, like peritoneal M phi, there was a decrease in the percentage of Mac+Ia+ M phi. Also, TBH splenic M phi showed a smaller but more uniform antigen density than normal host splenic M phi. Tumor growth modulated phenotypic alterations in peritoneal and splenic M phi subpopulations. Combined with earlier functional studies of M phi subpopulations, these data suggested a relationship between changes in M phi phenotype and tumor-induced dysfunction of M phi-modulated immune activity.     

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

Dendritic cell activating factor produced by mouse macrophage hybridoma

A mouse macrophage (M phi) hybridoma which produces a soluble factor responsible for the cooperation between M phi and spleen dendritic cells (DC) was constructed. The antigen-presenting activity and the stimulator cell activity in the allogeneic or syngeneic mixed leukocyte reaction of DC were significantly augmented when DC were incubated with the culture supernatant of the hybridoma treated with various stimulants including latex beads. The monokine present in the culture supernatant of the hybridoma, called dendritic cell-activating factor (DCAF), augmented the production of lymphocyte-activating factor by DC while Ia expression of DCAF-treated DC was not altered. DCAF had no effect on the antigen-presenting activity of peritoneal resident M phi or B cell blasts while the antigen-presenting activity of spleen M phi was enhanced, but the degree of the enhancement was much less than that of spleen DC. DCAF was found to have the following properties: its pI value is between pH 4 and 5; it is stable at pH 2 to 10; and it loses its activity on incubation at 75 C for 30 min. When the culture supernatant of the hybridoma stimulated with latex beads was subjected to gel filtration, the DCAF activity was detected in the 20 Kd to 25 Kd, 30 Kd to 40 Kd, and 50 Kd to 60 Kd molecular weight regions. The 30 Kd to 40 Kd fraction, which is the major peak fraction, was further purified by ion-exchange chromatography followed by gel-filtration chromatography. When each fraction was subjected to SDS-PAGE, a 30 Kd band corresponding to the DCAF activity was observed and DCAF was purified to about 90% purity.     

Cellular requirements for induction of human primary proliferative responses to trinitrophenyl-modified cells

Cellular requirements for induction of primary proliferative responses by human T cells to trinitrophenylated autologous stimulators have been characterized. Substantial proliferative responses were observed with each of the Ia+ stimulator populations tested. Nevertheless, major differences in the hapten specificity of such responses were observed. Thus purified macrophages/monocytes (M phi) when TNP-modified induced responses that were relatively modest in absolute magnitude, but were highly hapten specific. This reflected the very limited capacity of purified M phi to induce proliferation when unmodified, i.e., an autologous mixed leukocyte response (AMLR). In contrast, unmodified M phi-depleted B plus null cells were potent stimulators of AMLR, but hapten modification did not significantly enhance the responses induced by these cells. Moreover, when M phi were added to B plus null cell stimulators AMLR responses were reduced and, with TNP-modified stimulators, hapten-specific responses were restored. The data thus suggest that M phi may have important roles in induction of primary T cell responses to conventional antigens but function largely as regulators rather than stimulators of AMLR. Finally, we have introduced a novel antigen-presenting cell population, the irradiated Ia+ TNP-specific cloned T cell. The possibility that such cells may utilize autostimulatory positive feedback circuits for activation of naive T cells and in interactions between subpopulations of hapten-reactive T cells is discussed.     

The effects of phorbol ester on alloantigen presentation

B cells and Ia+ thyroid cells fail to stimulate alloreactive T cells in a primary mixed leukocyte reaction (MLR) and fail to activate some allo-class II (I-A) reactive T cell hybridomas. We now demonstrate that B cells can specifically stimulate a primary MLR in combination with the phorbol ester, PMA, but not with interleukin 1 (IL 1) or calcium ionophore. The primary MLR induced with B cells plus PMA can be blocked by either monoclonal anti-I-A or anti-L3T4 antibodies. In contrast, thyroid cells that can be induced to express Ia antigens after incubation with interferon-gamma fail to stimulate a primary MLR even in the presence of PMA or IL 1. We confirmed these observations by using the alloreactive T cell hybridoma, HTB-9.3, which does not react to stimulator B cells. In the presence of PMA, however, this I-Ab-specific hybridoma line was able to respond to relevant but not to control stimulator B cells. Furthermore, the response of HTB-9.3 to B cells plus PMA was also blocked by anti-I-A or anti-L3T4 antibody. In contrast to B cells, Ia+ thyroid cells could not activate HTB-9.3 even in the presence of PMA or IL 1. The data indicate that for primary class II restricted allo-responses, B cells provide signals that can be complemented with the phorbol ester PMA, whereas Ia+ thyroid cells do not, suggesting the existence of additional requirements for T cell activation.     

Macrophages and T cells do not express Mlsa determinants

In order to test the tissue distribution of Mlsa determinants, we have generated highly purified stimulator cell populations. First, Mlsa expression in bone marrow derived macrophages (M phi) of Mlsa genotype was tested in primary MLR and on Mlsa-specific T cell hybridomas (THy). Second, a similar experimental approach was used to analyze thioglycolate, peptone or Con A elicited peritoneal M phi. In all cases, these M phi cell populations were able to generate an excellent alloresponse, whereas no functional Mlsa determinants could be detected. Third, to further investigate whether the expression of Mlsa is lymphocyte specific, but dependent on expression of class II molecules, we have transfected I-Ek alpha and beta cDNA into a panel of thymomas of Mlsa genotype. Although we achieved a high level of surface I-Ek expression in all of these T cell tumors, none of them was able to trigger the Mlsa-specific THy. These results strongly suggest that Mlsa expression is limited to B cells. It is likely that Mlsa is a tissue-specific self-peptide that associates with class II molecules.     

The syngeneic mixed leukocyte reaction: the genetic requirements for the recognition of self resemble the requirements for the recognition of antigen in association with self

We have used cells from inbred strain 2 and strain 13 guinea pigs in order to define further the role of Ia antigens in the syngeneic mixed leukocyte reaction (MLR). The guinea pig syngeneic MLR resembled the autologous MLR in man in that it demonstrated both memory and specificity. The Ia antigens appeared to be the proliferative stimuli in that the primary stimulator cell was an Ia-positive adherent peritoneal exudate cell (PEC) and the reaction could be specifically inhibited by anti-Ia sera directed to the stimulator cell. We also demonstrated the existence of two (2 x 13)F1 T cell populations that were capable of reacting to one or the other parental PEC in the absence of any known exogenous antigen. These results suggest that the syngeneic MLR may represent T cell activation mediated through a receptor for self Ia.     

All human monocytes have the capability of expressing HLA-DQ and HLA-DP molecules upon stimulation with interferon-gamma

We have simultaneously studied expression of all three classes of human Ia (HLA-DR, DP, and DQ) on normal human B cells and monocytes (M phi) by using two-color immunofluorescence and flow cytometry. Expression was investigated on freshly isolated cells and after incubation of cells for 48 and 96 hr in interferon-gamma (IFN-gamma). All freshly isolated B cells express high levels of DR, DQ, and DP, and these levels are unchanged by incubation with IFN-gamma for 48 hr and 96 hr. In contrast, freshly isolated M phi are on the average 91% DR+, 32% DQ+, and 15% DP+. Incubation with IFN-gamma increases Ia expression on M phi to 98% DR+, 75% DQ+, and 58% DP+ at 48 hr, with virtually all cells becoming positive for all three Ia antigens at 96 hr. Furthermore, after the 96-hr incubation, antigen density increases 10-fold for DR, 15-fold for DQ, and 15-fold for DP in M phi to reach levels of expression comparable with B cells. These studies demonstrate that all peripheral blood monocytes have the capacity to become HLA-DQ and HLA-DP positive; IFN-gamma regulates expression of all three classes of human Ia in M phi; and IFN-gamma does not significantly modulate Ia expression in B cells.     

Failure of (SJL/J x PL/J)F1 hybrid mice to develop immunologic tolerance to V beta 17a+ T cells results in F1 anti-SJL mixed lymphocyte reaction.

It has been reported previously that spleen cells from (SJL x PL) F1 hybrid mice are not tolerant to SJL parental cells as assessed by a one-way MLR. The possibility that the F1 anti-SJL reaction was due to the effect of lymphokines produced by the irradiated SJL T cells in response to I-Eu expressed on the F1 hybrid cells was eliminated since inclusion of anti-I-E mAb was without effect. Cell separations showed the responder cells to be plastic and nylon wool nonadherent Ia- T cells. Separation of the SJL spleen cells showed that the stimulator cells were nonadherent, passed through a nylon wool column, and were Ia-. the F1-anti-SJL MLR was blocked 70 to 90% by inclusion of mAb KJ23a in the culture medium that indicated that the stimulatory cell population was V beta 17a+ T cells. This was confirmed by the use of V beta 17a+ and V beta 17a-T cell clones as stimulators. To determine whether failure to develop tolerance to this T cell subset in F1 hybrid mice might be responsible for the F1-anti-parent MLR, (SJL x PL)F1 mice were treated at birth and 48 h thereafter with anti-I-E mAb for 7 wk. Spleen cells from antibody-treated F1 mice were nonreactive with irradiated SJL parental cells in contrast to spleen cells from control mice which indicated that V beta 17a+ T cells were eliminated by negative selection before the development of tolerance.     

T cell recognition in the mixed lymphocyte response. I. Non-T, radiation-resistant splenic adherent cells are the predominant stimulators in the murine mixed lymphocyte reaction.

The ability of subpopulations of murine spleen cells to stimulate a mixed lymphocyte response (MLR) was studied. It was found that T cells (nylon-nonadherent spleen cells) and B cells [G-10 passed and treated with rabbit anti-mouse brain serum (RAMB) and complement (C)] were poor stimulators of an MLR. In contrast, whole spleen cells or B cells plus adherent cells (RAMB +C-treated spleen cells) produced good stimulation. However, a non-T, radiation-resistant splenic adherent cell (SAC) population was up to 20 to 50 times more efficient as a stimulator of an MLR on a per cell basis than an unseparated spleen population. These SAC were shown to express Ia determinants encoded by genes in I-A and I-E/C. These results suggest that Ia+ SAC may be the predominant stimulating cells in spleen cell populations, and the preferential target for T cell recognition in cell interaction events.     

A combined method for both endogenous myeloperoxidase and acid phosphatase cytochemistry as well as immunoperoxidase surface labelling discriminating human peripheral blood-derived dendritic cells and monocytes.

On light microscopical (LM) level dendritic cells (DC) isolated from lymphoid organs can be discriminated from macrophages (M phi) by the presence of acid phosphatase (APh) activity in a spot near the nucleus and constitutional expression of class II antigens. The aim of our study was to investigate whether DC and monocytes (Mo) enriched from human peripheral blood could be discriminated on the electron microscopical (EM) level. Therefore we developed a triple method by which we compared the presence of myeloperoxidase (MPO) containing vesicles, the localization of APh containing vesicles and expression of MHC class II and RFD1 (a DC-associated class II-like antigen) plasma-membrane antigens. DC, functionally characterized as potent stimulators in a MLR, are MPO-negative, whereas Mo show MPO in cytoplasmic granules. Although both DC and Mo show little APh activity at LM level, both types of cells show APh activity at the EM level but at different locations. In DC APh containing vesicles are present in a distinct juxtanuclear area, in contrast to Mo, which show APh activity in lysosomes scattered throughout the whole cytoplasm. Moreover, on both LM and EM level, DC are strongly class II positive, whereas Mo show variable labelling intensity for class II, while RFD1 was only found on DC.     

T lymphocyte heterogeneity in the rat. III. Autoreactive T cells are activated by B cells

Evidence has been presented to show that CD4+ autoreactive T cell lines (ATs)2 in the rat require periodic stimulation with syngeneic spleen cells for in vitro proliferation. This proliferation can be blocked by treatment of the stimulator (spleen) cells with mAb to Ia antigens. Although ATs are Ia+ and can activate the allogeneic MLR, they fail to be autostimulatory. Fractionation of the spleen cells revealed that ATs can be stimulated with B cells and not by macrophages, although the latter were efficient in several accessory cell functions, including antigen presentation, lectin-dependent T cell activation and allogenic MLR response. Moreover, B cells proliferated and differentiated in response to AT cells. These data are compatible with a model in which ATs respond to hitherto undetermined B cell membrane antigen(s) in association with MHC class II antigens. These results may have important implications in understanding autoimmune responses.