Functional analysis of cloned macrophage hybridomas. VII. Modulation of suppressor T cell-inducing activity

We have previously characterized a macrophage hybridoma clone, termed clone 59, which induced immunity but consistently failed to induce Ts responses. Macrophage 59 cells were cultured with supernatants from several activated T cell clones to determine if lymphokines could modulate the activity of this macrophage hybridoma to generate effector Ts. Culture supernatants from Th1 clones and from one atypical IL-4 and IFN-gamma-producing T cell clone successfully modulated clone 59 cells to induce effector Ts cells. In contrast, supernatants from activated Th2 cells failed to generate Ts-inducing activity in macrophage 59 cells. Culture with recombinant derived IFN-gamma was sufficient to cause modulation of Ts-inducing activity in macrophage 59 cells. The data imply that the differential functional activities ascribed to various macrophage hybridoma clones reflect macrophage heterogeneity instead of independent macrophage lineages. The suppression induced by clone 59 macrophages was genetically restricted to the putative I-J region. The ability of IFN-gamma containing supernatants to endow macrophage 59 with the capacity to induce effector suppressor cells was specifically abrogated by addition of an anti-IJk-idiotype antibody, which also reacts with IJ-interaction molecules, indicating that the mechanism of modulation most likely involves expression of IJ-interaction molecule determinants on antigen presenting cells.     

Components of the protein kinase C pathway induce Ia expression after injection into macrophages.

Macrophages are activated by a variety of microbial and cytokine stimuli. One feature of activation is the induction of class II Ag (Ia) on the cell surface. To understand the intracellular events that occur during activation, we investigated various agents with intracellular activities, and examined their effects on the induction of Ia. We first noted that several agents that activate protein kinase C (PKC) induced Ia, and that several inhibitors of PKC inhibited Ia induction by IFN-gamma. To directly test whether PKC induced Ia, we microinjected normal peritoneal macrophages with this enzyme and other intracellular mediators, then examined Ia expression. We observed that injection of PKC itself, or of other intracellular proteins thought to participate in the PKC pathway (Ras or phospholipase C gamma) strongly induced Ia expression. The Ia-inducing activity of transforming Ras protein was blocked by kinase inhibitor treatment of cells, suggesting that Ras signal transduction requires kinase activity. On the other hand, components of the protein kinase A pathway (phospholipase A2 and protein kinase A itself) did not induce Ia. Thus, the PKC pathway can control expression of macrophage surface Ia, possibly by regulating the genes of the MHC, and may play many other roles in the activation of macrophages.     

Pharmacologic evidence for the requirement of protein kinase C in IFN-induced macrophage Fc gamma receptor and Ia antigen expression.

Previous studies have implicated protein kinase C (PKC) as a mediator in the activation of macrophages by interferons. In order to probe further into the suspected role of protein kinase C in mouse peritoneal macrophage activation, the effects of protein kinase inhibitors in macrophage Fc gamma R and Ia Ag expression were studied. The protein kinase inhibitor, H7, reduced basal levels, and inhibited IFN-alpha-induced expression of Fc gamma R significantly. The concentration of H7 required to inhibit 50% of the Fc gamma R induction was approximately 12 microM, which reflects the previously reported affinity of this compound for PKC in vitro. H7 had only a minimal effect on IFN-gamma-induced Fc gamma R, suggesting different pathways of Fc gamma R induction by the two types of IFN. Ia induction by IFN-gamma was also inhibited by H7, indicating that both types of IFN can utilize PKC to mediate at least part of the signal required for Fc gamma R or Ia expression. HA-1004, a derivative of H7 which possesses high affinity for cyclic nucleotide-dependent protein kinases, but low affinity for PKC, did not alter induction, while H8, a slightly less effective PKC inhibitor than H7, was effective at higher concentrations. Another structurally distinct PKC antagonist, staurosporine, was also effective inhibiting IFN-alpha-induced Fc gamma R and IFN-gamma-induced Ia Ag expression, providing additional evidence that PKC is important. H7 was found to be effective when added as late as several hours after IFN treatment, indicating a prolonged or delayed requirement of PKC for optimal induction of Ia and Fc gamma R by IFN.     

Role of protein kinase C in IFN-mediated Ly-6E antigen induction.

The YAC T cell lymphoma normally does not express Ly-6E mRNA or Ly-6E surface molecules but can be induced to do so on incubation with either IFN-gamma or IFN-alpha/beta. This system afforded a model to assess the possible role of protein kinase C (PKC) in IFN-mediated Ly-6E induction. First, we used various pharmacologic agents known to interfere with the function of PKC or other kinases. The PKC inhibitors H-7 and phloretin were found to block Ly-6E induction by IFN-gamma or IFN-alpha/beta both at the mRNA and protein levels. In contrast, inhibitors of cyclic nucleotide-dependent kinases (HA1004), of myosin L chain kinase (ML-9, A-3) or of calmodulin (R24157, W-7) failed to suppress this induction. Next, we investigated the effects of the PKC activators PMA and mezerein (MEZ) on Ly-6E expression. Although neither PMA nor MEZ by themselves could induce Ly-6E in YAC cells, both agents enhanced by up to fivefold the induction of Ly-6 mRNA and Ly-6E surface expression triggered by IFN-gamma. However, the induction of Ly-6E expression caused by IFN-alpha/beta was only marginally increased by cotreatment of YAC cells with PMA or MEZ. Altogether, these observations demonstrate that PKC or a related kinase is involved in the transduction mechanisms that lead to Ly-6E induction. However, activation of PKC is not sufficient for this induction and requires other unidentified signal(s) provided by IFN. Our data also indicate that IFN-gamma and IFN-alpha/beta induce Ly-6E through overlapping but distinct intracellular pathways with different sensitivities to PKC activators.     

Transduction of the IFN-gamma signal for HLA-DR expression in the promonocytic line THP-1 involves a late-acting PKC activity

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7. H7 when added up to 20 hr after the initial stimulation with IFN-gamma prevented the further expression of HLA-DR. The general kinase inhibitors H8, H9, and HA1004, all less potent PKC inhibitors than H7, did not block the IFN-gamma-induced expression of HLA-DR at the concentrations employed. W7, a calmodulin antagonist, but not a PKC inhibitor, was also unable to prevent the IFN-gamma-induced expression of HLA-DR. Treatment of THP-1 with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, alone or with Ca2+ ionophore A23187, was unable to induce HLA-DR expression. However, pretreatment with PMA for 24 hr prior to IFN-gamma stimulation decreased the IFN-gamma-induced expression of HLA-DR without decreasing IFN-gamma receptor levels. These results suggest that PKC plays a significant role in the IFN-gamma-induced signal transduction pathway leading to the expression of HLA-DR in cells of the mononuclear phagocytic lineage, and that PKC activity is required throughout the course of events leading to the actual expression of HLA-DR.     

Activation of macrophages with N-formyl-methionyl-leucyl-phenylalanine: involvement of protein kinase C and tyrosine kinase

N-formyl-methionyl-leucyl-phenylalanine (fMLP) a potent chemotactic peptide stimulates immune responses by activating macrophages and other cells of the immune system. The present study reports inhibition of fMLP-induced activation of murine peritoneal and P388D-1 macrophage cell line by protein kinase C (PKC) inhibitors, H-7 and chelerythrine chloride. Similarly, tumoricidal activity was also downregulated by protein tyrosine kinase (PTK) inhibitors genestein and lavendustin A. Further, fMLP increased tyrosine phosphorylation of several proteins in murine macrophages, which were inhibited in presence of genestein and lavendustin A. These findings suggest the involvement of PKC and PTK in the activation of murine macrophages with fMLP.     

Protein kinase C-dependent and -independent mechanisms of cloned murine T cell proliferation. The role of protein kinase C translocation and protein kinase C activity

PMA can induce the proliferation of several CTL clones but not of several Th clones derived and tested in our laboratory. The PMA-stimulated proliferation of our CTL clones (which do not make IL-2 mRNA or protein) occurs independently of IL-2 and is not accompanied by lymphokine release. We now report, however, that protein kinase C (PKC) translocation is induced by PMA in CTL clones as well as in Th clones, which lack a proliferative response to PMA. These results suggest that PKC translocation itself is not a sufficient regulatory mechanism to account for cloned T cell proliferation. Moreover, IL-2 did not induce PKC translocation in a CTL clone, which proliferates when stimulated with IL-2. Thus, PKC translocation may not be necessary for activation of CTL proliferation. Nonetheless, cellular PKC activity appears to be required for the proliferative response of T cell clones after stimulation by PMA/PMA + calcium ionophore (A23187) or by triggering through the TCR: chronic PMA treatment, which depletes intracellular PKC activity, abrogates the proliferative response of T cell clones stimulated by PMA/PMA + A23187 or triggered through the TCR. T cell clones depleted of PKC activity, however, retain the ability to proliferate when challenged with IL-2. Murine T cell clones, therefore, possess PKC-dependent and PKC-independent pathways of proliferation that are not regulated by PKC translocation alone.     

Differential regulation of class II MHC determinants on macrophages by IFN-gamma and IL-4

Initiation of an immune response depends upon expression of class II MHC determinants on plasma membranes of APC. Murine peritoneal macrophages treated with either rIFN-gamma or rIL-4 display significantly more class II MHC determinants than untreated control cells. Analysis of the induction of macrophage Ia Ag by these cytokines showed considerable quantitative and qualitative differences. Maximal levels of Ia Ag induced in macrophages and detected by ELISA after IL-4 treatment at 48 h was about 80% of that induced by IFN-gamma. However, the frequency of Ia+ cells in replicate macrophage populations cultured for 48 h in excess concentrations of cytokine was 60 to 80% with IFN-gamma, 30 to 40% with IL-4, and 5% with medium alone. Thus, the subpopulation of macrophages able to respond to IL-4 for induction of Ia Ag expression was less than that able to respond to IFN-gamma. Expression of Ia Ag on macrophages continuously exposed to IFN-gamma was maximal at 48 h and remained at this high level through 6 days. Maximal Ia Ag expression for IL-4-treated cells was also detected at 48 h, but was not sustained with time in culture, and returned to base line by 4 days. A similar time course for levels of Ia-specific message in macrophages at various times after IFN-gamma and IL-4 treatment was detected by Northern dot blot analysis. Loss of Ia mRNA and Ag with time in culture in the IL-4 treated cells was not due to macrophage cell death, depletion of active cytokine, or presence of fluid-phase inhibitors. IL-4 unresponsive cells were fully capable of maximal response to IFN-gamma for Ia Ag induction. These findings suggest that IL-4 and IFN-gamma induce class II MHC determinants through different mechanisms which may provide discrete regulatory control of APC function.     

Combined effects of tumor necrosis factor-alpha, prostaglandin E2, and corticosterone on induced Ia expression on murine macrophages

Ia expression is an important marker of macrophage functional capacity. IFN-gamma induces Ia expression on perhaps all murine macrophages, whereas IL-4, granulocyte-macrophage CSF, and CSF-1 induce Ia on restricted sets of macrophages. Inhibitors of expression include PGE2, glucocorticoids, and IFN-beta. TNF has been found to augment Ia expression on several macrophage lineage cell lines but to inhibit expression on murine peritoneal macrophages. Our study shows that TNF can have opposite effects on Ia expression (induced by IFN-gamma) on thioglycollate-elicited peritoneal macrophages, depending on the length of time cells are treated and on the presence of other modulators. In particular, TNF augmented early expression induced by IFN-gamma but inhibited later expression. And although TNF synergized with PGE2 to markedly inhibit Ia induction on these cells, it partially antagonized the inhibition by corticosterone and IFN-beta. TNF and PGE2 also synergized to inhibit Ia expression induced on bone marrow-derived and splenic macrophages by either IFN-gamma or IL-4. In contrast to their effect on Ia expression, TNF and PGE2 had opposite effects on expression of gamma 2a FcR in macrophages. TNF blocked the increase in FcR expression due to any combination of PGE2, IFN-gamma, and IFN-beta. However, TNF and PGE2 both increased expression of gamma 2a FcR on WEHI-3 cells. If the different effects of TNF reflect the differentiation states of macrophages, its effects on Ia and FcR expression may vary with the progression of an immune response.     

Protein kinase C has both stimulatory and suppressive effects on macrophage superoxide production.

Unlike resident peritoneal macrophages (RPM) or tumor necrosis factor alpha (TNF alpha)-primed bone marrow-derived macrophages (BMM), unprimed BMM do not generate superoxide in response to the protein kinase C (PKC) activator, phorbol myristate acetate (PMA). However, these cells do contain significant levels of PKC activity. In contrast to PMA, zymosan induces the generation of superoxide in unprimed BMM, as well as in TNF alpha-primed BMM and RPM. Staurosporine, a potent PKC inhibitor, failed to affect the zymosan-induced production of superoxide by unprimed and TNF alpha-primed BMM and RPM, in spite of substantial inhibition of PMA-induced superoxide production by the primed BMM and RPM. However, when PKC was depleted from unprimed BMM by prolonged (24 h) treatment with phorbol dibutyrate (PdBt) (10(-7) M) the ability of zymosan to induce the production of superoxide was greatly diminished. Such a result could be interpreted as suggesting a role for PKC in the zymosan-induced response, a conclusion which contrasts with the inhibitor data. However, PKC depletion, in this case, is achieved via the PdBt-induced activation of PKC. It is thus possible that it is the initial activation of PKC, rather than its depletion, that suppresses superoxide production. Consistent with this interpretation, the co-stimulation of unprimed BMM with both zymosan and PMA resulted in a reduced superoxide release compared to zymosan alone. The activation of PKC therefore appears to have a suppressive effect on the generation of superoxide by unprimed cells. We thus conclude that PKC is not required for zymosan-induced superoxide production by either primed or unprimed macrophages and suggest that PKC may be involved in regulatory mechanisms restricting superoxide production by macrophages. However, since PMA alone can initiate the release of superoxide from primed BMM and RPM, it would appear that PKC can mediate both stimulatory and suppressive signals for macrophage superoxide production.     

Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.     

Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.     

Diminished protein kinase C-activated arachidonate metabolism accompanies rat macrophage differentiation in the lung

Alveolar macrophages (AM) differ from other macrophage (m phi) populations in their profile of eicosanoids synthesized from arachidonic acid (AA)3. Little information is available regarding possible differences in the regulation of AA metabolism among various m phi populations. In our study, we compared the ability of cultured resident rat AM and peritoneal m phi (PM) to release and metabolize AA in response to exogenous activators of protein kinase C (PKC). When stimulated with PMA, prelabeled PM released free [3H]AA in a dose-dependent manner over the concentration range 1 to 100 nM. As assessed by HPLC, PMA-stimulated PM metabolized AA to a variety of predominantly cyclooxygenase products. The dose-dependent synthesis of PGE2 by unlabeled PM stimulated with PMA was confirmed using RIA. The ability of PMA to trigger AA release and metabolism in PM was a function of its capacity to activate PKC, as indicated by the following: 1) an additional activator of PKC, oleoyl acetylglycerol, also triggered PM AA metabolism, whereas phorbol didecanoate, which lacks the ability to activate PKC, did not; 2) two structurally unrelated inhibitors of PKC activation (staurosporine and sphinganine) both abrogated PMA induced AA release in PM; and 3) pretreatment for 18 h with high dose PMA (used to deplete cellular PKC), but not phorbol didecanoate, rendered PM refractory to subsequent PMA stimulation of AA release. In contrast to PM, AM cultured in identical fashion failed to release or metabolize AA in response to either PMA or oleoyl acetylglycerol. PM and AM were also compared for their ability to release extracellular superoxide anion in response to PMA; once again, PM exhibited significantly greater release than did AM. Inasmuch as this unresponsiveness to activation of PKC distinguishes AM from other m phi populations, we conclude that it is a unique consequence of m phi differentiation in the lung. Moreover, because both AA metabolism and the respiratory burst are affected, this refractoriness appears to reflect a defect at some proximal level in PKC-mediated signaling.     

Direct evidence for an intracellular role for IFN-gamma. Microinjection of human IFN-gamma induces Ia expression on murine macrophages

An intracellular action for IFN-gamma was detected by using microinjection technology. Human IFN-gamma (huIFN-gamma) does not ordinarily act on murine cells because it fails to bind to murine cell surface receptors. However, when huIFN-gamma was microinjected into murine macrophages, a time and dose-dependent induction of Ia was detected by autoradiography on the surface of injected and neighboring cells. These results imply a direct role for internalized IFN-gamma and show that huIFN-gamma, although it fails to be recognized by murine cell surface receptors, can act internally on murine cells. The effect on Ia gene expression induced by microinjected huIFN-gamma was in part indirect: granulocyte/macrophage-CSF (GM-CSF) was released by IFN-gamma-injected macrophages, and this secondary mediator appeared to induce Ia on neighboring cells, inasmuch as anti-GM-CSF blocked Ia induction. Anti-GM-CSF also partially blocked Ia induction by extracellular murine IFN-gamma on murine macrophages. Thus, at least some of the Ia induction attributed to IFN-gamma was mediated by GM-CSF.     

A T cell-independent mechanism of macrophage activation by interferon-gamma

A primary interest in immunity to intracellular pathogenic microorganisms and tumors is to understand the mechanisms by which macrophages are activated for various functions. Two parameters of macrophage activation are the expression of the class II histocompatibility proteins or Ia molecules (1), and cytotoxic activity. The ability of T cells to induce these responses has been extensively documented and occurs via their secretion of interferon-gamma (IFN-gamma) after interaction with antigen (2-6). However, in a recent study using mice with the severe combined immunodeficiency (scid) mutation (7) which have no detectable T or B cell functions (7-9), we were surprised to find the induction of Ia expression on macrophages and the partial inhibition of bacterial growth after infection with Listeria monocytogenes (10). We have now utilized neutralizing monoclonal antibodies specific for murine IFN-gamma to investigate the mechanism of macrophage activation in scid mice. We show here that IFN-gamma can be produced by scid mice in the absence of lymphocyte-mediated immunity, and this IFN-gamma is important for macrophage activation during infection with Listeria. These results indicate the presence of an important T lymphocyte-independent mechanism of macrophage activation and IFN-gamma production in response to infection.     

Induction of macrophage Ia antigen expression by rIFN-gamma and down-regulation by IFN-alpha/beta and dexamethasone are mediated by changes in steady-state levels of Ia mRNA

In previous studies, the induction of Ia antigens on murine peritoneal exudate macrophages by recombinant IFN-gamma (rIFN-gamma) and the antagonism of rIFN-gamma-induced Ia expression by the inhibitors IFN-alpha/beta and glucocorticoids have been examined. In this report, these findings have been extended to an analysis of total or cytoplasmic mRNA from macrophage cultures treated with rIFN-gamma in the absence or presence of these two inhibitors. Recombinant IFN-gamma induced a 5.7- to 6.5-fold increase in steady-state levels of Ia (A alpha-specific) mRNA. Coordinate increases in steady-state mRNA for A beta, and E alpha were observed in response to rIFN-gamma. Maximum induction occurred 24 hr post-treatment and required the continued presence of rIFN-gamma. Induction of A alpha-specific mRNA was sensitive to the protein synthesis inhibitor cycloheximide. Simultaneous treatment of macrophage cultures with rIFN-gamma and IFN-alpha/beta or the glucocorticoid dexamethasone (DEX) resulted in a significant decrease in steady-state, A alpha-specific mRNA levels compared with treatment with rIFN-gamma alone. This analysis suggests that both the induction of Ia expression by rIFN-gamma, and the antagonism of rIFN-gamma-induced Ia gene expression by IFN-alpha/beta and DEX, are regulated by cognate changes in Ia mRNA.     

Role of protein kinase C and intracellular calcium mobilization in the induction of macrophage tumoricidal activity by interferon-gamma

These studies were designed to test the hypothesis that changes in intracellular Ca2+ levels and activation of the calcium ion- and phospholipid-dependent protein kinase C were required for the induction of macrophage tumoricidal activity by interferon-gamma (IFN-gamma). Phenothiazines and R24571, known antagonists of calcium-binding proteins and therefore nonspecific inhibitors of protein kinase C, blocked in a dose-dependent manner the induction of macrophage cytocidal activity by either natural or recombinant IFN-gamma. Macrophages depleted of intracellular Ca2+ by chelation with Quin 2, were also unresponsive to IFN-gamma. These treatments effected neither the binding of IFN-gamma to its cell surface receptor nor the normal intracellular processing of IFN-gamma. Activators of protein kinase C (such as phorbol esters) and Ca2+ ionophores when added alone did not effect the activation state of the macrophage population. However, macrophages exposed to both drugs in combination were elevated into the primed activation state such that in the presence of a second signal (lipopolysaccharide or heat killed Listeria monocytogenes), the cells were triggered to express full levels of tumoricidal activity. The capacity of phorbol esters to induce cellular activation correlated with their ability to bind and to activate protein kinase C. No synergistic effect was observed between IFN-gamma and protein kinase C activators and/or Ca2+ ionophores, indicating that the drugs could only prime and could not trigger macrophages for tumor cell killing. These results thus support the concept that protein kinase C activation and mobilization of intracellular Ca2+ are essential steps in the pathway of IFN-gamma-dependent induction of non-specific tumoricidal activity in macrophages.     

Priming of the respiratory burst of bone marrow-derived macrophages is associated with an increase in protein kinase C content.

The biochemical mechanism(s) underlying the priming of the macrophage for an enhanced PMA-induced respiratory burst is not understood. Because the cellular receptor for PMA is thought to be protein kinase C (PKC), we have investigated the effects of priming agents on cellular PKC levels. Sonicates from unprimed bone marrow-derived macrophages (BMM) were found to contain PKC activity (309 +/- 51 pmol 32P-incorporated/mg/min; mean +/- SE, n = 17) as measured by the phospholipid-, diacylglycerol-, and calcium-dependent phosphorylation of histone. Exposure of BMM to priming agents such as TNF-alpha, LPS, and granulocyte/macrophage-CSF resulted in a significant increase in both histone-phosphorylating activity and levels of immunoreactive PKC protein in these cells. A minimum of 6-h exposure, with an increasing effect up to 48 h, was required for a detectable increase in PKC level. The activity from primed BMM, like that of the untreated cells, was predominantly cytosolic. The kinetics and concentration dependence of the priming agent-induced increase in the PKC content of BMM closely paralleled the enhancing effects of these agents on the PMA-stimulated respiratory burst. Furthermore, CSF-1, a cytokine that does not prime BMM, failed to increase PKC activity. We propose that the exposure of BMM to priming agents leads to an increase in the expression of a stimulatory isozyme(s) of PKC, resulting in an enhanced ability to mount a respiratory burst in response to stimulation with PMA.     

Phorbol esters and calcium ionophore can prime murine peritoneal macrophages for tumor cell destruction

Murine macrophages from sites of inflammation develop toward tumoricidal competence by exposure to a macrophage-activating factor such as interferon-gamma (IFN-gamma). To explore the biochemical transductional events initiated by IFN-gamma, peritoneal macrophages from C57BL/6J mice elicited by various sterile irritants were treated in vitro with two pharmacologic agents that mimic the action of certain second messengers. Phorbol myristate acetate (PMA) and the ionophore A23187 cooperatively reproduced the ability of IFN-gamma to prime macrophages for tumoricidal function. Neither agent alone was able to prime macrophages. The two agents acted on the macrophages, and target susceptibility to kill was not altered by PMA and A23187. Only active phorbol esters, which are known to bind and stimulate protein kinase C, were able to cooperate with A23187 to induce priming. A cell-permeable synthetic diacylglycerol (sn-1,2-dioctanoyl glycerol) could also prime for cytolysis. In the presence of PMA, A23187, and EGTA, addition of Ca++ was sufficient for priming, whereas the addition of Mg++ was much less efficient. Priming by IFN-gamma, however, was not blocked by EGTA. Efflux of 45Ca++ from preloaded cells was significantly increased by A23187 and by IFN-gamma. Quin-2/AM, an intracellular chelator of Ca++, blocked priming by IFN-gamma. In summary, the data suggest that priming of macrophages for tumoricidal function by IFN-gamma involves, at least in part, alterations in protein kinase C and in levels of intracellular Ca++.     

Effect of interferon-gamma and human alpha 2-macroglobulin on peritoneal macrophage morphology and Ia antigen expression.

While the primary role of the plasma protein alpha 2-macroglobulin (alpha 2M) appears to be related to its proteinase inhibitory activity, alpha 2M has been reported to regulate the immune response in vitro. Previous studies have demonstrated that, although native alpha 2M has no effect on macrophage function, proteinase- or CH3NH2-treated alpha 2M antagonize the IFN-gamma-induced expression of class II major histocompatibility complex (Ia) antigens on mouse peritoneal macrophages. In this investigation, we examined the effects of alpha 2M-CH3NH2 on the IFN-gamma-induced expression of macrophage Ia antigens by indirect immunofluorescence microscopy, radioimmunoassay, and immunoprecipitation of biosynthetically-labelled Ia. While alpha 2M-CH3NH2 suppressed the IFN-gamma induced increase in the percentage of Ia-positive macrophages detected by immunofluorescence microscopy, alpha 2M-CH3NH2 had no effect on the average of number of Ia molecules expressed per cell as detected by radioimmunoassay. In addition, alpha 2M-CH3NH2 had no effect on the ability of IFN-gamma to induce biosynthesis of Ia. Microscopic examination of IFN-gamma-treated macrophages revealed that treatment with alpha 2M-CH3NH2 prevented IFN-gamma-induced changes in macrophage morphology. IFN-gamma-treatment of elongated inflammatory macrophages was associated with the generation of round cells which possessed few cytoplasmic projections. By contrast, addition of alpha 2M-CH3NH2 to the incubation prevented the IFN-gamma-induced morphological changes, and the cells remained elongated with irregular cytoplasmic borders. We postulate that alpha 2M-CH3NH2 decreases the IFN-gamma-induced expression of Ia by preventing morphological changes in macrophages, resulting in the distribution of existing Ia over a larger surface area. As a consequence of this, the perceived fluorescence intensity of the bound antibody is lowered and the cells appear to be Ia-negative.