Molecular mechanisms of signal transduction in macrophages

ImmunologyToday, vol. 8, No. 5, 1987

Molecular mechanismsof signal transduction in macrophages The mechanismsby which extracellularsignalsare receivedand transduced across the cell membrane are being illuminated by studies in macrophages. In this review Tom Hamilton and Dolph Adams discussmechanisms of signal transduction which either initiate rapid execution of function in macrophages or alter the cells' potential for taking such action.

ThomasA. Hamilton],3and DolphO. Adams

Activation of phospholipase C, in turn, hydrolyses phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-triphosphate and 1,2-diacylglycerol (for reviews of PIP2 hydrolysis, see Ref. 4). Murine mononuclear phagocytes bear at least 4 distinct receptors for the Fc portion of immunoglobulins, The general problem of how extracellular signals moduincluding a high-affinity receptor for immunoglobulins of late cell behavior is central to cell biology today. Progress the IgG2a isotype (FC~2aR)and a distinct receptor for in this area may ultimately identify pharmacologic agents immunoglobulins of the IgG1 and IgG2b isotypes (Fc~l~ capable of selectively modulating the function of various ~zbR)5. At least two genes for FcRs have been identified; cells. Macrophages are of particular interest, as they play these include a (expressed in macrophage-like cell lines) a significant role in host defense against microbes and and 13(expressed in ma~rophages and lymphocytes)6. ~, tumors, in homeostasis and in diseases such as atherrecently identified clone for the Fc~I/~2bRhas sequences ogenesis, carcinogenesis and chronic destructive identical to the [3 gene7. These genes s-8 encode transdisorders ~.2. Their function in all of these roles is strinmembrane proteins, which have homology with class II gently regulated by extracellular signals. Such signals can histocompatibility antigens and are thus putative memstimulate the immediate execution of various complex bers of the immunoglobulin superg_=nefamily. Of note, functions (e.g. destruction of microbes). Other exthe transmembrane and cytoplasmic domains encoded tracellular signals can alter the potential of mononuclear by the ¢x and 13 variants are quite distinct, raising the phagocytes so that competence to respond to extracellupossibility that the signal transduction mechanisms initilar signals of the first type is markedly enhanced or ated by occupancy of these two receptors may be diminished. different. The signal transduction mechanisms initiated by FC~zaR Mechanismsof signal transduction in rapid execution of and Fc~I/~2bR remain to be fully clarified. Ligation of function FC~2aRhas been reported to raise intracellular levels of cyclic AMP around 5-fold within 30 s in the P388D1 Receptors and second messengers macrophage-like cell line, while ligation of the Fc~l/~2bR n,,~pito t h , , f : r t t h : f mrtr.lr~rllirl,'~'~r nh:lt~t'~,e'lrl'~c h~r~., on this ceii iine leads to enhanced release of arachidonic around 50 surface receptors that are doubtless coupled acid (for review, see Ref. 9). Fc~l/~2bR,when isolated by to various second messengers~.2, this topic has been precipitation with a ,, c~cific monoclonal antibody (the extensively investigated only in a limited number of 2.4G2 MAb) is a ~,l,,r,Jprotein of M, 50-60000 s. The cases. We shall highlight recent progress in two protopredicted protein encoded by (xl would have an M, of typic systems: the chemotactic receptors recognizing 30000 while that encoded by 131would have an Mr of N-formylated peptides; and the receptors for immuno37000, before glycosylation or other post-translational globulins. modifications e. Suzuki and colleagues have isolated from The receptor for synthetic formyl-peptides, such the P388D1 cell line phosphatidylcholine (PC)-binding as N-formyl-methionyl-leucyl-phenylalanine (f-MET-LEUproteins, which also bind immunoglobulins of the IgG2b PHE), when occupied by its ligand, initiates chemotaxis, isotype and which have phospholipase A2 activity 9. Restimulates secretion of lysosomal enzymes and initiates cently, these investigators have shown that the major production of reactive oxygen intermediates ~. The transcomponent of the PC binding proteins has an Mr of ductional events set in play by engagement of this 8000 after glycosylation and reacts with the 2.4G2 receptor have been elegantly studied by Snyderman and MAb 1°. A 50-60 kDa protein isolated with the 2.4G2 colleagues (reviewed in Ref. 3). The receptor for t-METmonoclonal can be reconstituted in proteoliposomes or LEU-PHE is coupled to a guanine nucieotide regulatory piano-lipid bilayers and acts as a ligand-dependent, protein (N protein), which is itself of interest, since cation-selective ion channel ~~. These channels function it shares many characteristics with other guanine principally as monovalent cation channels, in which K+ is nucleotide regulatory proteins including Ns, N~, No and favored over Na +. Interestingly, cross-linking of Fc~l/~22bR transducin but is sufficiently distinct to be considered by the 2.4G2 MAb increases cytoplasmic levels of Ca novel and bear the designation No. The No-protein, when in J774 cells from ~ 90 nM tO 400 nM within 30 s, as c3n activated, stimulates a polyphosphoinositide-specific addition of specific ligand (e.g. IgG2b) to these cells12. phospholipase C by reducing the Ca2+ requirement of Under similar circumstances, the J774 macrophagc-like the enzyme to levels found in the normal resting cytosol. cell line undergoes changes in membrane potential, which can be inhibited by removing Na + from the ~2 Duke extracellular medium ~3. Other workers have, however, Departments of Pathology~ and Microbiology-Immunology., Medical Center, Durham, North Carolina 27710; Cleveland Clinic suggested that Fc-receptor-mediated phagocytosis can occur in macrophages without increases in overall cellu- 151 ResearchFoundation3, Cleveland,OH44106, USA (~ 1987, ElsevierPublications,Cambridge 0167 - 4919/87/$0200

Immunology Today. vol. 8, No. 5, 1987

fe.vte , lar caloum and that neither phagocytosis nor the respirato:-" burst i=~,".acrophages require fluxes of Na+~4.~s Initiation Of metabolic bursts

Leukocytes, including macrophages, when stimulated b~ a variety of ligands or particles, undergo a cyanidei-,sensitive respiratory burst and concomitantly secrete ,~active oxygen :.;~':ermediates (for reviews, see Refs 16-18~. During the respiratory burst, oxygen consumpt;on is greatly increased and molecular oxygen is reduced in ~r near the plasma membrane by an NADPHdependent oxldase complex to superoxide ion, which in turn is dismutated to hydrogen peroxide. This or a similar oxidase has been identified in macrophages (for example, see Ref. 19). Macrophages, at the same time, may undergo a smaller metabolic burst during which phospholipase Az is activated to cleave arachidonate from cellular pools of phospholipids; this may be subsequently oxidized by either cyclo-oxygenases or lipo-oxygenases to produce prostaglandins and HETEs or leukotrienes, respectivelY2°.21. Recent evidence indicates that these two metabolic bursts may be coincident but independent22. Measurement of the released products of these two metabolic bursts is a frequently used marker for analysis of signal transduction mechanisms leading to rapid responses. The oxidase complex is essentially dormant until leukocytes are stimulated, at which time the enzyme becomes activated and associated with the plasma membrane; activation of the oxidase, however, remains poorly understood in molecular terms ~6-~8. Monoclonal antibodies, produced against a subcellular preparation exhibiting NADPHoxidase activity and obtained from PMAstimulated neutrophils, identify an antigen of Mr 10000 that becomes associated with the superoxide ion-generating system of neutrophils; these monoclonal antibodies can initiate a respiratory burst23. In neutrophiis, phosphoryiation of a 48 kDa protein (or proteins) appears essential for the respiratory burst and to activation of the oxidase24. Initiation of arachidonate metabolism requires the acti'vation of phospholipase A2, a calcium-dependent enzyme (Refs 20, 21; and see also Ref. 25). The cascade begins by generation of free arachidonic acid. This step, which appears to represent a critical control point, may

be achieved either directly by activation of phospholipase A2 or indirectly through sequential actions of phospholipase C and diacylglycerol lipases. The former, which may be the more important regulatory point, appears to be initiated, at least in part, by rises in intracellular levels of Ca2÷ though the role of other regulatory molecules is not yet certain. What intracellular events, in macrophages, link ligation of surface receptors with these two metabolic bursts? (for reviews, see Refs 3, 17, 26, 27). Pharmacologic stimulation of leukocytes with stimulants of protein kinase C (PKc) such as phorbol myristic acetate (PMA), with the ionophore A23187 (in the presence of calcium), or both can initiate the release of reactive oxygen intermediates and metabolites of arachidonic acid. In regard to the release of ROI, PKc must not only be stimulated but translocated to the plasma membrane. It is thus not surprising that ligation of the receptor for formylated peptides, which generates heightened fluxes of intracellular calcium via the generation of IP3 and stimulation of protein kinase C (as well as its translocation to the membrane), stimulates secretion of 02- (see Ref. 3). As noted above, ligation of certain Fc receptors can lead to heightened intracellular levels of Ca2+; ligation of Fc receptors can also initiate heightened phosphate labelling of proteins in a pattern very closely resembling that initiated by PMA, suggesting that ligation of Fc receptors can stimulate protein kinase C28. Of interest, recent studies on Fc-receptor-initiated secretion of arachidonate imply initial generation of an Na+dependent signal, followed by synthesis of short-lived proteins, stimulation of PKc, generation of Ca2+ fluxes, and activation of phospholipase A229. Some basic events involved in these metabolic bursts are depicted in a highly simplified model (Fig. 1). It is to be emphasized that this is a minimal model and that other signal transduction and regulatory_ elements, as yet unidentified, are doubtless involved. These two metabolic bursts of macrophages are further regulated in several ways. In neutrophil~ ;,nd in macrophages, stimulation of these bursts can be primed (i.e., enhanced) by" previous exposure to stimuli which in themselves do not trigger a burst 16.17. Macrophage activation, whether achieved in vivo by stimulants such as BCG or in vitro by stimulants such as lymphokines,

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Immunology Today, voL 8, No. 5, 1987

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Fig.2. A schematicmodel of macrophagedevelopmentin responseto IFNyand LPSL Inflammatorymacrophagesrespondto IFN~and LPS,in sequence,by developingfirst to the primedand then to the fullyactivatedstages,respectively.Cellsin different stages of activation exhibit different physiologiccapacities,which often reflect their potential to execute different complex functions. Reprintedby permissionfrom Adams, D.O. and Hamilton, T.A. (1987)Immunol. Rev.(inpress).

I-A (classIIhistocompatibilityantigens),LFA-1(lymphocytefunction-associatedantigen-I),02- (superoxideanion),TFR(transferrinreceptor),CP(cytolyticprotease),TNF (tumornecrosisfactor). also heightens the degree of the respiratory burst (see Ref. 27 .for review). Several laboratories have reported that this is duP to increased affinity of the oxidase for NADPH27.3° although a recent report has suggested that the basis of regulation occurs earlier in the signal transduction cascade31. Translocation of PKc to the plasma membrane is stringently regulated4 and can be induced without activating PKc by altering intracellular levels of Ca2+ (Ref. 32). Finally, the respiratory burst can also be down-regulated, and this has been attributed both to changes in the oxidase (i.e. heightened Km and decreased Vmax)33 and to altered signal transduction 34. Mechanismsof signaltransductionin activation Signals that activate macrophages

The tissue macrophages, particularly recent arrivals from the blood, are multipotential and can develop along a variety of disparate routes ~.2. Traditionally, development of competence for microbicidal and/or turnoricidal function has been designated as the activation of macrophages, but one can alternatively define activation as acquisition of competence to perform any complex function, such as the ability to destroy tumor cells or to present antigen to thymus-derived lymphocytes ~. Whatever formal definition is employed, the developmental potential of macrophages is large and diverse, and some of the developmental options open to these cells appear to be mutually exclusive ~. Acquisition of competence for destruction of tumor

cells is a useful model for studying macrophage functional development 1. In murine macrophages, lytic function can be induced in vitro by a variety of signals, including the synergistic interaction between two distinct signals that operate in a defined sequence: (a) one of a class of lymphokines, operationally termed macrophageactivating factor(s) (MAF); and (b) a secondary signal, frequently supplied by bacterial cell wall products. The first signal lowers the dose requirement for the second. Of note, MAF and lipopolysaccharide (LPS) can work cooperatively but may also work independently, antagonistically and in parallel. The ability to analyse molecular mechanisms of signal transduction in macrophages stems from two developments. First, pure activating signals, defined in molecular terms, have become available (see below). Second, the developmental stages of macrophage activation have been characterized by a library of objective, quantitative and rapidly determined markers (see Fig. 2). Such markers often reflect the ability of various macrophages to execute specific functions 1. Signals that induce priming

The principal signal which primes macrophages for activation is MAF35. Interferon gamma (IFN~/) is a potent MAF in vitro and in vivo 36. Recent reports from several laboratories have described MAFs distinct from IFN~/, but the molecular identities of these factors remain to be established 35.36. Murine IFN~/is synthesized as a peptide of 17 kDa and post-translationally modified into active

153

ImmunologyToday,vol 8, No. 5, 1987

forms of 2n kDa or 25 kDa; both are glycosylated, though the glycosylated regions are apparently not essential for biological activity. The 3 ~.amino acids tn the COOH-terminus of the molecuie appear to be essential for receptor binding and macroohage activation, while a domain in the NH2-terminus appears necessaryfor antiviral responses (Ref. 35; see also Ref. 37). A specific receptor for !FN~/has been identified on murine macrophages and human monocytes36, These binding sites, which exhibit a Ka of --- 2 x 108 M -~ range from 3000-15000 per cell (Ref. 34; see also Refs 38-40). The number of receptors and their ligand binding affini*.y do not vary appreciably for macrophages in the several stages of activation36. It has been reported that the receptor for IFN~/on human monocytes differs from that on non-hematopoietic cells, and further recent evidence implies that the WEHI-3 macrophage-like cell line has, in addition to the classof binding sites previouslydescribed, a smaller class of receptors of much higher affinity (e.g. Kdof9 X 10-11 M)38-4°. Signals that push primed to activated macrophages A variety of signals push primed macrophages to the fully activated stage~.41. These 'second' signals include the lipopolysaccharide component of various bacterial cell walls, high concentrations of crude lymphokines, supematants of tumor cells, heat-killed Gram-positive microorganisms such as Listeria monocTtogenes, and liposome-encapsulated muramyl dipeptide. The molecular basis or bases of the interactions between LPS and a wide variety of host inflammatory and immune cells remain unresolved4Z.43. Although the intact molecule can apparently bind to macrophages via a portion of the carbohydrate element44, lipid A is the biologically active moiety4s. Binding of radiolabelled LPS or lipid A to cells can be demonstrated but convincing m~era~ton has not beer, forthcoming42.43. Recent evidence suggests that LPS may bind to the membrane protein leukocyte function-associated antigen 1 (LFA-1), though the functional significance of such binding remains to be established46. LPSappears to interact with membranes in two distinct steps: (1) a rapid temperature-independent attachment of LPS to the cells; and (2) a time- and temperature-dependent event, which may represent intercalation of the lipid moiety of LPS into the membranous bilayer42.43. Macrophages exhibit a cell-surface receptor which recognizes a diversity of polyanionic polymers, including proteins in which lysine residues have been altered bv acetylation or maleylation, complex carbohydrates sucl;i as the algal polysaccharide fucoidan and poly-inosiniccytidylic acid (poly IC)¢7. This 'scavenger' receptor, which has a molecular mass of 260 kDa, ts of considerable biological importance, because modified low density lipoproteins (LDLs)are taken up by macrophages via the receptor. Mononuclear phagocytes also bear a surface rec~.ptor which specifically recognizes maleylated bovine serum albumin (MaI-BSA) though not modified LDL48. Mal~ylated-BSA and the algal I~olysaccharide fucoidan trigger secretion of several proteases, including a cytolytic protease and tumoriciclal function in appropriately primed populations of macrophage49. Recent evidence suggests that these ligands are acting via the receptor for ~rllJ~,ru-~

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maleylated-BSA rather than the receptor for modified LDL (T.A. Hamilton, M. Haberland and D.O. AcJams, submitted). Signal transduction and IFN~I The functional consequences of IFNs in various cell ~,,pes are diverse (i.e., antiviral, antiproliferative, immunoregu!atory effects) and are observed from 8 h to 4d after application of the ligand so.s1. The most thoroughly characterized biochemical changes involve: (!) activation of a protein kinase, which phosphorylates the subunit of initiation factor ELF-2and a protein of 67 kDa; and (2) induction of 2'-5' oligoadenylate synthetase. Both responsesare slow (ie., peak at 6-8 h). The rapid intracellular messenger events which presumably couple occupancy of the receptor to these later changes remain obscure5o.sl. Several lines of evidence indicate that activation of the protein kinase and induction of 2',5'-oligoadenylate synthetase are separable events. Although the potential contribution of these changes to macrophage activation by IFN~/ remains to be established, IFN~/ does inhibit macrophage processing of ribosomal RNA, resulting in the accumulation of precursors that could activate the double-stranded RNAdependent enzymes induced by IFNlf in other cell types (for review, see Ref. 52). Because macrophages must be exposed to IFN,y for 2-4 h (at concentrations of 10 U or less per ml) for priming 34, attention has been focused L.n this period in attempts to identify changes generated by receptor occupancy. Two biochemical changes in macrophages which fit this criterion have been identified and are initiated by such co.,centrations of ligand. First, IFN~/ induces a rise in inttacellular levels of Ca2+, manifested by enhanced eff!ux of radiolabelled calcium from preloaded cellss3. This elevated efflux of Ca2+ initiated by physiologic doses of IFN~/ is first seen 5-10 min after initiation of treatment and is complete by 10-20 rain53. Furthermore, a mixture of MAF plus LPS likewise initiated quite slow rises in intracellular calcium levels as measured by 'Aequorin' in the RAW 264 macrophage-like cell line54. Second, IFN~ enhances the potential activity of PKc5s. This change (change of PKc to PKc*), which is induced by IFN~/but not by IFNa, IFNI3, colony stimulating factor 1 (CSF-1)or a variety of other signals, is seen as early as 1 h, peaks at 3-4 h and declines by 6-12 h. The change does not apparently result from an in~reased number of enzyme molecules, but rather from an increase in the catalytic efficiency of existing enzymes. Enzyme from IFN~/-treated macrophages phosphorylates substrat~ prct~in 2-4 *,imes more rapidly than ~iizyme from control cells, after stimulation with diacylglycerols6. The enhanced potential of macrophage PKc is manifested by enhanced phosphorylation of endogenous protein substrates, when the cells are stimulated with LPS or PMAs7. Fu thermore, the change has functional consequencess8. Finally, IFN3,transcriptionally regulates an early respons,_=protein of low molecular weight that is homologous to platelet protein 4 s9 and raises intracellular content of S-adenosyl-homocysteine6o. More immediate second messengers, acting over seconds to minutes, are probably involved in the effects of IFN~/but remain to be identified in macrophages. Interferons, at concentrations up to 20000 U/ml, cause in fibroblasts and Daucli cells rises in diacylglycerol and in the total pool of IP3 but

Irnmunology Today, voL 8, No 5, 1987

not in Ca2+ (Ref. 61). Two lines of evidence imply that these two transductional changes not only .accompany but are necessary for macrophage activation. First, several physioiogic responses induced by IFNlf can be pharmacologically mimicked by treating the cells with PMA (a stimulant of PKc) and an ionophore for divalent cations, such as A23187, in the presence of Caz+ (though not in the presence of other divalent cations). Specifically, PMA, A23187 and/or a combination of the two initiate competence for selective binc;ing of tumor cells, priming for cytolysis, down-regulation of the transferrin receptor and enhanced surface expression of class II histocompatibility antigens (ia molecules) 8-16 h later (Refs 53, 62-65; for additional references, see Ref. 51). Conversely, inhibitors of Ca2+ and nonspecific inhibitors of PKc block several effects of IFN~/s3.65-67.The evidence as to whether these changes are dependent upon calcium channels, as judged by use of inhibitors, remains controversial. Second, treatment of macrophages from A/J mice with IFN~/(which are hyporesponsive to IFNIf) does not modulate calcium metabolism or enhance PKc activity, despite the fact that such macrophages have receptors for IFNI~ of equal number and affinity as control macrophages68. Treatment of macrophages from AJJ mice with A23187 plus PMA restores functional development68. The totality of signalling events initiated by IFN~/ is doubtless much more complex than this. For example, IFN-y induces surface expression of la and LFA-1 antigens, but only the former response can be pharmacologically reproduced with A23187 and/or PMA64. Moreover, mice of the A/J strain, while hyporesponsive to IFN~/in regard to priming for cytolysis and induction of la antigens, respond normally to IFN~ by enhanced expression of surface LFA-164. These latter data raise the intriguing possibility that IFN~ may initiate several intracellular transductional pathways in macrophages. Signal transduction and LPS LPS and other second signals induce two types of functional response in macrophages: (1) rapid responses, such as the secretion of metabolites of arachidonic acid, neutral proteases and lysosomal hydrolases over a few hours (see above); and (2) developmental changes, such as induction of cytotoxic activity toward both microbes and tumor cells, over many hours or clays1.2. Two broad categories of transductional events in response to LPS have now been discerned, though the relationships t.o specific functions remain to be determined. LPS or lipid A, at concentrations that are effective as triggering signals (i.e., ~- ! 0 ng/ml) initiate, hydrolysis of phosphatidylinositol-zt,5-biphosphate (PIP2) and subsequent generation of the 1,4,5 isomer (inosltoi 1,4,5P3) of inositol triphosphate (IP3) in macrophages (Ref. 69 and V. Prpic, etal. submitted). It is this isomer which has been associated with rises !.~,intracel!ular calcium in other cells. The liDid moiety of L'S, thou~h not LPS itself, leads to large rapid elevations in intrac~llular levels of Ca2+ (i.e., from ~ 60 nM to ~ 600 nM Caa+) as estimated by Fura-II fluorescence; by this technique, LPS itself leads to rapid but smaller (i.e. from 60nM to 90nM) rises in Ca2÷. Certain stimulatory effects of LPS nn B lymphoma cell lines as well as the P388D1 macrophage-like cell lines may be transduced through one or more members of the

family of N-proteins, since certain stimulatory effects of LPS can be blocked tn these cell lines by Bordetella pertussis toxin 7o. Macrophages, treated with physiologically activating doses of LPS or lipid A, also exhibit a characteristic pattern of protein phosphorylationsT. Specifically, phosphorylation of five distinct proteins (i.e., pp28, pp33, pp38, pp67 and ppl03) is induced or enhanced in macrophages treated with LPS; the e|,hanced phosphonjlation is ol-.:,erved when the macrophages are assayed using a variety of radiolabelling protocols and followed by analysis with either one- or two-dimensional gel electrophoresis. The pattern of endogenous substrates phosphorylated in LPS-treated cells resembles closely that initiated by PMA. Moreover, partial proteolysis of 13p28, pp38 and pp67 indicates that both LPS and PMA ' eatment result in phosphorylation at similar sites, further supporting the possibility that LPS-induced protein phosphorylation is mediated via PKc. LPS also induces myristilation, another covalent modification of proteins, in a pattern which bears resemblance to that initiated by PMAz~. The above observations are interna!!y consistent with one another, in that initiation of polyphosphoinositide hydrolysis has been shown in many cell systems to trigger protein phosphorylation via PKc7z. The likely generation of diacylglycerol during hydrolysis of PIP2 is a reasonable candidate for the immediate stimulant of PKc in LPSinduced macrophage protein phosphonjlation72. Wightman and Raetz have, however, demonstrated that LPS, lipid A and biologically active precursors of lipid A can substitute for the phospholipid requirement of PKc, though these materials cannot stimulate the enzyme in the absence of diacylglycero173. The enhanced phosphorylatio~ may be essential to macrophage development induced by LPS. Alternate second signals, such as heat-killed L. monocytogenes. also induce similar protein phosphorylations7.74. Macrophages from C3H/HeJ mice are unable to initiate phosphorylation when treated with LPS and do not acquire lytic function. Treatment of C3HIHeJ derived macrophages with heat-killed L. rnonocytogenes restores both the biochemical response and functional development. The role of alterations in Ca2+ is less clear. IFN~/ and LPS both modulate levels of CAz+, though these appear to be mechanistically and temporally distinct. IFNI~ treatment results in a slow, prolonqed elevation of Ca2+ without PIP2 hydrolysis, while lipid A and LPS lead to rapid, transient spikes in intracellular concentrations of Ca2+, apparently associated with the generation of IP3. Chelators of Caz+ do, however, block several aspects of macrophage development, initiated by either IFN~ or LPS or both (see above and Ref. 75). Effects of LPSon early gene expressiov Stimulation of protein phosphoryl,:tion by PKc, while perhaps necessary, is not sufficient for a full functional response to LPS, because stimulants of PKc (such as PMA and diacylglycerol) are unable to replicate function fully $3. Changes in early gene expression in response to LPS may thus also contribute to the acquisition of competencez6-78. For example, modmdation of the c-los and c-myc prot~-oncogenes is induced in macrophages by LPSzs. Specifically, LPS causes a marked increase (5-20 fold) in mRNA levels for both genes peaking az

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about 30 min, followed by a dramatic decline in message levels by 60-90 min such that no c-los or c-myc sequences are detectable after this time. These levels gradually return to thosez9 seen in untreated cel~s by 18-24 h. Other so-called competence gen~=s(ie., JE and KC) (see Ref. 79 for review) are also induced by LPS in mononuclear phagocytes78. Enhanced expression or synthesis de nova of a set of polypeptides is also initiated by LPS8°.sl. By pulse labeling LPS-treated macrophages with 3sS-methionine, at least 7 proteins in the 38-85 kDa range are detected by electrophoretic analysis. These proteins are induced rapidly (i.e., within 30 rain) and are transiently expressed (optimally detected between 2 and 6 h after stimulation). Expression subsequently declines, such that by 8-12 h the response is no longer evioent; some of these proteins exhibit quite short haft-lives (1-3 h ). The early appearance, brief synthesis and short haft-lives of these proteins suggest that they do not mediate effector functions of activated macrophages, which are generally expressed from 6-24 h after stimulation and persist 24-48 h thereafter. Expression of these early proteins is apparently independent of the modu=ation of proto-oncogene expression induced by LPS,since PMA and several other

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stimuli of c-myc and c-fos expression do not induce expression of the early proteins8°.81. The identities of these early genes, their products and their functions remain to be defined. Nevertheless, several lines of evidence suggest that these proteins are important to activation. First, only those signals (taken from a large group of functionally distinct stimuli) which induce both pathways of gene expression also activate primed macrophagessT.so. Second, the activation of macrophages in response to LPS, in terms of tumoricidal function and suppression of la antigen expression, is dependent upon continuous protein synthesis (Ref. 80, 81 and T.J. Koerner, T.A. Hamilton and D.O. Adams, unpublished). This observation provides some support for a regulatory role of the early gene products in the development of lytic competence and in the suppression of surface expression of la (suppression by LPS described in Ref. 81). Interestingly, some of the early responses to IFN3t(e.g., cytolytic priming and induction of la) are not blocked by inhibiting protein synthesis and, in fact, are often enhanceds2.8o. These two pathways appear to be quite distinct. Several lines of evidence indicate that events associated with the breakdown of PIPz are independent of the

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Fig. 3. A modelof signal transductionin macrophageac~vation. IFI~I, a~ng via putative early messengers, alters Ca2+ and the potentialof PKc Second signals, including LPS, heat killed L. m0n0cyt0genes(HKLM), po~/-inosinic-cytJdylicadd (potyIC) and malelyatedbovineserum albumin (MaI-BSAJ,induce hydrolysisof PIP2and expressionof 'early'proteins, Theseevents, in turn. mayregulate transcriptionflTanslationof genes for effector molecules suchas la or INF. Reprintedby permissbn from Adams, D.O. and Hamilton, T.A. (1987) ImmunoL

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iii

events which initiate synthesis of the 7 short-lived proteins 8°. For example, expression of mRNA for the competence genes c-fos, c-myc, and JE appears to be associated with breakdown of PIP2, while expression of mRNA for the competence gene KC (as well as synthesis of the 7 polypeptides) appears to be associated with the other LPS-dependent pathway 78.

Interrelationships between signal paths induced by IFN~I and LPS At least three biochemical cascades are initiated by IFNIt and LPS in responsive macrophages (Fig. 3). IFN~/ modulates both metabolism of Ca2+ and the potential activity of PKc. LPS initiates at least ~,~0 independent pathways: (1) hydrolysis of PIP2, stimulation of PKcmediated protein phosphorylation and expression of mRNA for c-rnyc, c-fos, and JE; (2) the independent expression of other genes, manifested by rapid and transient synthesis of a set of short-lived proteins plus mRNA for KC. Cooperation between these three paths is evident in several ways. First, pretreatment of macrophages with IFN~/ heightens the subsequent phosphorylation of proteins induced by LPS or PMAs8. Second, macrophages pretreated with IFN~/ require -100 fold less LPS to initiate synthesis of the early proteins 80. Third, complex regulatory interactions between these pathways control expression of some genes important for function (see below). Gene regulation in macrophage activation In addition to the transient regulatory changes in gene expression described above, it has been well documented that the protein composition of the macrophage plasma membrane undergoes extensive remodeling, as does the character of secretory products (for reviews, see Refs 1, 2, 82) during macrophage activation in response to LPS, IFN~, or both. Some of these chang,,~ can hp detected by two-dimensional gel electrophoresis83.~. It should be emphasized that chan'~es in secretory products, membrane proteins or other cellular proteins are all complexly regulated during activation: (1) some proteins are increased, while others are decreased; (2) changes in either direction can be initiated by LPS or IFN~/or by LPS plus IFN~/; and (3) IFN~/ and LPS can act in opposing directions. With the cloning of many of these proteins, emphasis is beginning to be placed on the regulation of the genes encoding these proteins. IFN~/inducesat least 10 distinct proteins including class I, class II, and class III histocompatibility molecules8s.86. LPS increases secretion of interleukin-1 (IL-1) ard tumor necrosis factor (TNF)87.88. One major regulatory focus appears to be level of mRNA for the appropriate I~rutein, since this often correlates closely with surface expression and/or secretion. For example, !FN~/induces mRNA for class II histocompatibility molecules (see Ref. 85), while LPS induces mRNA for TNF8s. These two signals, furthermore, interact. IFN~/ enhances the expression of mRNA for TNF initiated by LPS (T.J. Koerner, D.O. Adams and T.A. Hamilton, unpublished). LPS, which suppresses surface expression of la89, also suppresses expression of mRNA for such class II histocompatibility antigens induced by IFN~/(T.J. Koerner, T.A. Hamilton and D.O. Adams, unpublished). These two changes closely correlate with enhanced secretion of TNF or suppressed surface expression of la respectively.

Whether these alterations in mRNA levels are due to altered transcription of the genes, stability of message or both and how regulation of message levels relates to early messenger events remain to be established. Suppressive effects of LPS on expression of IFN-y induced la molecules can be blocked with cycloheximide81 raising the possibility that early gene expression initiated by LPS participates in suppressive effects on the la gene. Conclusionsand perspectives The current data highlight some of the possible mechanisms by which extracellular signals regulate the functional potential of mononuclear phagocytes. Clearly, these preliminary observations are but a prelude to the undoubtedly complex story that will ultimately emerge. It is nevertheless interesting to compare the known signalling events in macrophage development with those already observed in other models of cellular development such as acquisition of competence for proliferation in fibroblasts or in lymphocytes9°-92 or of memory in neurons93. Likewise, studies of the receptor for chemotactic peptides have already become a model for analysing rapid stimulus-response coupling. Analysis of both models has already revealed substantial complexities in the interactions between various transductional events. Elements of the simplified events depicted in models of rapid transduction are already presumed to be interacting (see Fig. 1). Likewise, IFNlf not only induces important functional genes but also regulates other genetic responses induced by LPS. It will thus be of interest to determine whether the patterns of gene regulation described above are general features of cellular development or are uniquely related to the macrophage with respect to its stringent regu!ation and diverse potential for development. ~/~ arP jll~t h p n i n n l n n tn ~dress the fa~i.nati,ng questions posed by these models. Over the coming years details of the various cascadesand perhaps others as yet unidentified will doubtless emerge, as will specific details of how these pathways interact. Topics of particular interest will be how suppressive agents impinge upon these signal mechanisms and how rapid mechanisms, such as those depicted in Fig. 1, are changed during development. We already know that rnany suppressive agents such as a2-macroglobulin-protease complexes or prostaglandins can shut down several macrophage functions and that activated macrophages in some caseshave decreased contents of cyclic AMP-dependent protein kinase (i.e., PKa)94. It already seems reasonable to predict that the diversity of macrophage function and potential for development will ultimately be equalled by the diversity of signal pathways and their interactions. The authors' work describedin this review was supported in pa~ by USPHSGrantsCA 29589, CA 16784, CA 39621 and ES 02922, a grant from the Council for Tobacco ResearchUSA, Inc., and a gift from RJR/Nabisco. References 1 Adams,D.O.and Hamilton,T.A. (1984)Ann. Rev.Immunol. 2, 283 2 Nathan,C.F.and Cohn,Z.A. (1984) in Textbookof Rheumatology(Kelly,W., Harris,E., Ruddy,S.and SledgeR., eds),W.B. Saunders 3 Snyderman,R.. Smith, C.D.and Verghese,M.W. (1986)

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