Chemoattractant-induced activation of c-fos gene expression in human monocytes.

Human monocytes use the products of phosphoinositide hydrolysis (1,2-diacylglycerol and inositol 1,4,5-triphosphate) as second messengers to trigger rapid cellular activation during the occupancy of chemoattractant receptors. The effect of chemoattractants on modulation of gene expression in monocytes was examined in this study. The chemoattractants FMLP and platelet-activating factor induced the progressive increase of c-fos RNA to 6-15-fold over those of control within 30 min after treatment. Similar kinetics of c-fos gene activation was also observed when cells were treated with PMA or sn-1,2-dioctanoylglycerol, but not with the calcium mobilizer ionomycin, suggesting a role for protein kinase C in gene regulation by chemoattractant receptors. Activation of c-fos gene expression by FMLP is mediated through a pertussis toxin-sensitive G protein, since pertussis toxin treatment of the cells blocked the induction of the c-fos gene by FMLP but not PMA. The level of c-myc RNA was slightly decreased after 1 h of treatment with chemoattractants, but not with PMA or diacylglycerol. This implies that chemoattractant receptor occupancy generates signals beyond protein kinase C activation that are capable of selectively downregulating monocyte gene expression. The effect of FMLP and PMA on the accumulation of c-fos RNA appears to result from altering both the rate of transcription and message stability. These observations indicate that signals generated through chemoattractant receptor occupancy may regulate monocyte function at the genetic level.

Interactions of the complement system with endotoxic lipopolysaccharide. Generation of a factor chemotactic for polymorphonuclear leukocytes.

Endotoxic lipopolysaccharide has recently been shown to fix large amounts of the complement components related to the biologic activities mediated by that system. The present study sought to determine whether the generation of chemotactic factor by endotoxin in serum was dependent upon complement system activation. Preheating serum, incubating at 0 degrees C, or incubating in the presence of EDTA, all prevented chemotactic factor generation as well as complement fixation by endotoxin. "Endotoxoids" deficient in complement-firing activity were also deficient in chemotactic factor generation. Chemotactic factor could not be generated by endotoxin in sera of mice congenitally deficient in the C'S component of complement, while chemotactic factor was generated by endotoxin in the sera of coisogenic mice with normal complement levels for that species. The chemotactic factor induced by endotoxin was heat stable and nondialyzable. Molecular sieve chromatography and sucrose density gradient ultracentrifugation demonstrated that the chemotactic factor was a relatively low molecular weight product (15,000-30,000) and as such different from previously scribed C' system-derived chemotactic factors. These experiments demonstrate that generation of chemotactic factor by endotoxin in serum is dependent upon C' system activation involving at least C'5. Furthermore, the relatively low molecular weight of this factor suggests that it might be derived from activation of a single complement component rather than from complexing of multiple complement components.

The chemotactic attraction of human fibroblasts to a lymphocyte-derived factor.

A quantitative assay that measures fibroblast chemotaxis in vitro is described. Application of this technique has revealed that peripheral blood lymphocytes stimulated by antigen or mitogen in vitro produce a factor that is chemotactic for human dermal fibroblasts. This lymphocyte-derived chemotactic factor for fibroblasts (LDCF-F) is different from the lymphokine that is chemotactic for monocytes or macrophages. Macrophages are required for the generation of LDCF-F by T lymphocytes stimulated by phytohemagglutinin. The fibroblast chemotactic factor is heat stable (56 degrees C for 30 min), trypsin sensitive, and neuraminidase resistant. LDCF-F could function to attact connective tissue fibroblasts to sites at which cell-mediated immune reactions are occurring in vivo.

Human malignant and mitogen-transformed cells contain retroviral P15E-related antigen.

Virus-related oncogenes have been demonstrated in human tumor cells and may play a role in neoplastic transformation. Cancerous effusions contain inhibitors of monocyte function and are absorbed by monoclonal antibodies to the immunosuppressive retroviral structural protein, P15E. We therefore examined eight human malignant cell lines for P15E-related antigens, by indirect immunofluorescence. Up to 87% of fixed malignant cells were reactive with two different monoclonal anti-P15E antibodies, while under identical conditions approximately 7% of freshly isolated human mononuclear cells were positive. Differentiation of two tumor cell lines with dibutyryl cyclic AMP resulted in decreased anti-P15E reactivity. Blast transformation of human mononuclear cells with mitogens induced reactivity with anti-P15E. Thus human malignant and blast-transformed cells contain antigens related to P15E. Expression of this viral-related gene may occur during rapid cell division and be abnormally regulated in cancer cells, thus rendering them more resistant to immune destruction.

Biological activity of complement in vivo. Role of C5 in the accumulation of polymorphonuclear leukocytes in inflammatory exudates.

The importance of C5 in the generation of complement (C)-dependent chemotactic activity in vitro is well recognized. However, the actual role C5 may play in the accumulation of polymorphonuclear leukocytes (PMN) at inflammatory sites in vivo has not been established. Injection of glycogen or endotoxin into the peritoneal cavities of guinea pigs resulted, shortly thereafter, in the local accumulation of PMN. Preceding the influx of leukocytes, the peritoneal fluid became chemotactic for rabbit PMN in vitro. The majority of this activity could be attributed to a cleavage product of C5 (C5a). Similarly, injection of endotoxin into the peritoneal cavity of C5-normal mice resulted in the generation of a chemotactic factor for mouse PMN which was followed by the accumulation of PMN in the peritoneal fluid. In contrast, injection of endotoxin into the peritoneal cavity of C5-deficient mice resulted in the generation of virtually no detectable chemotactic activity and a markedly depressed accumulation of PMN during the first 24 hr after injection. The data suggest that C5 plays an important role in the early phases of PMN accumulation in response to inflammatory stimuli. The rapid accumulation of PMN in response to an inflammatory stimulus such as bacterial endotoxin would be expected to be a major factor in host defense against proliferation and dissemination of infectious agents.

Murine malignant cells synthesize a 19,000-dalton protein that is physicochemically and antigenically related to the immunosuppressive retroviral protein, P15E.

Murine tumors contain low molecular weight factors that inhibit macrophage accumulation at inflammatory foci. Certain oncogenic murine leukemia viruses contain similar inhibitory activity and the active component of the retroviruses was shown to be the envelope protein P15E. A number of murine malignant and nonmalignant cell lines, as well as primary tumors, have now been examined to determine whether production of retroviral P15E or a related protein is characteristic of neoplastic cells. Tumor lines examined included the Hep 129 hepatocarcinoma, BP8 fibrosarcoma, RL1 lymphoma, and three variants of the B16 melanoma. Tumor lines were virus negative by electron microscopy. Nonmalignant cells examined included ST0, 3T3/BALB, and 3T3/L1 fibroblasts and unstimulated, as well as mitogen-stimulated murine splenocytes. Cells were pulse-labeled with [35S]methionine, proteins immunoprecipitated with two monoclonal antibodies to P15E and analyzed by SDS-PAGE and gel fluorography. All tumor lines synthesized a approximately 19,000-dalton protein that co-migrated with retroviral P15E on SDS-PAGE. None of the nonmalignant cells synthesized this protein. Two-dimensional gel electrophoresis of the proteins precipitated from two B16 melanoma lines by monoclonal anti-P15E showed them to be physicochemically similar to P15E from Rauscher leukemia virus. A competition ELISA assay for P15E was developed and confirmed the results obtained by metabolic labeling and demonstrated P15E-related antigens in the tumor cell lines and also in the ascites fluid of mice injected with Hep 129 cells. More importantly, P15E antigens were expressed in both a spontaneous mammary adenocarcinoma and in a primary methylcholanthrene-induced fibrosarcoma. Nonmalignant tissues from animals bearing these tumors contained no detectable P15E antigen. Extracts from the primary fibrosarcomas, when injected into the thighs of mice, inhibited the intraperitoneal accumulation of inflammatory macrophages. The inhibitory activity was specifically removed by absorption with monoclonal antibody to P15E. These results suggest that synthesis of the immunosuppressive retroviral protein P15E, or a very similar protein, routinely occurs during the growth of murine neoplastic cells. This P15E-related protein is present in spontaneous murine primary tumors as well as in all murine tumor cell lines tested. The expression of such proteins by transformed cells in vivo could confer a selective advantage for their sustained growth since they would be more likely to escape immune surveillance.

Development of specific receptors for N-formylated chemotactic peptides in a human monocyte cell line stimulated with lymphokines.

A human monocyte-like cell line, U937, when grown in continuous culture, does not secrete lysosomal enzymes or migrate towards chemotactic factors. When the cells are stimulated by lymphokines, however, they develop the ability both to migrate directionally and to secrete enzymes in response to several types of chemoattractants. The development, by stimulated cells, of chemotactic and secretory responses to one class of chemoattractants, the N- formylated peptides, is accompanied by the appearance on the cells of specific binding sites for these substances. Using tritiated N-formyl- methionyl-leueyl-phenylalanine (fMet-Leu-[(3)H]Phe) as a ligand, it was determined that unstimulated U937 cells possess no detectable binding sites. However, after stimulation with lymphocyte culture supernates for 24, 48, and 72 h, they developed 4,505 (+/-) 1,138, 22,150(+/-) 4,030, and 37,200 (+/-) 8,000 sites/cell, respectively. The dissociation constants for the interaction of fMet-Leu-[SH]Phe with the binding sites were approximately the same regardless of stimulation time and ranged between 15 and 30 nM. The binding of fMet-Leu-[(3)H]Phe by stimulated U937 cells was rapid and readily reversed by the addition of a large excess of unlabeled peptide. The affinity of a series of N-formylated peptides for binding to U937 cells exactly reflected the potency of the peptides in inducing lysosomal enzyme secretion and chemotaxis. The availability of a continuous human monocytic cell line that can be induced to express receptors for N-formylated peptides will provide a useful tool not only for the characterization of such receptors but also for the delineation of regulatory mechanisms involved in cellular differentiation and the chemotactic response.

Resistance of neoplasms to immunological destruction: role of a macrophage chemotaxis inhibitor.

Several tissue culture lines of 6C3HED, a murine lymphoma, were more susceptible to immunologic destruction in vivo than the highly virulent 6C3HED line maintained by serial intramuscular transplantation. The attenuated tissue culture cells were rejected by normal syngeneic recipients, but thymectomized mice were unable to reject attenuated cells. In such mice, the growth rate of attenuated cells was equivalent to the growth rate of virulent cells in normal syngeneic mice. The increased susceptibility of attenuated cells to destruction by syngeneic hosts was shown to correlate with decreased production by the tumor cells of a macrophage chemotaxis inhibitor, and not with altered antigen density. In addition, when inhibitor isolated from virulent cells was administered to mice challenged with attenuated cells, the latter cells became virulent in vivo. When attenuated and virulent cells were administered simultaneously in the same host, the attenuated cells were able to develop into progressively growing tumors. The data suggest that the successful growth of neoplastic cells in normal may require tumor cells to produce factors which subvert the ability of the host to mobilize macrophages rapidly at the tumor site.

Targeted disruption of the leukotriene B(4) receptor in mice reveals its role in inflammation and platelet-activating factor-induced anaphylaxis.

Leukotrienes are derived from arachidonic acid and serve as mediators of inflammation and immediate hypersensitivity. Leukotriene B(4) (LTB(4)) and leukotriene C(4) (LTC(4)) act through G protein-coupled receptors LTB(4) receptor (BLTR) and Cys-LTR, respectively. To investigate the physiological role of BLTR, we produced mice with a targeted disruption of the BLTR gene. Mice deficient for BLTR (BLTR(-/-)) developed normally and had no apparent hematopoietic abnormalities. Peritoneal neutrophils from BLTR(-/-) mice displayed normal responses to the inflammatory mediators C5a and platelet-activating factor (PAF) but did not respond to LTB(4) for calcium mobilization or chemotaxis. Additionally, LTB(4) elicited peritoneal neutrophil influx in control but not in BLTR(-/-) mice. Thus, BLTR is the sole receptor for LTB(4)-induced inflammation in mice. Neutrophil influx in a peritonitis model and acute ear inflammation in response to arachidonic acid was significantly reduced in BLTR(-/-) mice. In mice, intravenous administration of PAF induces immediate lethal anaphylaxis. Surprisingly, female BLTR(-/-) mice displayed selective survival (6 of 9; P = 0.002) relative to male (1 of 11) mice of PAF-induced anaphylaxis. These results demonstrate the role of BLTR in leukotriene-mediated acute inflammation and an unexpected sex-related involvement in PAF-induced anaphylaxis.

Interactions of the complement system with endotoxic lipopolysaccharides in immunoglobulin-deficient sera.

Bacterial lipopolysaccharides (LPS) derived from a variety of organisms effectively induced C consumption in humans, bovines, and porcines with developmental agammaglobulinemia; birds with experimental agammaglobulinemia; and humans with agammaglobulinemia syndromes. This interaction proceeded even in precolostral piglet sera which contained less than 2.5 x 10(-6) mg/ml gamma globulin, and led to generation of neutrophil chemotactic factor and anaphylatoxin in these sera. Hence, the LPS-C interaction can proceed in sera markedly deficient in immunoglobulin. The question of whether immunoglobulins can be bypassed in the LPS-C interaction, or whether they are regularly utilized in a way so efficient that their participation is masked, was considered.

Opiates transdeactivate chemokine receptors: delta and mu opiate receptor-mediated heterologous desensitization.

An intact chemotactic response is vital for leukocyte trafficking and host defense. Opiates are known to exert a number of immunomodulating effects in vitro and in vivo, and we sought to determine whether they were capable of inhibiting chemokine-induced directional migration of human leukocytes, and if so, to ascertain the mechanism involved. The endogenous opioid met-enkephalin induced monocyte chemotaxis in a pertussis toxin-sensitive manner. Met-enkephalin, as well as morphine, inhibited IL-8-induced chemotaxis of human neutrophils and macrophage inflammatory protein (MIP)-1alpha, regulated upon activation, normal T expressed and secreted (RANTES), and monocyte chemoattractant protein 1, but not MIP-1beta-induced chemotaxis of human monocytes. This inhibition of chemotaxis was mediated by delta and micro but not kappa G protein-coupled opiate receptors. Calcium flux induced by chemokines was unaffected by met-enkephalin pretreatment. Unlike other opiate-induced changes in leukocyte function, the inhibition of chemotaxis was not mediated by nitric oxide. Opiates induced phosphorylation of the chemokine receptors CXCR1 and CXCR2, but neither induced internalization of chemokine receptors nor perturbed chemokine binding. Thus, inhibition of chemokine-induced chemotaxis by opiates is due to heterologous desensitization through phosphorylation of chemokine receptors. This may contribute to the defects in host defense seen with opiate abuse and has important implications for immunomodulation induced by several endogenous neuropeptides which act through G protein-coupled receptors.

Alterations of new methylated phospholipid synthesis in the plasma membranes of macrophages exposed to chemoattractants.

Chemotactic factors have been shown to inhibit the methylation of phosphatidylethanolamine in macrophages without affecting total phospholipid synthesis. It would thus be anticipated that newly synthesized membranes of macrophages exposed to chemoattractants would have an increased ratio of phosphatidylethanolamine to its methylated derivatives. These ratios were measured directly in newly synthesized phospholipids of plasma membranes isolated from guinea pig peritoneal macrophages. The phosphatidylethanolamine: methylated phospholipid ratio in such plasma membranes was increased by 53 to 111% upon exposure of the cells to chemotactic factors. This increase was due to decreased synthesis of methylated phospholipids and not to altered formation of phosphatidylethanolamine or activation of phospholipases. Methylated phospholipid ratios were also studied in the leading front lamellipodia isolated from macrophages migrating under chemotactic and nonchemotactic conditions. The phosphatidylethanolamine:methylated phospholipid ratios were increased up to fourfold in lamellipodia of macrophages migrating towards chemotactic agents when compared to those from cells migrating randomly. Biophysical changes in the plasma membrane produced by an increase in the ratio of phosphatidylethanolamine:methylated phospholipids as a result of exposure of cells to chemoattractants may be required for sustained directed migration.

A chemoattractant receptor on macrophages exists in two affinity states regulated by guanine nucleotides.

The binding characteristics of the oligopeptide chemoattractant receptor on guinea pig macrophages and macrophage membrane preparations were characterized using detailed binding studies and computer analysis. Viable macrophages bound the radiolabeled chemoattractant N-formyl-methionyl-leucyl-[3H]phenylalanine with single dissociation constant (KD) of 18.4 +/- 4.6 nM with 15,300 +/- 1,800 sites per cell. Binding data from membrane preparations indicated the presence of two classes of binding sites with KD of 1.5 +/- 0.4 nM and 25.5 +/- 11.0 nM. Approximately 23% of the receptors were in the high affinity state. In the presence of added guanine nucleotide di- or triphosphates, the high affinity receptors in the membrane preparations were converted to low affinity states with no change in the total receptor number. Nonhydrolyzable derivatives of GTP were most potent in converting the receptor from its high to low affinity state. These data suggest that the affinity state of the oligopeptide chemoattractant receptor in macrophages is regulated by guanine nucleotides and GTPase, implying that the transduction mechanisms of this receptor may be controlled by a guanine nucleotide regulatory unit.

N-Formylmethionyl peptide receptors on equine leukocytes initiate secretion but not chemotaxis.

The chemotaxis of leukocytes appears to be initiated by the binding of chemotactic factors to the surface of these cells. N-Formylated peptides induce chemotaxis and lysosomal enzyme secretion of leukocytes; because these peptides are available in a purified radiolabeled form, they have been useful in the characterization of receptors for chemotactic factors. Equine polymorphonuclear leukocytes secrete lysosomal enzymes but do not exhibit chemotaxis in respone to the N-formylated peptides, even though they have a high-affinity cell surface receptor for these agents. The specificity of the equine receptor resembles the specificity of the receptor on chemotactically responsive leukocytes from other species. Equine polymorphonuclear leukocytes may thus be an excellent model for the study of the events that lead to a biological response following receptor occupancy.

Molecular and cellular mechanisms of leukocyte chemotaxis.

The application of modern scientific methods to the study of leukocyte function has begun to reveal the molecular and cytostructural bases of the chemotactic responses of these cells. Leukocyte chemotaxis is initiated by the binding of chemoattractants to distinct plasma membrane receptors; this finding alters transmembrane potential and activates ionic fluxes. The subsequent sequence of metabolic processes leads to a rearrangement of cytoskeletal elements that is manifested by orientation and migration of the cells toward the source of the chemotactic gradient.

Calcium influx requirement for human neutrophil chemotaxis: inhibition by lanthanum chloride.

Calcium fluxes of human neutrophils measured in the presence of chemotactically active serum showed a marked stimulation of calcium-45 uptake from the media. Chemotactically inactive serum did not cause an influx of calcium. The magnitude of the calcium influx due to activated serum is sufficient to trigger contractile systems previously described in muscle cells. Lanthanum chloride inhibited the chemotactic response of human neutrophils to activated serum. Lanthanum in concentrations that suppressed chemotaxis also inhibited the calcium influx caused by activated serum. Lanthanum in concentrations that suppressed chemotaxis also inhibited the calcium influx caused by activated serum. The data support a direct role of calcium influx in chemotaxis of neutrophils.

An inhibitor of macrophage chemotaxis produced by neoplasms.

The accumulation of macrophages at neoplastic sites may be an important event in immunologically mediated tumor killing. The implantation of syngeneic neoplasms in mice, however, was found to depress the animal's ability to localize macrophages at inflammatory sites. A low-molecular-weight (6,000 to 10,000) factor released by growing neoplasms that inhibits the accumulation of macrophages in vivo and chemotactic responsiveness in vitro was identified. The factor is active in the inhibition of macrophages and is ineffectual at retarding the migration of polymorphonuclear leukocytes. Neoplastic cells may thus abrogate immunosurveillance by releasing products that prevent potentially tumoricidal macrophages from accumulating at sites of developing malignancies.

Receptor-coupled activation of phosphoinositide-specific phospholipase C by an N protein.

Cleavage of phosphatidylinositol 4,5-bisphosphate by phospholipase C results in the production of two important second messengers: inositol-1,4,5-trisphosphate and 1,2-diacylglycerol. Although several receptors promote this cleavage, the molecular details of phospholipase C activation have remained unresolved. In this study, occupancy of a Ca2+-mobilizing receptor, the oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte plasma membranes, was found to lead to the activation of a guanine nucleotide regulatory (N) protein by guanosine 5'-triphosphate. The activated N protein then stimulated a polyphosphoinositide-specific phospholipase C by reducing the Ca2+ requirement for expression of this activity from superphysiological to normal intracellular concentrations. Therefore, the N protein-mediated activation of phospholipase C may be a key step in the pathway of cellular activation by chemoattractants and certain other hormones.

A potential second messenger role for unsaturated fatty acids: activation of Ca2+-dependent protein kinase.

Arachidonate and other unsaturated long-chain fatty acids were found to activate protein kinase C from human neutrophils. Kinase activation by arachidonate required calcium and was enhanced by diolein but did not require exogenous phosphatidylserine. Submaximal levels of arachidonate also enhanced the affinity of the kinase for calcium during activation by phosphatidylserine. Thus the release of arachidonate, which is triggered in many cell types by ligand-receptor interactions, could play a second messenger role in the regulation of cellular function by activation of protein kinase C.

Bone marrow-derived lymphoid cells (B cells): functional depletion with cobra factor and fresh serum.

Treatment of rat spleen cells with cobra factor and fresh rat serum provided a simple, rapid means of functionally eliminating complement receptor lymphocytes. Cells able to differentiate into plaque-forming cells in a syngeneic, irradiated host were diminished, but cells able to induce a graft-versus-host reaction were not diminished. There was no effect on plaque-forming cells from an immune spleen.