Monocyte Chemotactic Responsiveness Research Articles

Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine-beta (or C-C) family of cytokines. Murine MCP-1, first identified as the JE gene, differs from human MCP-1 in molecular size and extent of glycosylation. We have used Chinese hamster ovary cells to express recombinant murine MCP-1 and find that its predominant form is a microheterogeneous protein of M(r) approximately 25,000. Most of MCP-1's microheterogeneity is due to variable amounts of sialic acid that are terminally attached to a constant number of O-linked oligosaccharide chains per molecule. This carbohydrate, along with a small amount of N-linked carbohydrate, accounts for 50% of the apparent molecular size of murine MCP-1 and is not required for in vitro monocyte chemoattractant activity. Mutational analysis shows that most of the carbohydrate is added to a 49-amino acid C-terminal domain that is not present in human MCP-1 and is not required for in vitro biologic activity, suggesting that murine MCP-1 consists of an N-terminal domain containing monocyte chemoattractant activity and a heavily glycosylated C-terminal domain of as yet unknown function. MCP-1 produced in COS cells contains a small amount of sulfate, but Chinese hamster ovary-produced MCP-1 does not. The absence of sulfate does not alter MCP-1's in vitro chemoattractant properties. In vitro, highly purified murine MCP-1 attracts monocytes, but not neutrophils, with a specific activity similar to human MCP-1 (EC50 approximately 0.5 nM). Equilibrium binding experiments with human monocytes reveal the presence of approximately 3000 binding sites per cell with a Kd of 0.77 nM. In vivo, injection of up to 1 micrograms murine MCP-1 in a variety of murine strains induces the appearance of a sparse mixed inflammatory infiltrate. The disparity between MCP-1's in vitro and in vivo effects suggests that other factors may be required to elicit a full-blown monocyte chemotactic response to MCP-1 in vivo.

Monocyte chemotactic and phagocytic responses in migraine and tension-type headache patients

Monocyte chemotactic and phagocytic responses were assessed in two groups of migraine patients (with and without aura) and in two groups of tension-type headache patients (episodic and chronic). The chemotactic but not the phagocytic response, assessed interictally, is significantly lower in migraine patients (p < 0.006) and in episodic tension-type headache patients, though not so significantly in the latter (p < 0.05), than in the control individuals. The chemotactic response tends to increase significantly during attack in migraine patients both with and without aura (p < 0.008 and p < 0.007 respectively). The same was evident for the phagocytic response in both migraine patient groups (p < 0.007 and 0.0004). No modifications of monocyte functions were found during attacks neither in episodic nor chronic tension-type headache patients. These findings suggest that one or more mediators of neurogenic inflammation having phagocytic and chemotactic enhancing properties (substance P, prostaglandin E and thromboxane A2 etc.) are implicated in the modification of monocyte function. The demonstration of a defect in monocyte function during the interictal period in migraine patients confirms the results of recent research which evidenced reduced capacity of monocyte to phagocyte and kill microorganisms in the course of migraine.

Production and characterization of mouse monoclonal antibodies against human monocyte chemoattractant protein-1.

We developed five different hybridoma cell lines that produced mAb against human monocyte chemoattractant protein-1 (MCP-1). The subclass of all five antibodies was IgG1. All five mAb formed complexes with metabolically labeled MCP-1 that could be demonstrated by immunoprecipitation. The antibodies were specific for MCP-1. They did not cross-react by immunoprecipitation with structurally related host defense cytokines present in metabolically labeled PHA- or LPS-stimulated mononuclear cell culture fluids, nor did they cross-react in a direct ELISA with neutrophil attractant/activation protein-1, with crude platelet lysate proteins, or with pure platelet proteins that have amino acids sequences similar to that of MCP-1. The mAb also reacted with rMCP-1 expressed in Escherichia coli, suggesting that they recognize protein structure rather than the glycosylated portion of human MCP-1. When the mAb were mixed with MCP-1, the monocyte chemotactic response to MCP-1 was inhibited. A sandwich ELISA was developed to detect MCP-1 in biologic fluids containing relatively high concentrations of other proteins. The sensitivity was 300 pg/ml, or 30 pg/ELISA well. An anti-MCP-1 mAb column was used in an improved method of MCP-1 purification. Approximately 240 micrograms of MCP-1 were purified from 5 liters of FCS-containing U-105MG cell culture supernatant. The yield was at least 60%. In addition to two forms of MCP-1 reported previously by us, two more forms of MCP-1 were found in a mixture of culture supernatants of PHA- and LPS-stimulated human PBMC.

Analysis of the monocyte chemotactic response to lysophosphatidylcholine: role of lysophospholipase C

Previously, we reported that lysophosphatidylcholine (lyso-PtdCho), a component of oxidized low-density lipoprotein, was a monocyte chemoattractant (M.T. Quinn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2805–2809). Monocyte chemotaxis was also stimulated by lyso-platelet activating factor but not by platelet activating factor itself. In the present studies, we used other analogs of lyso-PtdCho to determine structural and metabolic features required for chemotactic activity. Although both d- and l-lyso-PtdCho stimulated chemotaxis, suggesting a lack of stereospecificity, studies using propanediol and ethanediol analogs of lyso-PtdCho suggested that a free hydroxyl moiety or an ester-linked fatty acid vicinal to the phosphocholine group of the lysophospholipid was required for the expression of activity. Incubation of [ 3H]choline-labeled lyso-PtdCho with monocytes resulted in the formation of labeled PtdCho, glycerophosphocholine (GPC), phosphocholine, and free choline, while resident peritoneal macrophages, cells which we show do not respond chemotactically to lyso-PtdCho, metabolized the labeled substrate to generate only labeled PtdCho and GPC; no labeled phosphocholine was found, suggesting a possible role for lysophospholipase C activity in the monocyte chemotactic response. Although monoacylglycerol, the product of lysophospholipase C hydrolysis of lyso-PtdCho, was not chemotactic for monocytes, diacylglycerol demonstrated chemotactic activity, suggesting that the subsequent acylation to diacylglycerol may be involved in the monocyte chemotactic response to lyso-PtdCho. Indeed, monocytes incorporated [ 3H]glycerol from [ 3H]glycerol-labeled lyso-PtdCho into di- and triacylglycerol. Based on these results, a model is proposed whereby the monocyte chemotactic response to lyso-PtdCho involves a sequence of metabolic steps which includes hydrolysis of lyso-PtdCho to monoacylglycerol and phosphocholine by lysophospholipase C followed by acylation of monoacylglycerol to diacylglycerol. Diacylglycerol would then act as an intracellular second messenger that could activate or facilitate the chemotactic response.

Immune dysfunction in multiple myeloma. Reduced natural killer cell activity and increased levels of soluble interleukin‐2 receptors

Multiple myeloma (MM) is characterized by an increased susceptibility to infections and to other malignancies. Selected related immune functions were studied. Spontaneous and interleukin-2-stimulated natural killer (NK) cell activities were normal in 19 patients with MM compared with 62 controls. In contrast, interferon-stimulated NK cells had a significantly lower increase in activity in MM than in controls. The normal improvement in lytic NK cell activity after addition of indomethacin to the mononuclear cell cultures (to inhibit prostaglandin-mediated suppression) was not observed in cultures from MM patients. As reported for other lymphoproliferative disorders, the levels of soluble interleukin-2 receptors in serum were significantly higher in MM (600 U/ml median value) compared with controls (317 U/ml median value), P less than 0.0001, and the concentration of interleukin-2 receptors was significantly correlated with the concentration of monoclonal immunoglobulin in serum. Blood monocyte chemotactic responsiveness was significantly lower in MM patients with both zymosan-activated serum and f-Met-Leu-Phe as cytotaxins, suggesting reduced ability to accumulate at inflammatory foci. In contrast, release of reactive oxygen radicals, believed to be associated with the killing ability of monocytes, was normal after in vitro stimulation.

Immunosuppressive retroviral-related factors in sera of patients with head and neck cancer

The chemotactic responsiveness of mononuclear phagocytes has often been found defective in patients with various malignancies. We have previously reported a defective chemotactic responsiveness in patients with head and neck cancer. Low-molecular-weight factors (LMWFs) have been isolated from tumors and can be held responsible for the inhibitory effect on monocyte chemotactic responsiveness. It is an intriguing new finding that these LMWFs can be neutralized by antibodies reactive to P15E, a structural envelope protein of murine leukemia retroviruses. In this report we describe a relatively easy and rapid method for the detection of immunosuppressive P15E-like factors in the sera of patients with head and neck cancer. The test is based on the monocyte polarization assay. Although only nine head and neck cancer patients were included in this study, the findings indicate that the test might be of value for clinical application. An early detection of a recurrence after treatment might be possible by the finding of a reappearance of the P15E-like factors in patients' sera during follow-up.

Blood monocyte oxidative burst activity in acute P. falciparum malaria

The release of superoxide anion from blood monocytes was studied in eight patients with acute primary attack P. falciparum malaria. Before treatment a significant enhancement of the oxidative burst prevailed, which contrasts with previous findings of a depressed monocyte chemotactic responsiveness. During treatment and after clinical recovery the activity of superoxide anion release normalized in all patients.

Chemotactic Activity of Recombinant Human Granulocyte Colony-Stimulating Factor

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induced migration across polycarbonate filters of human polymorphonuclear leukocytes (PMN). rhG-CSF was active in inducing PMN migration at concentrations greater than or equal to 10 to 100 U/mL (7 to 70 ng/mL). rhG-CSF did not contain appreciable levels of endotoxin contamination as assessed by Limulus amebocyte assay, and Polymixin B did not affect the chemotactic activity of rhG-CSF. A monoclonal anti-G-CSF antibody blocked the induction of migration by G-CSF, thus establishing that the cytokine was responsible for the activity of the recombinant preparation. Checkerboard analysis was performed by seeding different concentrations of G-CSF above and/or below the filter and revealed that the migratory response to this cytokine was best observed in the presence of a positive concentration gradient between the lower and upper compartments of the chamber, thus indicating an actual chemotactic effect. When different migrating cells were examined, rhG-CSF was inactive on large granular lymphocytes and endothelial cells under conditions in which appropriate reference attractants were active. In contrast, rhG-CSF elicited a chemotactic response in monocytes inhibited by specific antibody. Thus, G-CSF is a chemotactic signal for phagocytes. This cytokine, when produced at inflammatory sites, may contribute to the recruitment of phagocytes from the blood compartment to amplify resistance against certain noxious agents.

Chemotactic activity of recombinant human granulocyte colony- stimulating factor

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induced migration across polycarbonate filters of human polymorphonuclear leukocytes (PMN). rhG-CSF was active in inducing PMN migration at concentrations greater than or equal to 10 to 100 U/mL (7 to 70 ng/mL). rhG-CSF did not contain appreciable levels of endotoxin contamination as assessed by Limulus amebocyte assay, and Polymixin B did not affect the chemotactic activity of rhG-CSF. A monoclonal anti-G- CSF antibody blocked the induction of migration by G-CSF, thus establishing that the cytokine was responsible for the activity of the recombinant preparation. Checkerboard analysis was performed by seeding different concentrations of G-CSF above and/or below the filter and revealed that the migratory response to this cytokine was best observed in the presence of a positive concentration gradient between the lower and upper compartments of the chamber, thus indicating an actual chemotactic effect. When different migrating cells were examined, rhG- CSF was inactive on large granular lymphocytes and endothelial cells under conditions in which appropriate reference attractants were active. In contrast, rhG-CSF elicited a chemotactic response in monocytes inhibited by specific antibody. Thus, G-CSF is a chemotactic signal for phagocytes. This cytokine, when produced at inflammatory sites, may contribute to the recruitment of phagocytes from the blood compartment to amplify resistance against certain noxious agents.

Phenotypic and functional abnormalities in monocytes from patients with haemophilia A treated with factor VIII concentrates.

The phenotype and functions of monocytes in patients with haemophilia A and age-matched controls were studied. Fourteen male haemophiliacs were classified in three categories according to the mean number of units of factor VIII received during the last 5 years. Eleven patients were positive for antibodies to human immunodeficiency virus but none of our patients were homosexuals or drug abusers, nor do they fulfill the criteria of acquired immunodeficiency syndrome. Patients treated with high amounts of factor VIII concentrates (greater than 3 x 10(5) U/year) showed a significantly lower percentage of monocytes expressing HLA-DR, LFA-1 and CR3 antigens as compared with patients receiving lower amounts of factor VIII (less than 2 x 10(6) U/year) or controls. Kinetics of DR, LFA-1 and CR3 in cultured monocytes showed tht they were lost faster by monocytes from haemophiliacs treated with large amounts of factor VIII than by control monocytes. Adherence ability and chemotactic response of monocytes from patients treated with less than 3 x 10(5) U/year of factor VIII were also impaired. Although phagocytic indices were in normal ranges in haemophiliacs, a significant difference was observed between percentages of phagocytic monocytes from haemophiliacs treated with the largest doses of factor VIII and normal controls. Tests for respiratory burst activity, measured by chemiluminescence and superoxide anion generation, and Staphylococcus aureus killing were in normal ranges in haemophiliacs' monocytes.

Defective monocyte chemotactic responsiveness in patients with active tuberculosis

Monocyte migration was studied in 15 patients with newly diagnosed tuberculosis and 17 healthy control subjects matched for age, sex and ethnic group, using a raft modification of the Boyden chamber. Compared with matched controls, patients with tuberculosis exhibited impaired in vitro monocyte random locomotion and directional locomotion (chemotaxis) in response to 2% casein. However, no correlation was found between in vitro monocyte migration and monocyte count, radiological extent of disease or in vitro dermal reactivity to tuberculin-PPD. The observed defects were corrected during the course of chemotherapy.

Suppression of blood monocyte and neutrophil chemotaxis in acute human malaria.

The host response to Plasmodia includes the production of enlarged populations of peripheral blood monocytes and tissue macrophages in the spleen and the liver. Since the hyperplasia of the mononuclear phagocyte system is believed to arise as a consequence of an enhanced blood monocyte influx, we tested monocyte chemotactic responsiveness in 19 patients with acute primary attack malaria. In addition, the neutrophil chemotaxis was measured in 12 patients. Before the initiation of antimalarial treatment a significant depression of monocyte chemotaxis was observed in approximately half of the patients when compared with healthy control subjects. The depression was found in Plasmodium falciparum malaria as well as in P. vivax or P. ovale malaria patients. The defective responsiveness was not receptor specific, since the responses towards casein and zymosan activated serum proved to be equally suppressed. The monocyte chemotaxis was followed in 14 of the patients, during treatment and after complete recovery. After 3 days of treatment the response had improved in most of the patients, and after 7 days all patients had a normal monocyte chemotaxis, which remained normal after one month. No significant differences between P. falciparum and P. vivax/ovale malaria was observed with respect to blood monocyte chemotactic responsiveness. Neutrophil chemotaxis in patients with P. falciparum infections was similarly suppressed before treatment (54% of controls), was still defective after 3 days of treatment, and nearly normalized after 7 days (87% of controls). Furthermore, monocyte phagocytic and candidacidal activities were assessed in the same patients on admission and during the follow-up. In contrast to chemotaxis, these functions were normal in all of the patients whenever measured. In conclusion, not all cell functions were altered in concert, and the previously unreported suppression of chemotactic migration might reflect a change in blood leucocyte subpopulations, deactivation in vivo or a direct suppressive effect of plasmodia induced products.

Immunosuppressive retroviral P15E-related factors in head and neck carcinomas.

We report on the effect of low molecular weight factors (LMWFs) derived from 14 different head and neck carcinomas on the chemotactic responsiveness of healthy donor monocytes, as measured by their polarization activity. The factors inhibited the polarization of the monocytes significantly (61.5% to 94.5% vs 12.5% to 29% in cases where the LMWF was derived from healthy oral mucosa). The inhibitory effect exerted by these LMWFs could be neutralized only by absorption with one of three different murine monoclonal antibodies or a rabbit polyclonal antibody to the murine retroviral envelope protein P15E. This shows that retroviral-related material is present in head and neck carcinomas and that this material is responsible for the observed defective polarization that probably underlies the earlier described defects on monocyte chemotactic responsiveness.

Enhancement of defective monocyte function during immunotherapy with recombinant interferon.

The influence of immunotherapy with high dose (50 X 10(6) units/m2) recombinant leukocyte A interferon on blood monocyte functions was studied in eight patients with bronchogenic carcinoma. Monocyte chemotactic responsiveness (MCR) was initially depressed (9.8 +/- 1.6 cells/field) compared to healthy controls (17.6 +/- 5.1 cells/field), P less than 0.01. Recombinant interferon was administered three times weekly, and after 7 days a significant improvement in chemotaxis was observed (16.6 +/- 3.0 cells/field), P less than 0.05. The MCR remained normal until cessation of interferon therapy (greater than 1 month). Phagocytic and candidacidal activities were normal in the patients and were not influenced by treatment with interferon. In conclusion, high dose recombinant interferon given to cancer patients caused a normalization of defective blood monocyte chemotaxis, which persisted for greater than 1 month.

Effects of tumor growth on host defenses.

Monocytes and macrophages play an important role in host defense against neoplasia. Studies from our and other laboratories have demonstrated that patients with a variety of cancers have a defect in monocyte chemotactic responses. Tumor-bearing mice are also inhibited in their ability to accumulate macrophages to inflammatory foci. We have shown that extracts prepared from murine tumors, as well as the plasma and urine of tumor-bearing mice, contain anti-inflammatory proteins which are antigenically and physicochemically related to the immunosuppressive retroviral envelope protein p15E. Similarly, proteins capable of inhibiting monocyte chemotactic responses are present in human cancerous effusions and can be specifically absorbed by monoclonal antibodies to p15E. Furthermore, we have demonstrated that human malignant and mitogen-transformed cells contain p15E-related antigens. These findings led us to propose a two stage model of tumorigenesis: the first stage involves neoplastic transformation of a cell while the second stage involves activation of a gene coding for a p15E-like protein which allows the transformed cell to escape immune surveillance and go on to become a tumor. Support for this model has come from recent studies which have identified within the human genome an endogenous retrovirus sequence whose envelope gene is partially homologous to a highly conserved region of p15E. Using a synthetic peptide, termed CKS-17, we have shown that this region may be responsible for many of the biological activities of p15E and is capable of suppressing lymphocyte and natural killer cell immune functions as well as those of monocytes and macrophages. Thus tumors may be capable of evading host defense mechanisms by activation of a normal gene related to the immunosuppressive retroviral protein p15E.

Relationship between immune system and gram-negative bacteria: Monocyte chemotaxis induced by supernatants from human peripheral blood OKT8 + lymphocytes stimulated with smooth and rough Salmonella strains

It has been recently reported that smooth (S) Salmonella typhimurium LT-2 and rough (R) mutants, S. minnesota R345 (Rb) and R595 (Re), spontaneously adhere to human peripheral blood mononuclear cells (PBMC). The binding is confined to T cells and is mediated by the lipopolysaccharide (LPS) moiety of the bacteria outer membrane. In this study, we have investigated the monocyte chemotactic responsiveness induced by supernatants recovered from human PBMC stimulated with either S or R Salmonella strains. All supernatants were able to trigger monocyte chemotaxis, even if to a different degree according to the bacterial strain used. These data were further supported by experiments which showed that stimulation of PBMC by purified homologous LPS led to the release of a chemotactic lymphokine (CLK) for human monocytes. Moreover, this CLK seems to be released by T cells, and in particular OKT8 + cells, since OKT8 − PBMC failed to produce CLK.

Mononuclear phagocyte function in head and neck cancer. Chemotactic responsiveness of blood monocytes in correlation between histologic grade of the tumor and infiltration of these cells into the tumor area.

The chemotactic responsiveness of peripheral monocytes and the acid-phosphatase activity of tumor-infiltrating macrophages, as well as the ultrastructural appearance, were studied in 40 patients with squamous cell carcinoma of the head and neck. The chemotactic responsiveness was found to be decreased in carcinoma patients, and this value appeared to be positively correlated in individual patients with the number of tumor-infiltrating macrophages, as well as with the histologic grade of the tumor. Patients with poorly differentiated malignancies showed impaired monocyte chemotactic responsiveness and low numbers of tumor-infiltrating macrophages. Macrophages present in the parenchyma of the tumor showed a weak and diffuse pattern of acid phosphatase activity. The acid phosphatase activity of stromal macrophages was much stronger and distributed in foci. Electron microscopic examination of the parenchymal macrophages revealed low numbers of lysosomes and the presence of tumor cell debris in the cytoplasm of the cell, without any sign of a surrounding phagosomal membrane. Together with the weak cytochemical reactivity, this probably indicates the poor functional state of the phagocyte when infiltrated in the parenchyma of the tumor. Low molecular weight factors derived from the tumor are known to decrease chemotactic responsiveness of peripheral monocytes. The poor functional state of the macrophages infiltrated within tumor parenchyma might be explained by assuming that a high concentration of such factors in the near vicinity of malignant cells causes toxic effects in macrophages.

Potentiation of the defective monocyte chemotaxis in Hodgkin's disease by in vitro tuftsin treatment.

In vitro pre-incubation of monocytes derived from patients with Hodgkin's disease with tuftsin (50 micrograms/ml) significantly restored the deficient chemotactic responsiveness of these cells to the complement-derived factor C5a, as demonstrated by a monocyte migration assay based on the Boyden technique. Potentiation of the chemotactic responsiveness of monocytes was most significant after elective splenectomy. The results indicate that the specific receptors required for tuftsin activities may be available on the monocyte membranes in Hodgkin's disease. Since tuftsin is a natural, non-immunogenic tetrapeptide that can also be produced synthetically, it may provide a new therapeutic approach in Hodgkin's disease to at least partial restoration of the defective cellular immunity.

Heterogeneity of human neutrophil and monocyte chemotactic responsiveness.

Heterogeneity of human neutrophil and monocyte chemotactic responsiveness.

Immune functions in methylmalonicaciduria.

A variety of phagocytic cell and lymphocyte assays were employed to evaluate the immune status of four patients with methylmalonicaciduria . One patient had a depressed absolute granulocyte count and two patients had depressed neutrophil and monocyte chemotactic responses. All subjects had normal neutrophil phagocytic and bactericidal activities. One patient had a decreased T-cell number; blastogenic responses to phytohaemagglutinin and pokeweed mitogen were normal in all subjects. B lymphocyte measurements were variably abnormal; two children had decreased B-cell numbers; two had marginally decreased IgG levels; a third had an undetectable rubella titre; and two had elevated serum IgE concentrations. In vitro exposure of normal cells to methylmalonic acid concentrations up to 50 mg/100 ml did not affect chemotactic or lymphoproliferative responses. In conclusion, although B-cell function may be affected, no consistent abnormality of lymphocyte or phagocytic cell functions could be attributed to the metabolic disorder.