Monocyte-derived Macrophage Cultures Research Articles

Viral phenotype and host-cell susceptibility to HIV-1 infection as risk factors for mother-to-child HIV-1 transmission

To investigate the role of maternal HIV-1 isolate phenotype and a child's cell susceptibility/resistance to viral infection in mother-to-child HIV-1 transmission. Forty-nine women were studied at the time of delivery. Primary isolates, obtained by culturing patient peripheral blood mononuclear cells (PBMC) with PBMC from healthy donors, were characterized for tropism and syncytium-inducing capability in monocyte-derived macrophages (MDM), peripheral blood lymphocytes (PBL), and in the MT-2 and MOLT-3 T-cell lines. Seven women transmitted HIV-1 to their children. Primary isolates were obtained from six and 28 transmitting and non-transmitting mothers, respectively. All primary isolates from transmitting mothers and their infants but only 50% of those from non-transmitting mothers replicated in MDM, regardless of their replication capacity in T-cell lines. PBL and MDM cells from six uninfected children were exposed to the corresponding maternal isolates. Polymerase chain reaction analysis of HIV-1 DNA in cells and p24 antigen assay in culture supernatants disclosed that two PBL and five MDM cultures were resistant to viral infection; two other PBL cultures, although HIV-1-infected, were negative for p24 production. Depletion of CD8+ cells only partially restored productive infection in CD4+ cell cultures. Moreover, all six PBL but only one MDM cultures were productively infected by an isolate obtained from a transmitting mother, thus suggesting that MDM resistance to HIV-1 infection is not viral isolate-restricted. Our findings strongly suggest that mother-to-child HIV-1 transmission is influenced by both monocyte-macrophage tropism of the maternal isolate and susceptibility of the child's target cells, in particular monocyte-macrophages, to HIV-1 infection.

Viral phenotype and host-cell susceptibility to HIV-1 infection as risk factors for mother-to-child HIV-1 transmission

Objective: To investigate the role of maternal HIV-1 isolate phenotype and a child's cell susceptibility/resistance to viral infection in mother-to-child HIV-1 transmission. Patients and methods: Forty-nine women were studied at the time of delivery. Primary isolates, obtained by culturing patient peripheral blood mononuclear cells (PBMC) with PBMC from healthy donors, were characterized for tropism and syncytium-inducing capability in monocyte-derived macrophages (MDM), peripheral blood lymphocytes (PBL), and in the MT-2 and MOLT-3 T-cell lines. Results: Seven women transmitted HIV-1 to their children. Primary isolates were obtained from six and 28 transmitting and non-transmitting mothers, respectively. All primary isolates from transmitting mothers and their infants but only 50% of those from non-transmitting mothers replicated in MDM, regardless of their replication capacity in T-cell lines. PBL and MDM cells from six uninfected children were exposed to the corresponding maternal isolates. Polymerase chain reaction analysis of HIV-1 DNA in cells and p24 antigen assay in culture supernatants disclosed that two PBL and five MDM cultures were resistant to viral infection; two other PBL cultures, although HIV-1-infected, were negative for p24 production. Depletion of CD8+ cells only partially restored productive infection in CD4+ cell cultures. Moreover, all six PBL but only one MDM cultures were productively infected by an isolate obtained from a transmitting mother, thus suggesting that MDM resistance to HIV-1 infection is not viral isolate-restricted. Conclusions: Our findings strongly suggest that mother-to-child HIV-1 transmission is influenced by both monocyte-macrophage tropism of the maternal isolate and susceptibility of the child's target cells, in particular monocyte-macrophages, to HIV-1 infection. AIDS 1995, 9:427-434

Cystamine inhibits HIV type 1 replication in cells of monocyte/macrophage and T cell lineages.

The effects of cysteamine (2-aminoethanethiol, MEA) and its disulfide, cystamine, on the human immunodeficiency virus (HIV-1) expression in chronically infected promonocytic cells (U1), T cell line (ACH-2), and peripheral blood monocyte-derived macrophages (MDM) were investigated. U1 and ACH-2 cells constitutively express low levels of virus, which is increased by the addition of tumor necrosis factor (TNF-alpha), interleukin 6 (IL-6), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and other inducers. Cystamine, in noncytotoxic doses, suppressed in a concentration-dependent fashion the induction of HIV-1 expression mediated by TNF-alpha, IL-6, GM-CSF, and monokine-enriched monocyte culture supernatants in both U1 and ACH-2 cells as determined by HIV-1 reverse transcriptase (RT) activity. Similarly, HIV-1 expression was substantially reduced in the cystamine-treated primary MDM cultures compared with the untreated control cultures. The addition of cystamine into HIV-1 chronically infected MDM (12 days after infection was established) also suppressed 80-90% of RT activity in comparison to the untreated controls. HIV-1 (Bal) infected MDM cultures (without cystamine treatment) demonstrated giant syncytium formation, whereas cystamine-treated cultures lacked the giant syncytia induced by HIV-1 infection. Cystamine also inhibited LPS-induced TNF production in MDM. In contrast to cystamine, cysteamine showed no significant effects on either the monokine-induced HIV-1 expression in U1 or ACH-2 or acute and chronic HIV-1 infection in MDM.(ABSTRACT TRUNCATED AT 250 WORDS)

The Biological Phenotype of HIV-1 Is Usually Retained during and after Sexual Transmission

The biological phenotype of HIV-1 isolates from 21 subjects with primary HIV-1 infection was determined in primary lymphocytes and monocyte-derived macrophages as well as in three T-cell lines (MT-2, HUT78, and C8166). Seven isolates (33%) replicated on at least one cell line and were classified as rapid/high ( n = 6) or intermediate ( n = 1), while 14 isolates (67%) did not replicate in cell lines and were classified as slow/low. All isolates replicated in primary monocyte-derived macrophage cultures. Studies on 10 pairs consisting of index case and seroconverting sexual partner showed that the biological phenotype of HIV-1 was unchanged in nine transmissions, including 5 pairs where a rapid/high virus was transmitted. Most individuals (5 of 6) infected by a rapid/high virus retained this phenotype after seroconversion. These individuals also had significantly lower CD4 lymphocyte counts 1-3 years after infection. Thus, in contrast to what has been suggested by others, we found that HIV-1 variants with rapid/high phenotype were usually not suppressed, neither during sexual transmission nor after seroconversion in the new host. These findings have important implications for the understanding of HIV-1 transmission and pathogenesis as well as for vaccine design.

Infection of human macrophages with an endogenous tumour necrosis factor-alpha (TNF-alpha)-independent human immunodeficiency virus type 1 isolate is unresponsive to the TNF-alpha synthesis inhibitor RP 55778.

Monocyte-derived macrophages (MDM) were demonstrated to be susceptible to productive infection by the monocytotropic human immunodeficiency virus type 1 (HIV-1) strain HIV-1/Ba-L and by three primary HIV-1 isolates, HIV-1/DAS, HIV-1/PAR and HIV-1/THI. Production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-1 beta was monitored between days 3 and 26 after MDM infection. TNF-alpha and IL-6 were detected in cell culture supernatants from days 16 to 21 following HIV-1/DAS, HIV-1/PAR and HIV-1/Ba-L infection, at the time of high viral replication. IL-1 beta was not found at the same time points. TNF-alpha mRNA expression occurred around the peak of both TNF-alpha levels and supernatant RT activities. In HIV-1/THI-infected macrophage cultures no endogenously produced TNF-alpha was observed, despite high levels of HIV-1 in MDM. This result demonstrates that a primary isolate may replicate independently of TNF-alpha in MDM. To investigate the relationship between TNF-alpha and viral replication we used a TNF-alpha synthesis inhibitor, RP 55778. Treatment throughout the course of cell culture resulted in a significant decrease in both TNF-alpha levels and viral production in HIV-1/DAS-, HIV-1/PAR- and HIV-1/Ba-L-infected MDM cultures. This phenomenon is reversed by adding recombinant human TNF-alpha to the RP 55778-treated cell cultures from day 14 post-infection. No effect of RP 55778 was observed in MDM cultures infected with the primary isolate HIV-1/THI, whose replication is independent of TNF-alpha production and therefore remained unchanged after RP 55778 treatment. We conclude that the clinical value of such a drug is directly dependent on the ability of the HIV-1 strains involved to induce TNF-alpha production at the time of viral replication.

Inhibition of phagocytosis of human macrophages induced by airborne particulates

Human monocyte-derived macrophages isolated from peripheral blood were treated with different extracts of airborne particulates collected in the highly industrialized Rhine-Ruhr area. All tested extracts showed a substantial impairment of phagocytosis by inhibition of phagocytic activity as well as phagocytic capacity, while cell viability was rather well maintained. Significant reduction of phagocytosis already appeared at a concentration equivalent to extracted particulates from 3.8 m 3 air. Having properties of alveolar macrophages, human monocyte-derived macrophage cultures may offer a reliable in vitro model for assessment of pulmonary toxicity by respirable pollutants.

Tumour Promoters/Protein‐Kinase C Activators Augment the Survival and Function of Human Monocyte‐Derived Macrophages in Long‐Term Cultures

In this study the effect of various Protein kinase C (PKC) activators/tumour promoters on the maturation and activity of human peripheral blood monocytes was examined. Monocytes were cultured in the absence or presence of various PKC activators for up to 2 weeks, and examined for the number of adherent cells, expression of myeloperoxidase enzymes, CD14 antigens, mannose/N-acetylglucosamine (Man/GlcNAc) receptors, and the production of TNF-alpha. The presence of PKC activators in cultures of monocyte-derived macrophages (HuMoDM) prevented the loss in the number of initially plated monocytes, otherwise observed in long-term tissue cultures with time of incubation. This effect of PKC activators on monocyte survival was diminished in the presence of PKC inhibitors. HuMoDM obtained in the presence of PKC activators maintained a normal differentiation pattern, as was evident by the loss of granular myeloperoxidase enzymes and CD14 antigens, and the acquisition of membrane Man/GlcNAc receptors. HuMoDM which differentiated in the presence of PKC activators also released TNF-alpha in comparable amounts to freshly harvested human monocytes. PKC activators/tumour promoters augmented the viability of long-term cultures of human monocyte-derived macrophages. Such macrophages may facilitate cell and molecular biology studies of differentiated human macrophages.

Treatment of human monocyte-derived macrophages with a TNFα synthesis inhibitor prior to HIV1 infection: consequences on cytokine production and viral replication

Human monocyte-derived macrophages (MDM) were infected with the viral strain HIV1/Ba-L and with the clinical isolates HIV1/DAS and HIV1/PAR. Kinetics of tumour necrosis factor alpha (TNF alpha) and interleukin-6 (IL6) production were investigated for 28 days after infection. At the early stages of infection we observed significant TNF alpha and IL6 secretion 2 to 10 h after infection, whatever the viral strain we used. During the late events of MDM infection, TNF alpha and IL6 were detected over 16 to 21 days following HIV1 infection, at the time of high viral replication. Pretreatment of MDM with a TNF alpha synthesis inhibitor, RP 55778, 4 h prior to HIV infection induced a modified cytokine pattern during the first ten hours of infection: TNF alpha production was totally inhibited despite comparable amounts of IL6. At the late phases of the cell culture, a decrease in magnitude of both viral and cytokine production as well as a delay in the appearance of reverse transcriptase activity and cytokine secretion peaks were observed in RP-55778-pretreated and HIV1-infected MDM cultures. Similar results were obtained after pretreatment of HIV1/DAS-infected MDM cultures with an anti-TNF alpha monoclonal antibody.

HIV-1 pseudotype virus containing a Cocal virus genome and an HIV envelope: construction, assay and use

A method is described for the production and assay of pseudotype viruses between human immunodeficiency virus type 1 (HIV-1) and Cocal virus (COV), containing an HIV-1 envelope and a COV genome (COV(HIV)). COV(HIV) pseudotype virus is a useful tool for the investigation of a variety of questions regarding HIV entry into susceptible cells, including steps in virus binding, fusion, and internalization, and the role of molecules which inhibit entry. COV, a rhabdovirus closely related to vesicular stomatitis virus (VSV), replicated and caused cytopathic effect in primary cultures of human peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDM), and in human cell lines of lymphocytoid or monocytoid origin, making it an ideal candidate for pseudotype production. 174XCEM cells, which were permissive for selected macrophage-tropic strains as well as most lymphocyte-tropic strains of HIV-1, were used to produce stocks of putative pseudotype virus. To neutralize parental COV in these stocks, a rabbit antiserum was produced which had a neutralization index of > 10 7 at a dilution of 1:100. Using these methods, pseudotype viruses were produced with a titer of about 10 4 PFU per ml; these same stocks contained HIV-1 at a titer of about 10 5 TCD50 per ml and COV at a titer of about 10 8 PFU per ml. CD4-expressing HeLa cells were used to assay pseudotype stocks made with lymphocyte-tropic strains of HIV-1. The authenticity of the pseudotype stocks was validated by several controls, including their failure to register on congenic CD4-negative HeLa cells and their inhibition by monoclonal anti-CD4 antibodies such as Leu 3a.

Infection of human monocyte‐derived macrophages by human immunodeficiency virus mediated by cell‐to‐cell transmission

We have infected ten-day-old primary cultures of human monocyte-derived macrophages (MDM) with HIV-1 by cocultivation with chronically infected monocytic cell lines. This work has involved the U-937 monocytoid cell line, chronically infected with the HIV-IIIB strain of HIV-1 (U-937HIV IIIB) as well as a number of cell clones, termed UHC, which were derived from U-937HIV IIIB by limiting dilution. Cell-free virus, derived from each of U-937HIV IIIB cells and the UHC1 clone were noninfectious for MDM, as determined by failure to express viral p24 antigen (Ag). In contrast, viral p24 Ag production was detected in MDM that had been cocultivated with U-937HIV IIB, and with each of three UHC clones that produced infectious virus. Infection, in each case, was confirmed by polymerase chain reaction detection via the amplification of proviral DNA. In contrast, cocultivation with the UHC15.7 clone, which fails to cleave viral gp160 to its gp120 and gp41 products or the UHC8 clone, which lacks functional reverse transcriptase, did not lead to infection of MDM. Pretreatment of MDM for 2 hr with 1 microM AZT completely prevented infection by culture fluids containing HIVada, a macrophage-tropic virus, but did not affect infection mediated by cocultivation. These results suggest that cell-to-cell transmission of HIV-1, among monocyte-derived macrophages, can be mediated by proviral DNA. Moreover, gp120 at the surface of infected cells may play an important role in this process, since cell-to-cell HIV transmission could not be demonstrated with the UHC clone that is defective in cleavage of the viral envelope glycoprotein gp160.

Human monocyte-derived macrophage cultures: An alternative test system for the detection of pulmonary toxicity induced by inhaled particulate pollutants

Alveolar macrophages are major target cells for toxicity from inhaled particulates. To investigate pulmonary toxicity induced by airborne particulates a phagocytosis assay using human monocyte-derived macrophage cultures was used. Monocytes were isolated from peripheral blood and cultured for 10–14 days. During this period the monocytes differentiated to macrophages. After treatment with various concentrations of different samples of airborne particulate matter, phagocytosis was induced by the addition of Polychromatic Fluoresbrite Microspheres at a cell/particle ratio of 1:10. Phagocytosis was assessed from the determination of phagocytic activity (% cells showing phagocytosis) and of phagocytic capacity (number of phagocytized particles/cell). A concentration-dependent reduction of phagocytic activity and capacity was observed, while cell viability was not greatly affected as compared with the control. These results were in good agreement with those obtained from in vivo inhalation experiments with rodents as well as with those using rat alveolar macrophages obtained after bronchoalveolar lavage and treated in vitro with extracts of particulate matter. Therefore, it is suggested that the use of human monocyte-derived macrophage cultures represents a promising in vitro test system for the evaluation of pulmonary toxicity induced by particulate pollutants.

Vitamin D3 compounds regulate human immunodeficiency virus type 1 replication in U937 monoblastoid cells and in monocyte-derived macrophages

We studied the effects of vitamin D3 compounds on the replication of human immunodeficiency virus type 1 (HIV-1) in the monoblastoid cell line U937 and in primary monocyte-derived macrophage cultures to understand how modulators of monocyte/macrophage effector function might affect the pathogenesis of HIV-1 infection. U937 cell cultures exposed to 1, alpha 25-dihydroxyvitamin D3 prior to HIV-1 infection showed enhanced virus replication that was apparently due to increased cellular resistance to viral cytopathic effects; a marked inhibition of virus replication was noted in cells exposed to 1 alpha,25-dihydroxyvitamin D3 subsequent to infection. Exposure of blood-derived monocyte/macrophages to vitamin D3 compounds prior to infection also affected virus growth; in most cases, substantial inhibition of HIV-1 replication was noted in vitamin D3-treated macrophage cultures. Our results demonstrate that vitamin D3 compounds with recognized abilities to induce cellular differentiation can modulate HIV-1 infection of human macrophages.

Uptake of exogenous free cholesterol induces upregulation of tissue factor expression in human monocyte-derived macrophages.

Lipid-laden macrophages present as foam cells may contribute to the hyperthrombotic state of human atherosclerotic lesions by the production of tissue factor (TF). We investigated the effect of exogenous nonlipoprotein cholesterol on the expression of TF by human monocyte-derived macrophages in culture. Nonlipoprotein cholesterol at 50 micrograms/ml increased TF activity 4-fold; TF induction was dose- and time-dependent. Expression of TF activity was positively correlated with the free cholesterol content of monocyte-derived macrophages, was increased upon inhibition of cholesterol esterification, and reflected de novo synthesis of TF protein. TF expression in cholesterol-loaded macrophages remained sensitive to stimulation (approximately 12-fold) by bacterial lipopolysaccharide, indicating that intracellular free cholesterol and lipopolysaccharide act by distinct mechanisms in inducing TF procoagulant activity. Our results suggest that loading human monocyte-derived macrophages with free cholesterol induces upregulation of TF expression, thereby contributing to thrombus formation at sites of plaque rupture.

The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus

Although the immune response to Brucella abortus is multifaceted, the key event in contending with this pathogen appears to be the interaction of the organism with cells of the mononuclear phagocyte system. A cell culture system was developed which allowed the long-term maintenance of blood monocyte-derived macrophages in Teflon culture vessels in a relatively unstimulated state. The assay system was optimized for timing of bacteria-macrophage interaction and numbers of bacteria and macrophages used in each assay. Interaction of B. abortus strain 2308 with bovine mononuclear phagocytes from animals phenotypically resistant and susceptible to infection with B. abortus was investigated. This cell culture and assay system should provide a useful model for the investigation of intracellular parasitism in cattle.

Interferon-gamma and interleukin-4 down-regulate soluble CD14 release in human monocytes and macrophages.

CD14 is a 53-kd glycoprotein that is mainly expressed in myeloid cells and exists in two forms. The membrane-bound form represents the receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein. The function and regulation of the soluble form are unknown. In the present study we investigated the release of soluble CD14 (sCD14) in cultures of human mononuclear leukocytes, elutriated monocytes, and monocyte-derived macrophages. The release of sCD14 into the medium of the cells cultured for 15 and 45 h was investigated in the absence or presence of selected cytokines. sCD14 release occurred constitutively and correlated with cell number. In monocytes differentiating into macrophages, cumulative release of sCD14 was linear from day 1 to day 7. Spontaneous sCD14 release after 15 h of culture (2 x 10(6) cells/ml) was higher in the supernatant from monocytes (314 +/- 58 ng/ml) than that from mononuclear leukocytes (68 +/- 10 ng/ml) and similar to that from macrophages (469 +/- 79 ng/ml). Cycloheximide and actinomycin D inhibited sCD14 release. Recombinant interferon-gamma (rIFN-gamma) and recombinant interleukin-4 (rIL-4) directly decreased sCD14 release in mononuclear leukocyte, monocyte, and macrophage cultures. rIL-2 and rIFN-alpha reduced sCD14 release into the supernatants of mononuclear leukocytes only. Use of anti-IFN-gamma antibodies indicated that the down-regulation of sCD14 release by rIL-2 and rIFN-alpha was partially due to induction of endogenous IFN-gamma. The down-regulation of sCD14 release by all four cytokines was both time and dose dependent. rIFN-gamma and rIL-4 added simultaneously had a synergistic effect on sCD14 down-regulation. In conclusion, sCD14 release may have an immunomodulatory role in circulating monocytes, is apparently not related to the process of macrophage differentiation, and is selectively down-regulated during an immune response when levels of IFN-gamma and IL-4 are high.

Comparative binding and degradation of lipoprotein(a) and low density lipoprotein by human monocyte-derived macrophages.

The binding and degradation of equimolar concentrations of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) isolated from the same individual were studied in primary cultures of human monocyte-derived macrophages (HMDM). At 4 degrees C, LDL receptor-mediated binding of both Lp(a) and LDL was of low affinity, being 0.8 and 0.23 microM, respectively. Competitive binding studies indicated that the binding of Lp(a) to HMDM was competed 63% by excess LDL. In contrast to the 4 degrees C binding data, the degradation of Lp(a) at 37 degrees C was mainly nonspecific because the amount of Lp(a) processed by the LDL receptor pathway in 5 h was 17% that of LDL. According to pulse-chase experiments, this phenomenon may be accounted for by the facts that less Lp(a) is bound to HMDM at 37 degrees C and that Lp(a) has a lower intrinsic degradation rate and was not due to increased intracellular accumulation or retroendocytosis of the lipoprotein. Degradation of both lipoproteins was primarily lysosomal and only modestly affected by up- or down-regulation of the LDL receptor. The rate of retroendocytosis in HMDM was approximately equal to the degradation rate and appeared to be independent of the type of lipoprotein used, up- or down-regulation of the LDL receptor, or the presence of the lysosomotropic agent chloroquine. Overall, the results indicate that HMDM degrade Lp(a) mainly via a nonspecific pathway with only 25% of total Lp(a) degradation occurring through the LDL receptor pathway. As both 37 degrees C degradation and 4 degrees C binding of LDL are mainly LDL receptor specific, the different metabolic behavior observed at 37 degrees C suggests that Lp(a) undergoes temperature-induced conformational changes on cooling to 4 degrees C that allows better recognition of Lp(a) by the LDL receptor at a temperature lower than the physiological temperature of 37 degrees C. How apo(a) affects these structural changes remains to be established.

Phagocytosis of Mycobacterium leprae by human monocyte-derived macrophages is mediated by complement receptors CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) and IFN-gamma activation inhibits complement receptor function and phagocytosis of this bacterium.

We have examined phagocytosis of Mycobacterium leprae by human monocyte-derived macrophages (MDM). Compared with monocytes, MDM exhibit greatly enhanced adherence of M. leprae (6.5 +/- 2-fold increase). MDM adherence of M. leprae is serum dependent and requires heat-labile serum components because heat inactivation of serum reduces adherence by 70 +/- 3%. mAb against C receptors CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) inhibit phagocytosis of M. leprae in fresh nonimmune serum. Single mAb against each receptor inhibit M. leprae adherence by 25 +/- 4% - 33 +/- 6%. Single mAb used in combination against all three receptors inhibit M. leprae adherence by 51 +/- 6%. Most significantly, pairs of mAb used in combination against all three receptors inhibit by 80 +/- 4%. By electron microscopy, MDM ingest all M. leprae that adhere in fresh nonimmune serum. In the presence of mAb against CR1, CR3, and CR4, the percentage of MDM cross-sections that contain intracellular bacteria is reduced 66 +/- 3% and the mean number of bacteria per cross-section is reduced 78 +/- 10%. MDM activated by IFN-gamma exhibit markedly reduced adherence (by light microscopy) and ingestion (by electron microscopy) of M. leprae. MDM in culture for 5 days inhibit M. leprae adherence by 83 +/- 2% and ingestion by 88% when activated for 5 days. Paralleling this, IFN-gamma-activated MDM exhibit markedly reduced C receptor function, reflected by markedly decreased adherence and ingestion of C3b- and C3bi-coated E. Decreased C receptor function by IFN-gamma-activated MDM correlates with decreased surface expression of CR1 but not CR3 or CR4. CR1 expression on MDM in culture for 5 days is reduced by 32 +/- 9% and 75 +/- 3% after IFN-gamma activation for 5 and 2 days, respectively. This study demonstrates that MDM have an enhanced capacity to phagocytize M. leprae, and that in addition to CR1 and CR3, phagocytosis involves CR4, whose expression on MDM is highly maturation-dependent. This study also demonstrates that IFN-gamma activation markedly reduces the capacity of MDM to phagocytize M. leprae, and it provides a molecular mechanism for this phenomenon-decreased C receptor function.

The influence of oxidatively modified low density lipoproteins on expression of platelet-derived growth factor by human monocyte-derived macrophages

Platelet-derived growth factor (PDGF) is secreted by several cells that participate in the process of atherogenesis, including arterial wall monocyte-derived macrophages. Macrophages in human and non-human primate lesions have recently been demonstrated to contain PDGF-B chain protein in situ. In developing lesions of atherosclerosis, macrophages take up and metabolize modified lipoproteins, leading to lipid accumulation and foam cell formation. Oxidatively modified low density lipoproteins (LDL) have been implicated in atherogenesis and have been demonstrated in atherosclerotic lesions. The effects of the uptake of various forms of modified LDL on PDGF gene expression, synthesis, and secretion in adherent cultures of human blood monocyte-derived macrophages were examined. LDL oxidized in a cell-free system in the presence of air and copper inhibited the constitutive expression of PDGF-B mRNA and secretion of PDGF in a dose-dependent fashion. Oxidatively modified LDL also attenuated lipopolysaccharide-induced PDGF-B mRNA expression. These changes were unrelated to the mechanism of lipid uptake and the degree of lipid loading and were detectable within 2 h of exposure to oxidized LDL. The degree of inhibition of both basal and lipopolysaccharide-induced PDGF-B-chain expression increased with the extent of LDL oxidation. Monocyte-derived macrophages exposed to acetylated LDL or LDL aggregates accumulated more cholesterol than cells treated with oxidized LDL, but PDGF expression was not consistently altered. Thus, uptake of a product or products of LDL oxidation modulates the expression and secretion of one of the principal macrophage-derived growth factors, PDGF. This modulation may influence chemotaxis and mitogenesis of smooth muscle cells locally in the artery wall during atherogenesis.

Intracellular regulation of lipoprotein lipase in human monocyte-derived macrophages

The intracellular pathway of lipoprotein lipase (LPL) has been examined in human monocyte-derived macrophages in culture. These cells were previously shown to synthesize and constitutively secrete LPL. The secretion is dependent on new enzyme synthesis. 6-d-old human monocytes have stores of mRNA for linear release of LPL up to 24 h. Enzyme activity in cells and in culture medium was almost completely inhibited by 24 h treatment with tunicamycin, an inhibitor of glycosylation. In monensin-treated cells a pronounced increase in enzyme activity was found, whereas the secreted activity was markedly reduced. This indicates that LPL in human monocytes is processed through a pH sensitive part of the Golgi complex and that the terminal glycosylation is not needed for the expression of its catalytic activity. Our results suggest that lysosomal function is not important in secretion of the enzyme, whereas vesicular transport seem to be involved in regulating LPL in human monocyte-derived macrophages in culture.

Human immunodeficiency virus-1 infection of macrophages in vitro neither induces tumor necrosis factor (TNF)/cachectin gene expression nor alters TNF/cachectin induction by lipopolysaccharide.

The synthesis of tumor necrosis factor (TNF)/cachectin was assessed in primary monocyte-derived macrophage (MDM) cultures after in vitro infection with a macrophage-tropic strain of HIV-1 (HTLV-IIIBa-L/85). Productive and cytopathic infections in MDM cultures were established using a high multiplicity of infection (m.o.i. = 3) under conditions that minimized endotoxin contamination. Culture supernatants were tested for TNF/cachectin activity by L929 cell cytotoxicity assay, and TNF/cachectin mRNA was assessed by a sensitive PCR amplification technique that could detect between 1 and 10 cells fully activated for TNF/cachectin expression. Unstimulated MDM cultures produced no detectable levels of TNF/cachectin activity or mRNA, consistent with previous demonstrations that production of this cytokine by macrophages is an inducible and not a constitutive event. HIV-1 infection failed to induce detectable TNF/cachectin activity or mRNA in these unstimulated cultures. In addition, the responsiveness of macrophages to lipopolysaccharide (LPS) induction of TNF/cachectin production was assessed in dose-response and kinetic experiments. No differences between infected and uninfected cultures were discernable. These results demonstrate that productive and cytopathic infection with a macrophage-tropic strain of HIV-1 does not alter the regulation of TNF/cachectin expression in macrophages.