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Monocytes treated with 500 IU/ml human recombinant interferon-γ (rIFN-γ) 1 day before and continuously after human immunodeficiency virus (HIV) infection showed no evidence of virus replication 7 days after addition of the viral inoculum. There was no HIV- associated cytopathic effect, no reverse transcriptase (RT) activity or p24 detected in culture fluids, and no HIV RNA or DNA in cell lysates. Furthermore, no evidence of HIV infection was evident in replicate cultures in which all IFN-γ was removed at 7 days and the cells were cultured for an additional 3 weeks without IFN-γ. The 50% inhibitory dose for reduction of maximum RT activity in HIV-infected monocyte cultures was about 1 IU/ml IFN-γ. No increase in HIV replication was evident in monocytes treated with IFN-γ at any concentration (0 to 5000 IU/ml) or at any time (7 days before to 10 days after HIV infection). In side-by-side experiments with identical monocytes and HIV-1 stock, rIFN-γ was 10 to 20 times more effective than rIFN-α2b for induction of antiviral activity. With both interferons, significant antiviral activity was evident with monocytes treated 1 day before, at the time of, or up to 3 days after infection. At 7 to 10 days after infection (a time at which less than 20% of total cells were infected with HIV) addition of even high concentrations of IFN-α or IFN-γ had no effect on virus replication. These data suggest that the principal action of IFN-α and IFN-γ was directed against the fluid-phase virus. Cell-cell spread of infection within the HIV-infected monocyte culture and extent of virus replication in HIV-infected cells were not affected by interferon treatment. J. Leukoc. Biol. 56: 362–368; 1994.

Natural IFN-alpha n3, a purified mixture of many different natural IFN alpha species, was 10- to 100-fold more effective than equal concentrations of human rIFN-alpha 2b or rIFN-alpha 2a for inhibition of HIV replication in primary human monocytes. This difference was highly reproducible in multiple side-by-side experiments using the identical HIV-1 inoculum and the same monocyte target cells: natural IFN-alpha n3 was more effective than rIFN-alpha 2b at lower concentrations for protection against a constant HIV-1 inoculum; cells treated with natural IFN-alpha n3 were protected against a greater HIV-1 challenge than were cells treated with the same concentration of rIFN-alpha 2b. Fractionation of natural IFN-alpha n3 by reversed-phase high-pressure liquid chromatography (RP-HPLC) showed that most antiviral activity for HIV localized to discrete and reproducible peaks. The RP-HPLC peak that contained purified natural IFN-alpha 2b was the least effective fraction. These data suggest heterogeneity among IFN-alpha species for antiviral activity against HIV and may provide a molecular basis for more effective IFN-alpha therapy.

To investigate the expression of human immunodeficiency virus (HIV) genes in human monocytes, a DNA transfection system was developed and characterized using cultured primary monocytes. Monocytes that were cultured 6-7 days in an adherent monolayer were efficiently recovered and transfected by electroporation with an expression vector containing the Escherichia coli lacZ gene under control of the cytomegalovirus immediate-early promoter. Successful transfection was detected by expression of beta-galactosidase activity and by histochemical staining for beta-galactosidase in cells that were allowed to readhere to plastic following transfection. Over 30% of the surviving adherent monocytes expressed the transfected beta-galactosidase gene. In the same manner, monocytes were transfected with HIV provirus clones pIIIB and pIIB/PB. The provirus pIIIB/PB differs from pIIIB only in that it contains a small sequence from the env gene of a macrophage tropic HIV-1. Virus derived from pIIIB will not replicate in monocytes whereas virus derived from pIIIB/PB will. Monocytes transfected with either provirus DNA expressed high levels of p24 antigen within 1 day of transfection, and cell-free supernatants contained virus that was infectious for T cells. In contrast, only supernatants from pIIIB/PB transfections contained virus capable of infecting monocytes. Thus, proviral DNA of T cell tropic HIV efficiently completes the retroviral life cycle in monocytes in a manner indistinguishable from that of macrophage tropic HIV, and progeny virus retain their T cell tropism.

Levels of human immunodeficiency virus (HIV) DNA, RNA, or p24 antigen and reverse transcriptase activity in T-cell cultures treated with 500 IU of recombinant alpha interferon (rIFN alpha) per ml were comparable to those in control cultures. Radioimmunoprecipitation analysis of proteins in lysates of IFN-treated T cells documented a marked accumulation of HIV proteins. Localization of gp120 by immunofluorescence showed a diffuse pattern in IFN-treated cells quite distinct from the ring pattern in untreated control cells. That large quantities of gp120 in aberrant cell compartments might affect HIV morphogenesis was confirmed in infectivity studies: virions from IFN-treated cells were 100- to 1,000-fold less infectious than an equal number of virions from control cells. Direct examination of IFN-treated and control HIV-infected cells by transmission electron microscopy showed little difference in the number or distribution of viral particles. However, quantitation of gp120 by immunogold particle analysis revealed a marked depletion of envelope glycoprotein in virions released from IFN-treated cells. This defect in gp120 assembly onto mature viral particles provides a molecular basis for this loss of infectivity.

The frequency of human immunodeficiency virus (HIV)-infected monocytes that spread on a model basement membrane was about twofold greater than that of an equal number of uninfected control cells through the initial 12 to 18 h of culture. By 24 h, virtually all HIV-infected and uninfected control cells spread on the basement membrane gel. The frequency of spread cells in the uninfected control population was < 10% of total cells by 12 days. In contrast, 30 to 40% of HIV-infected monocytes remained spread through this time interval and formed a dense interdigitated network of cell processes on and into the gel matrix. Invasion of the basement membrane matrix by HIV-infected monocytes suggested increased secretion of proteases able to digest the gel. Indeed, levels of neutral protease activity in culture fluids from HIV-infected monocytes were significantly higher than those from equal numbers of uninfected control cells. High levels of protease activity in culture fluids of HIV-infected monocytes required productive virus infection and were not observed with cells exposed to T cell-tropic HIV isolates. The predominant protease activity in these cultures was a 92-kd neutral metallogelatinase. HIV-induced changes in monocyte metalloprotease activity may be important for extravasation of infected cells to tissue or for the development of AIDS-associated neuropathology, carcinogenesis, and opportunistic infection.

Macrophages and microglia are the principal target cells for human immunodeficiency virus (HIV) in brain, and as such, are likely participants in the neuropathology of HIV infection. In a model system for this process, we found that fluids from human monocyte cultures enhanced survival and differentiation of the neurons in fetal rat brain explants. In contrast, fluids from HIV-infected monocyte cultures were strongly toxic to neurons and paradoxically enhanced the proliferation of glial cells. Further, neuronotoxic activity in these fluids was mediated through activation of NMDA binding receptors on the neurons and was inhibited by any of several different NMDA antagonists. Neuronotoxic activity was directly related to contamination of the HIV virus stock with Mycoplasma arginini and M. hominis. Pure cultures of mycoplasma, bacterial lipopolysaccharide (LPS), or murine recombinant tumor necrosis factor alpha (rTNF alpha) each induced neuronotoxicity which exactly mirrored that induced by the contaminated HIV stock. It is likely that mycoplasma or components of the mycoplasma plasma membrane stimulate TNF alpha production by the glial cells in the brain explants. Indeed, careful depletion of glial cells in these explants prevented mycoplasma or LPS-mediated neuronotoxicity. No neuronotoxicity was evident with HIV-1 virus stock, HIV-1 gp120, or culture fluids from HIV-infected T cells or monocytes when these preparations were free of contamination by mycoplasma and LPS. These findings suggest caution in interpretation of those experiments in which similar contamination has not been rigorously excluded.

Components of the host immune response that constrain virus replication and affect long-lasting antiviral immunity following HIV infection are incompletely defined. IFNs are critical participants in host antiviral processes. While IFN induces significant anti-retroviral activities, they also serve as harbingers for poor clinical outcomes. Moreover, monocytes, a major cellular source of IFN and HIV in man, are poor producer cells for IFN following HIV infection. Indeed, HIV infection of monocytes results in a diminished production and induction of IFN. IFN is only produced during cell to cell contact between HIV-infected cells and uninfected PBMC. Analysis of the biologic activity of HIV-induced IFN(s) shows that it poorly restricts HIV replication. Thus, the role of IFN in HIV disease is complex and seemingly paradoxical. The diminished capacity of HIV-infected monocytes to produce IFN and the production of defective IFNs likely reflect specific viral adaptive mechanisms for persistent infection.

CD4 is the surface receptor for HIV envelope. Some evidence exists, however, that other cell surface receptors may be involved in viral entry subsequent to the initial binding of gp120 to CD4. Antibodies to leukocyte integrin LFA-1, a major component of intercellular adhesive interactions, have been shown to inhibit HIV-induced syncytia formation. Using a stringent system for in vitro HIV infection of human leukocytes, we examine the ability of some monoclonal antibodies (mAb) against various adhesion-related molecules to block or partially inhibit productive viral replication. HIV-1 infection of target monocytes or T cells by cell-free virus was blocked completely or partially by some mAb that prevent cell-cell interactions (CD4, HLA-DR, LFA-1, LFA-3), but not by others (ICAM-1, MAC-1, gp150.95, CD2, CD3, CD14). The capacity for mAb to block HIV infection appears to be epitope-specific, and does not relate to the ability to block homotypic adhesion. HIV transmission from infected cells was more difficult to block than was infection by cell-free virus. Adhesion molecules may be involved in facilitating early stages of HIV infection, following gp120/CD4 binding but prior to viral integration, in a manner distinct from cell-cell adhesion.

Cells of monocyte-macrophage lineage are susceptible targets and important tissue reservoirs of human immunodeficiency virus (HIV) type 1. In comparison to the productive HIV infection of macrophages in the brain, lungs, and lymph nodes, epidermal Langerhans cells do not serve as principal viral reservoirs.

Resident tissue and inflammatory macrophages produce low but detectable levels of nitrogen oxides de novo, and serve successfully as host cells for Leishmania major, a protozoan parasite that replicates in phagolysosomes (Fortier et al., 1983). In contrast, activated macrophages release markedly increased levels of NO 2 and NO~ (Stuehr and Marietta, 1987; Drapier et al., 1988), and the intracellular environment of these cells is extremely inhospitable for L. major survival (Nacy et al., 1981). We recently correlated the intracellular microbicidal capacity of activated macrophages with production of nitrogen oxides (Green et al., 1990a): in all cases, macrophages that kill intracellular amastigotes release high levels of NO 2 into the culture medium. The effector molecule for destruction of the parasite is NO, the highly reactive and unstable intermediate of the nitrogen oxidation of L-arginine (Hibbs eta!., !987; Green et al., 1990a; Liew et al., 1990a). A competitive inhibitor of L-arginine, N~'-monomethyl-L arginine (NGMMLA), completely blocks synthesis of NO and inhibits killing of L. major (Green et al., 1990a).

Human recombinant interferon-alpha (IFN alpha) restricted viral replication in human immunodeficiency virus- (HIV) infected T cells and monocytes. With T cells, reverse transcriptase (RT) activity in culture fluids was reduced threefold from that of control infected cells by IFN treatment, but HIV p24 antigen levels were unchanged. In contrast, levels of p24 antigen and RT activity in lysates of IFN-treated infected cells were threefold greater than those of controls. These differences suggest that the mechanism for IFN-induced antiviral effects in HIV-infected T cells resides in the terminal events (assembly and release) of the virus replication cycle. Monocytes treated with IFN at the time of virus challenge showed no p24 antigen or RT activity, no HIV-specific mRNA, and no proviral DNA in cells for up to 3 weeks after infection. IFN treatment of chronically infected monocytes also decreased virus replication, as assessed by p24 antigen, mRNA and RT detection assays. However, levels of proviral DNA in the IFN-treated and control HIV-infected cells were indistinguishable. The presence of large quantities of proviral DNA in cells with little or no evidence for active transcription documents a situation approaching true microbiological latency.

Viral isolates were recovered by cocultivation on macrophage colony-stimulatingfactor (MCSF)-treated monocyte target cells from peripheral blood mononuclear cells (PBMCs) in 25 out of 27 patients seropositive or at risk for HIV infection. Frequency of virus recovery was independent of the patient's age, sex, numbers of CD4+ T cells, clinical stage or zidovudine (azidothymidine) therapy. Sixteen out of 19 HIV isolates were serially passaged in MCSF- treated monocytes. Five out of five virus isolates were also passaged in phytohemagglutinin/interleukin-2 (PHA/IL-2)-treated lymphoblasts. In lymphoblasts, no qualitative or quantitative differences were observed between these isolates and human T-cell leukemia virus IIIB (HTLV-IIIB) for (1) release of p24 antigen reverse transcriptase, and infectious virus, (2) induction of typical cytopathic effects (cell syncytia in 3-10% of cells) and cell lysis, (3) frequency of infected cells (5-20% of PBMC) as detected by in situ hybridization for HIV RNA, (4) down-modulation of T cell plasma membrane CD4, and (5) site of progeny virion assembly and budding (plasma membrane only with no intracytoplasmic accumulation of virus). Progeny virus recovered from infected lymphoblasts was fully infectious for other lymphoblasts, but failed to infect MCSF-treated monocytes. Detailed analysis of target cell tropism among HIV isolates showed that HIV isolated in monocytes infected both monocytes and lymphoblasts; progeny virus isolated in lymphoblasts infected only T cells. HIV interacts differently with monocytes and T cells. Understanding this interaction may more clearly define both the pathogenesis of HIV disease and strategies for therapeutic intervention.

Macrophages are an important in vivo reservoir for HIV. Conclusive evidence that CNS, pulmonary, lymph node and blood-derived mononuclear phagocytes harbor and support HIV replication is supported by numerous independent studies. HIV variants which preferentially replicate in macrophages have been recovered from infected individuals, suggesting that these cells and variant viruses are involved in the establishment and progression of HIV-related disease.

Lymphokine-activated macrophages are cytotoxic for larvae of the helminth parasite Schistosoma mansoni. That soluble secreted factors may mediate this cytotoxicity was suggested by the observation that culture supernatant fluids from stimulated macrophages also exhibited larvacidal activity. These fluids contain the monokine tumour necrosis factor (TNF). Several observations indicated that TNF is directly toxic to schistosome larvae. Cytotoxic sera taken from BCG- or S. mansoni-immunized mice after endotoxin challenge killed schistosomula in vitro, and upon gel filtration the larvacidal factor(s) in the sera co-eluted with the tumoricidal activity defined as TNF. Recombinant-derived TNF exhibited direct toxicity to schistosomula at high concentrations, or at lower concentrations in the presence of IFN gamma. The larvacidal activity of macrophage supernatant fluids was abrogated by addition of either anti-TNF antisera or Zn+2, which has been shown to inhibit TNF-induced damage of tumour cells. Anti-TNF and Zn+2 likewise suppressed schistosomulum killing by lymphokine-activated peritoneal macrophages or the IC-21 macrophage line, indicating that TNF also plays a role in the effector mechanism of larval killing by whole cells.

The safety and tolerance of interferon alfa-n3 (IFN-alpha n3) was tested in 20 adults with asymptomatic human immunodeficiency virus type 1 (HIV-1) infection (> 400 CD4 lymphocytes/mm3). IFN-alpha n3 was self-injected three times per week for 3-6 months: 5 patients received 1 mega-IU (MIU)/dose, 10 received 5 MIU/dose, and 5 escalated to their maximum tolerated dose. Subjects were evaluated every 2-4 weeks through 2 months after cessation of treatment. Neuropsychological tests were given at 3-month intervals. Markers of IFN activity, anti-IFN neutralizing antibodies, and antiviral response were measured monthly. IFN-alpha n3 was safe and well tolerated: influenza-like symptoms were uncommon, laboratory toxicity was minimal, no adverse neurobehavioral side effects were evident, and no patient developed neutralizing antibodies against IFN. IFN-alpha n3 induced IFN-specific biologic responses and dose-related antiviral activity against HIV-1. Subjects showed stabilization of CD4 cells for > 20 months. IFN-alpha n3 should be studied in combination with other antiretroviral agents and in persons with more advanced HIV-1 infection.

HIV-infected monocytes form highly invasive network on basement membrane matrix and secrete high levels of 92-kd metalloproteinase (MMP-9), an enzyme that degrades basement membrane proteins. In the present study, using matrigel as a model basement membrane system, we demonstrate that treatment of human immunodeficiency virus (HIV)-infected monocytes with interferon-γ at 50 U/ml inhibited the ability of infected monocytes to form an invasive network on matrigel and their invasion through the matrigel matrix. These effects were associated with a significant reduction in the levels of MMP-9 produced by HIV-infected monocytes treated with interferon-γ 1 day prior to infection with HIV as compared with that of untreated HIV-infected monocytes. Monocytes treated with interferon-γ 1 day after HIV infection showed the presence of integrated HIV sequences; however, the levels of MMP-9 were substantially lower than those produced by monocytes inoculated with live HIV, heat-inactivated HIV, or even the control uninfected monocytes. Exposure of monocytes to heat-inactivated HIV did not result in increased invasiveness or high MMP-9 production, suggesting that regulation of metalloproteinase by monocytes was independent of CD4-gp120 interactions and required active virus infection. Furthermore, addition of interferon-γ to monocytes on day 10 after infection inhibited MMP-9 production by more than threefold with no significant reduction of virus replication. These results indicate that the mechanism of interferon-γ–induced down-regulation of MMP-9 levels and reduced monocyte invasiveness may be mediated by a mechanism independent of antiviral activity of IFN-γ in monocytes. Down-regulation of MMP-9 in HIV-infected monocytes by interferon-γ may play an important role in the control of HIV pathogenesis.

Murine macrophages supported exponential intracellular growth of Francisella tularensis LVS in vitro with a doubling time of 4 to 6 h. LVS was internalized and remained in a vacuolar compartment throughout its growth cycle. The importance of endosome acidification to intracellular growth of this bacterium was assessed by treatment of LVS-infected macrophages with several different lysosomotropic agents (chloroquine, NH4Cl, and ouabain). Regardless of the agent used or its mechanism of action, macrophages treated with agents that blocked endosome acidification no longer supported replication of LVS. Over several experiments for each lysosomotropic agent, the number of CFU of LVS recovered from treated macrophage cultures was equivalent to the input inoculum (approximately 10(4) CFU) at 72 h. In contrast, over 10(8) CFU was consistently recovered from untreated cultures. Pretreatment of macrophages with these endosome acidification inhibitors did not alter their ingestion of bacteria. Further, the effects of the inhibitors were completely reversible: inhibitor-pretreated LVS-infected macrophages washed free of the agent and cultured in medium fully supported LVS growth over 72 h. Endosome acidification is an important cellular event essential for release of iron from transferrin. The growth-inhibitory effects of both chloroquine and NH4Cl were completely reversed by addition of ferric PPi, a transferrin-independent iron source, at a neutral pH but not by addition of excess holotransferrin. Thus, intracellular localization in an acidic vesicle which facilitates the availability of iron essential for Francisella growth is a survival tactic of this bacterium, and iron depletion is one mechanism that macrophages use to inhibit its growth.

We have previously shown that the treatment of monocytes with interferon-gamma (IFN-gamma) prior to exposure with human immunodeficiency virus type-1 (HIV) results in complete inhibition of HIV infection of monocytes. In the present report, we have extended this study to obtain information on the mechanism(s) underlying IFN-gamma-induced inhibition of HIV infection of monocytes. To examine the effect of IFN-gamma on HIV entry, the first event in the infectious cycle of the virus, we amplified HIV-gag sequences in the genomic DNA and RNA of IFN-gamma treated monocytes, and found no evidence for the presence of either proviral DNA or HIV RNA sequences. These results were consistent with the absence of intracellular HIV particles either in the latent or actively replicating state as determined by flow-cytometric analysis of these cells. Furthermore, no HIV-induced cytopathic effects, such as multinucleated giant cell formation or cell death, were observed in IFN-gamma-treated monocytes after their exposure to HIV. Stimulation of IFN-gamma-treated monocytes 6 days postinfection with tumor necrosis factor-alpha (TNF-alpha), which is known to augment HIV replication in the infected cells, did not result in the induction of the HIV indicating the absence of latent HIV infection in IFN-gamma-treated monocytes. Treatment of monocytes with IFN-gamma, TNF-alpha, or with a combination of the two agents which is known to induce antimicrobial free radical nitric oxide (NO2- in the murine system did not induce NO2- production human monocytes suggesting the antiviral activity of IFN-gamma to be independent of NO2(-)-mediated killing of HIV or HIV-infected monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

The hallmark of human immunodeficiency virus (HIV) infection is the progressive loss of CD4+ T-cells over a prolonged interval. In the infected individual, two types of cells are infected by HIV: CD4+ T-cells and tissue macrophages. Levels of HIV in blood and tissues are dependent upon and change with the stage of the infection. Acute infection, usually lasting weeks to months after initial exposure to the virus, is characterized by a substantial viremia in which HIV actively replicates within blood leukocytes and high titers of free virus are found in plasma (more than 10 000 infectious virions/ml blood) (Clark et al. 1991; Daar et al. 1991). The chronic subclinical phase of infection is notable for low levels of plasma viremia and of virus-infected cells (less than 100 infectious virions/ml blood) (Ho et al. 1989). The major reservoirs for HIV in blood are CD4+ T-cells which are infected at a frequency of about 0.1 to 1% (Schnittman et al. 1989). HIV-infected CD4+ T-cells have on average only one proviral DNA copy integrated into genomic DNA. Less than 0.1% of these infected cells is transcriptionally active at any given time (Harper et al. 1986; Simmonds et al. 1990). During subclinical infection, the frequency of blood cells that express HIV mRNA and presumably produce infectious virus is only 0.01% (Harper et al. 1986; Clarke et al. 1990; Daar et al. 1991). During subclinical disease when very few cells are producing virus in blood, it is likely that cells in tissue provide most of the actively replicating virus that maintains infection during the long latent interval of 8 to 12 years (Lifson et al. 1988).

An IgM monoclonal antibody (mAb) recognized surface antigens specific to Francisella tularensis wild-type (Schu4) and live vaccine strain (LVS), and reacted with both in ELISA and slide agglutination tests. This mAb also reacted with LVS microorganisms in tissues of infected mice as assessed by an indirect fluorescence technique. Western blot analysis showed the mAb to react with antigens associated with F. tularensis LPS. J. Leukoc. Biol. 53: 112–116; 1993.

NFχB is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocyte leukemias. There is little information available on the response of NFχB to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NFχB binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NFXB in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NFχB from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SPI was unaffected by addition of TPA. The disappearance of NFXB from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NFXB species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NFχB. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NFχB and HIV-1 replication induced by phorbol esters in promonocyte leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes.

Interferon (IFN) plays an important role in the treatment and pathogenesis of HIV disease. Recent studies show beneficial effects of IFN alpha in the treatment of HIV-associated Kaposi's Sarcoma and early HIV-infection. Moreover, cell culture studies support these beneficial effects. HIV infection of monocytes is blocked by IFN alpha administered at the time of viral challenge. The IFN alpha-treated cells show no evidence of HIV infection. Viral gene products produced in monocytes infected with HIV then treated with IFN alpha gradually decrease to baseline. Large quantities of proviral DNA are seen in the HIV-infected IFN alpha-treated cells with little evidence for viral transcription suggesting true microbiological latency. While most viral infections of cells result in IFN production, HIV is a notable exception. Indeed, HIV does not induce monocytes to produce IFN alpha and blocks its production following poly(I).poly(c) stimulation. This allows HIV yet another mechanism to evade an important host antiviral response. Paradoxically, the appearance of IFN activity in sera of HIV-infected patients is associated with disease progression, not resolution. Recent observations showing that the interaction between HIV-infected monocytes and PBMC results in the production of IFN alpha s with reduced anti-HIV activity may help explain this paradox. Thus, IFN alpha plays an important but complex role in HIV disease. The elucidation of cellular factors that regulate the antiretroviral effects of IFN alpha may lead to the development of novel therapeutic strategies for HIV infection.

HIV infection produces symptomatic disease only after a relatively long incubation period which can exceed 10 years (1). During the interval of subclinical infection, virus replication is apparently held in check by host immune reactions and certain regulatory factors intrinsic to the viral genome. Recent studies document high level viremia (10 to 104 TCID50/ml plasma) in acute HIV infection that rapidly (6 to 8 weeks) subsides to undetectable levels (2). The frequency of productively infected cells in blood after this early and formidable viremia is 0.01 to 0.001 %. This improbably low frequency remains constant through end-stage disease (3). Components of the host immune response that constrain virus replication after the acute viremic interval are still incompletely defined. Vigorous humoral and cellular immune reactions are easily demonstrated against both structural and regulatory HIV gene products (4).

The live vaccine strain (LVS) of Francisella tularensis caused lethal disease in several mouse strains. Lethality depended upon the dose and route of inoculation. The lethal dose for 50% of the mice (LD50) in four of six mouse strains (A/J, BALB/cHSD, C3H/HeNHSD, and SWR/J) given an intraperitoneal (i.p.) inoculation was less than 10 CFU. For the other two strains tested, C3H/HeJ and C57BL/6J, the i.p. log LD50 was 1.5 and 2.7, respectively. Similar susceptibility was observed in mice inoculated by intravenous (i.v.) and intranasal (i.n.) routes: in all cases the LD50 was less than 1,000 CFU. Regardless of the inoculation route (i.p., i.v., or i.n.), bacteria were isolated from spleen, liver, and lungs within 3 days of introduction of bacteria; numbers of bacteria increased in these infected organs over 5 days. In contrast to the other routes of inoculation, mice injected with LVS intradermally (i.d.) survived infection: the LD50 of LVS by this route was much greater than 10(5) CFU. This difference in susceptibility was not due solely to local effects at the dermal site of inoculation, since bacteria were isolated from the spleen, liver, and lungs within 3 days by this route as well. The i.d.-infected mice were immune to an otherwise lethal i.p. challenge with as many as 10(4) CFU, and immunity could be transferred with either serum, whole spleen cells, or nonadherent spleen cells (but not Ig+ cells). A variety of infectious agents induce different disease syndromes depending on the route of entry. Francisella LVS infection in mice provides a model system for analysis of locally induced protective effector mechanisms.

Fixed macrophages within tissues and the mononuclear phagocytes cabled to sites of inflammation are premier scavenger cells able to eliminate most infectious threats by a wide array of toxic effector molecules and hydrolytic enzymes. Paradoxically, a variety of protozoa, bacteria, fungi, and viruses preferentially infect and replicate within these same scavenger cells. Sites of replication include not only the phagobysosome, but also the cytoplasm of infected cells. In many instances, these microorganisms can also be killed by the infected macrophage host cell. Complex signals generated through the cytokine network of local and systemic immune reactions induce a state of activation in the infected cell which kills the intracellular parasite (presumably without killing the infected macrophage). Effector molecules that such activated macrophages use to kill and eliminate these pathogens have always been a mystery. In this review, we examine the experimental approaches that identified nitrogen oxides derived from L-arginine as essential components in the microbicidab activity of cytokineactivated macrophages, and discuss the fascinating, but complex interactions of host cells, cytokines, and infectious pathogens that regulate production and action of toxic nitrogen oxides. Details of the biochemical pathways for nitrogen oxidation of L-arginine and its regulation within mammabian cells are now emerging [1-3]. Although not fully characterized, it is known that nitric oxide (NO) is a short-lived intermediate product of this novel pathway

Functionally diverse subpopulations of macrophages and lymphocytes, a wide array of stimulatory signals, and an enormous effector repertoire of activated macrophages keeps this field dynamically active. We review new advances in the identification of cytokines that interact to activate macrophages, and in the discovery of effector molecules used by activated macrophages to destroy their targets.

Tumor necrosis factor is important in systemic and cutaneous defense, homeostasis, and many disease states. The numerous and diverse effects of tumor necrosis factor are best understood when considered as concentration-dependent, with normal homeostasis progressing to defense followed by toxic effects. Understanding tumor necrosis factor is important for the dermatologist as more studies appear in our literature and potential clinical uses of tumor necrosis factor (and possible anti-tumor necrosis factor agents) are realized.

Monocytes treated with interferon-alpha (IFN-alpha) at virus challenge show no evidence of human immunodeficiency virus (HIV) infection: no p24 antigen or reverse transcriptase (RT) activity, no viral mRNA and no proviral DNA. Levels of p24 antigen and RT activity in monocytes infected with HIV 1-3 weeks before IFN-alpha treatment gradually decrease to baseline. HIV-induced cytopathic changes are markedly reduced, as are levels of HIV mRNA: the frequency of productively infected cells is less than or equal to 1%. But, levels of proviral DNA in the IFN-alpha-treated and control HIV-infected cells are indistinguishable, and remain so through 3 weeks. Large quantities of proviral DNA in IFN-alpha-treated cells with little active transcription suggest true microbiological latency. The major potential source for IFN-alpha in HIV-infected patients is the macrophage. With any of 15 virus isolates, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, IFN-omega or IFN-beta are not detected nor the mRNA expressed in HIV-infected or uninfected monocytes. Both uninfected and HIV-infected monocytes produce high levels of these cytokines after treatment with synthetic double-stranded RNA (poly-I:C). Uninfected monocytes also produce high levels of IFN-alpha after treatment with Poly-I:C, Newcastle disease virus or herpes simplex virus. In marked contrast, HIV-infected monocytes express no IFN-alpha activity or mRNA before or after treatment with any of these agents. The markedly diminished capacity of HIV-infected monocyte to produce IFN-alpha reflects a specific transcriptional block and may be an adaptive mechanism of virus to alter basic microbicidal functions of this cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Human immunodeficiency virus (HIV-1) infected and replicated in primary cultures of normal human ileal and colonic epithelial cells. Monocyte-tropic strains (ADA, 24, and 36) were better able to replicate in the gastrointestinal (GI) cells than the T-cell-tropic HIV strain HTLV-IIIB. In some cultures, virus replication persisted through several months. Intestinal epithelium may be an initial target and reservoir for HIV and a vector for virus dissemination and transmission.

Recombinant hematopoietic colony-stimulating factors have profound effects on developing and mature granulocytes, macrophages, and lymphocytes. Use of these agents for treatment of disease may result in a variety of adverse cutaneous reactions. The recent discovery of colony-stimulating factor production by keratinocytes and dermal cells suggests that these agents may also be significant in cutaneous homeostasis and in the pathogenesis of cutaneous diseases.

Nitric oxide (NO) produced by cytokine-treated macrophages and hepatocytes plays a vital role in protective host responses to infectious pathogens. NO inhibits iron-sulfur-dependent enzymes involved in cellular respiration, energy production, and reproduction. Synthesis of L-arginine-derived nitrite (NO2-), the oxidative end product of NO, directly correlates with intracellular killing of Leishmania major, an obligate intracellular protozoan parasite of macrophages: the level of NO2- production is a quantitative index for macrophage activation. The competitive inhibitor of NO synthesis, monomethylarginine (NGMMLA), inhibits both parasite killing and NO2- production. For Leishmania, the parasite itself participates in the regulation of this toxic effector mechanism. This participation is mediated by parasite induction of tumor necrosis factor alpha (TNF alpha), an autocrine factor of macrophages: NO synthesis by interferon-gamma (IFN-gamma)-treated cells can be blocked by monoclonal antibodies to TNF alpha. NO production by IFN gamma-treated hepatocytes is of special interest in malaria infections: sporozoite-infected hepatocytes kill the intracellular malaria parasite after treatment with IFN gamma; this killing is inhibited by NGMMLA.

Mononuclear phagocytes are major participants in human immunodeficiency virus (HIV) disease. These cells function as susceptible targets, persistent reservoirs for virus in tissue and key immunoregulatory elements that control the level of virus replication and the extent of disease. In this review, the second of the series, Monte Meltzer and colleagues review the distinct interactions between HIV and monocytes and between HIV and T cells. Understanding this dualism may more clearly define both the pathogenesis of HIV disease and strategies for therapeutic intervention.

Interferon alpha (IFN-alpha) induces significant antiretroviral activities that affect the ability of human immunodeficiency virus (HIV) to infect and replicate in its principal target cells, CD4+ T cells and macrophages. A major endogenous source of IFN-alpha during any infection is the macrophage. Thus, macrophages have the potential to produce both IFN-alpha and HIV. In this study, we examined the production of IFN-alpha and other cytokines by macrophage colony-stimulating factor (M-CSF)-treated cultured monocytes during HIV infection. Tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), IL-6, IFN-omega, or IFN-beta were not detected nor was the mRNA expressed in either uninfected or HIV-infected monocytes. However, both uninfected and HIV-infected monocytes produced high levels of each of these cytokines after treatment with synthetic double-stranded RNA [poly(I).poly(C)]. Uninfected monocytes also produced high levels of IFN-alpha after treatment with poly(I).poly(C), Newcastle disease virus, or herpes simplex virus. In marked contrast to the preceding observations, HIV-infected monocytes produced little or no IFN-alpha before or after treatment with any of these agents. The absence of detectable IFN-alpha activity and mRNA in poly(I).poly(C)-treated HIV-infected monocytes was coincident with high levels of 2',5' oligoadenylate synthetase and complete ablation of HIV gene expression. The antiviral activity induced by poly(I).poly(C) may be a direct effect of this synthetic double-stranded RNA or secondary to the low levels of IFN-beta and IFN-omega produced by infected cells. The markedly diminished capacity of HIV-infected monocytes to produce IFN-alpha may reflect a specific adaptive mechanism of virus to alter basic microbicidal functions of this cell. The inevitable result of this HIV-induced cytokine dysregulation is virus replication and persistence in mononuclear phagocytes.

Peritoneal macrophages from C3H/HeN mice treated i.p. with T cell mitogens or viable BCG organisms were cytotoxic to syngeneic tumor cells in vitro. Macrophages from endotoxin-unresponsive C3H/HeJ mice treated with BCG or T cell mitogens, however, were not tumoricidal. Furthermore, unlike cells from C3H/HeN mice, macrophages from C3H/HeJ mice could not be activated for tumor cytotoxicity after in vitro treatment with bacterial endotoxins or with lymphokine-rich supernatants. The subnormal induction of cytotoxic macrophages after in vitro or in vivo treatments in C3H/HeJ mice appears to be a highly selective defect. Macrophage responses (yield, phagocytosis, or peroxidase staining) in inflammatory exudates induced by BCG, T cell mitogens, or heterologous serum in C3H/HeJ or C3H/HeN mice were identical. C3H/HeJ macrophages also responded normally in vitro to chemotactic lymphokines. Thus, C3H/HeJ macrophages possess a profound and selective defect in tumoricidal capacity. This defect was not dependent upon exogenous endotoxins. Defective macrophage cytotoxic responses may reflect non-LPS related functions regulated by the LPS gene.

The in vitro chemotactic response of peritoneal macrophages from mice infected intraperitoneally (ip) with Mycobacterium bovis, strain BCG, was greater than the response of macrophages from uninfected mice in terms of rate, magnitude, and sensitivity. This increase in chemotactic responsiveness induced after a single ip injection of BCG was apparent in macrophages harvested 15 hr later, became maximal by 3 wk, and persisted through 6 wk. Peritoneal macrophages from mice treated with one of several macrophage activation agents (endotoxin, polynucleotides, or concanavalin A) had chemotactic responses to both lymphocyte and complement-derived chemotactic factors comparable to that seen with BCG-activated macrophages. Macrophages from peritoneal exudates induced by oil or thioglycollate, however, were less responsive to chemotactic stimuli than were macrophages from untreated mice. Increased chemotactic responsiveness was a functional property of the activated macrophage.

Although macrophages from Mycobacterium bovis, strain BCG-infected C3H/HeN mice were highly cytotoxic to tumor cells in vitro, cells from BCG-infected A/J mice were not tumoricidal. Varying the dose of BCG or the time of infection did not evoke cytotoxic activity with A/J macrophages. Inflammatory responses to BCG infection in A/J mice were relatively normal: the yield of macrophages was about ¼ that from C3H/HeN mice; however, no differences between strains were detected for inflammation-induced increases in macrophage phagocytic capacity or in percentage of peroxidase-positive cells. Similarly, production of macrophage activation factor activity by tuberculin-stimulated BCG-immune spleen cell cultures was also intact in A/J mice. Thus, despite adequate inflammatory responses and production of active lymphokines, macrophages from BCG-infected A/J mice were not activated for tumor cytotoxicity. Under certain conditions, however, these macrophages could develop cytotoxic activity. Macrophages from BCG-infected but not control A/J mice developed tumoricidal activity after further in vitro treatment with lymphokines, bacterial lipopolysaccharides (LPS), or certain plant lectins. Peritoneal exudate macrophages first treated with lymphokines also developed cytotoxic activity but only after further treatment with LPS or lectins. The phenotypic expression of the A/J genetic defect in macrophage cytotoxic activity was very similar to the defect of lipid A-unresponsive C3H/HeJ mice except for the concentration of LPS required for cytotoxic activity by lymphokine-treated cells. Other responses of A/J mice to LPS were also different from those of the unresponsive C3H/HeJ mice. Responses of A/J mice to the lethal toxicity of LPS or of A/J macrophages to direct toxicity of LPS in vitro, responses influenced by the Lps gene, were normal. On the other hand, macrophage cytotoxic activity and spleen cell proliferative responses to LPS in vitro, responses also controlled by the Lps gene, were abnormal. We conclude that the genetic basis for the macrophage tumoricidal defect of A/J mice represents either a previously undescribed allelic change at the Lps locus or, what is more likely, gene mutation(s) at entirely different loci.

Culture fluids from a phorbol myristate acetate-stimulated EL-4 thymoma cell line were previously found to activate mouse macrophages to become nonspecifically tumoricidal. By gel filtration, 23,000- and 45,000-MW peaks were identified. In this study we compared EL-4 culture fluid activity with that obtained from antigen-stimulated mouse spleen cells. By four criteria, functional activity from the two sources was comparable: 1) Macrophage activation could be separated temporally into two steps, priming and triggering. 2) Macrophages activated by the fluids exhibited peak cytotoxic activity within 5–9 hr; no cytotoxic activity was demonstrable if addition of target cells was delayed 14 hr. 3) Mouse peritoneal exudate macrophages, but not resident macrophages, became cytotoxic after a 5-hr incubation with culture fluids. 4) Macrophages from certain strains of mice were incapable of activation; the same pattern of strain unresponsiveness was observed with both EL-4 and spleen cell culture fluids. Thus, it is likely that the two EL-4 products and the mouse spleen cell lymphokine activate the same cytotoxic mechanism.

The effects of prolactin on lactation and reproductive organs are well known. However, the other possible target organs and physiological consequences of altered levels of circulating prolactin remain poorly understood. In this study, mice were treated with bromocryptine, a dopamine receptor agonist that inhibits pituitary prolactin secretion. Bromocryptine treatment prevented T-cell-dependent induction of macrophage tumoricidal activity after the intraperitoneal injection of Listeria monocytogenes or Mycobacterium bovis. Coincident treatment with ovine prolactin reversed this effect. Of the multiple events leading to macrophage activation in vivo, the production by T-lymphocytes of gamma-interferon was the most impaired in bromocryptine-treated mice. Lymphocyte proliferation after stimulation with mitogens in vitro was also depressed in spleens of bromocryptine-treated mice, and coadministration of prolactin also reversed this effect. Bromocryptine treatment also reduced the number of deaths resulting from inoculation of mice with Listeria; exogenous prolactin significantly reversed this effect. The critical influence of pituitary prolactin release on maintenance of lymphocyte function and on lymphokine-dependent macrophage activation suggests that, in mice, lymphocytes are an important target tissue for circulating prolactin.

Mouse strains were surveyed for susceptibility to the lethal effects of Rickettsia akari infection: A/HeN, A/J, C3H/HeJ, C57BL/10ScCR, C57BL/10ScN, and NZW/N were highly susceptible: AL/N, BALB/cN, CBA/N, C3H/HeN, C57BL/6N, C57BL/10J, C57L/N, DBA/2N, and P/J were resistant. These data show that resistance to R. akari infection in mice can be influenced by genetic factors. That this influence may, in part, be mediated through the activated macrophage was suggested by observations that animals from strains derived from the A strain or from strains with the Lps d gene, strains with previously characterized and genetically different defects in development of nonspecific macrophage microbicidal and tumoricidal activities, were highly susceptible.

Peritoneal macrophages from mice with syngeneic transplantable footpad tumors were less responsive in vitro to chemotactic stimuli than were macrophages from mice without tumors. The macrophage chemotactic defect was about half the normal response and was evident in mice with nonpalpable 3 to 5-day tumors. Depressed macrophage chemotaxis persisted until the death of the mouse at 6 to 8 weeks. The number of peritoneal exudate cells elicited in tumor-bearing mice 8 to 14 days after intraperitoneal infection with BCG was also depressed. This inflammatory defect was detected 1 week after tumor transplantation and persisted for more than 5 weeks. The yield of peritoneal exudate macrophages induced by BCG infection in mice with tumors was about half the normal yield. In contrast to resident peritoneal macrophages, peritoneal exudate macrophages induced by BCG in tumor-bearing mice were as responsive to chemotactic stimuli as BCG-activated macrophages from control mice. BCG-activated macrophages from tumor-bearing or control mice were cytotoxic to tumor cells in vitro; no differences in tumoricidal responses of these BCG-activated macrophages were evident. The number of BCG organisms necessary to induce activated tumoricidal macrophages in mice with or without tumors was also identical. Peritoneal exudate macrophages from tumor-bearing mice infected with BCG at a dose that did not induce tumoricidal cells showed chemotactic defects similar to those of the resident macrophages. These results suggest that the response to BCG infection in tumor-bearing mice was defective. The defect was quantitative: a sequela of abnormal inflammatory responses. BCG-activated macrophages from tumor-bearing and control mice were functionally indistinguishable.

Peritoneal macrophages from Schistosoma mansoni -infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro . In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro . However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ( 1 3 to 1 5 those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni -infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni -activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.

Peritoneal macrophages (Mφ) from C3H/HeN mice became cytotoxic for 1023 tumor cells after incubation with lymphokine (LK) for 8–12 hr and lost tumoricidal activity by 22 hr in the continued presence of LK; bacterial endotoxin (LPS) was ineffective in inducing tumoricidal activity. Mφ from C3H/HeJ mice were not activated for tumor cytotoxicity after treatment with LK or LPS. C3H/HeN Mφ acquisition of tumoricidal activity was accompanied by unique changes in Mφ lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acids (UFA) increased two- to threefold when the cells showed maximal tumoricidal activity and returned to control levels when the Mφ lost tumoricidal activity. LPS treatment of C3H/HeN Mφ and LK or LPS treatment of C3H/HeJ Mφ did not cause characteristic Mφ lipid alterations. To determine at what stage during Mφ activation the correlative CHOL and UFA compositional changes were occurring, C3H/HeN Mφ were primed with LPS or low concentrations of LK and triggered with LPS or LK; Mφ lipid and fatty acid composition was monitored at each stage. LK was shown to be able to prime and trigger whereas LPS could only trigger LK-primed Mφ for tumor cytotoxicity. In all cases, the increase in Mφ CHOL and UFA content occurred at the triggering step for tumor cytotoxicity rather than at the priming step. These data suggest that there is a correlation between the effects of endogenous and exogenous factors that control expression of Mφ tumoricidal activity and their effects on Mφ CHOL and UFA content; the establishment of these changes in Mφ lipid composition occurs at a time when the cells are triggered for tumor cytotoxicity.

Macrophages from endotoxin (LPS)-unresponsive C3H/HeJ mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that induce cytotoxicity with cells from LPS-responsive C3H/HeN mice. Under certain conditions, however, these macrophages could become tumoricidal: macrophages from in vivo immune reactions such as those induced by BCG infection, but not cells from irritant-induced peritoneal exudates, developed full cytotoxic capability after further exposure in vitro to microgram per milliliter concentrations of LPS or to supernatants from antigen-stimulated leukocyte cultures (lymphokines). Capacity of macrophages from BCG-infected C3H/HeJ mice to become tumoricidal after LPS exposure was gradually lost with time in culture and was absent by 24 hr. Requirements for at least two activation stimuli for cytotoxic activity with C3H/HeJ macrophages could also be fulfilled entirely in vitro: cells cultured in lymphokines plus LPS were fully tumoricidal; macrophages cultured in either agent alone were not cytotoxic. Similar interactions between lymphokines and LPS could be demonstrated with macrophages from LPS-responsive C3H/HeN mice. Tumoricidal activity by C3H/HeN macrophages cultured in lymphokines plus nanogram per milliliter concentrations of LPS was considerably greater than that by cells cultured in either agent alone. LPS and lymphokines were synergistic in their action on macrophage cytotoxicity. The synergistic effect of LPS on cytotoxicity by lymphokine-activated macrophages was evident after a 15-min pulse and was not dependent upon fluid-phase LPS. Synergistic action, however, was dependent upon 1.) treatment sequence: LPS was active only if given simultaneously with or after lymphokine treatment; LPS exposure before lymphokine treatment was ineffective, and 2.) treatment interval: capacity of lymphokine-treated macrophages to express cytotoxic activity after LPS exposure decayed with time in culture and was lost by 24 hr. Thus, macrophage activation for tumor cytotoxicity in both C3H/HeJ and C3H/HeN mice was the final result of a cascade of short-lived intermediary reactions in a defined sequence. Tumoricidal activity of fully activated cells and responsiveness of each noncytotoxic precursor cell to activation stimuli were all short lived macrophage reactions. Present evidence suggests that none of these reactions was reversible. Completion of each state of macrophage activation was completely dependent upon the simultaneous presence of localized activation signals and macrophage precursors able to respond.

Peritoneal cells from mice infected ip with Mycobacterium bovis, strain BCG, were cytotoxic to syngeneic tumor cells in vitro. Cytotoxicity was estimated by measurement of release of tritiated-thymidine (3-H-TDR) from prelabeled target cells. The cell responsible for tumor cytotoxicity was the macrophage. Macrophages from uninfected mice or from oil-, starch-, or thioglycollate-induced peritoneal exudates had little effect on labeled tumor monolayers. Tumoricidal macrophages were present at 3-7 days and persisted through 6 weeks after a single BCG injection. Two neoplastic/nonneoplastic cell-line pairs, all four of the cell lines derived from a cloned syngeneic embryo cell line, were used as target cells for BCG-activated macrophages. Both tumor cell lines released significantly more 3-H-TDR than did the two nonneoplastic lines. In a mixed neoplastic/nonneoplastic target cell population, BCG-activated macrophages selectively destroyed the neoplastic cells; nonneoplastic cells were not affected as "innocent bystanders".