Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants.

An adjuvant role for certain short bacterial immunostimulatory DNA sequences (ISSs) has recently been proposed on the basis of their ability to stimulate T helper-1 (Th1) responses in gene-vaccinated animals. We report here that noncoding, ISS-enriched plasmid DNAs or ISS oligonucleotides (ISS-ODNs) potently stimulate immune responses to coadministered antigens. The ISS-DNAs suppress IgE synthesis, but promote IgG and interferon-gamma (IFN-gamma) production. They furthermore initiate the production of IFN-gamma, IFN-alpha, IFN-beta, and interleukins 12 and 18, all of which foster Th1 responses and enhance cell-mediated immunity. Consideration should be given to adding noncoding DNA adjuvants to inactivated or subunit viral vaccines that, by themselves, provide only partial protection from infection.

Failure of dideoxynucleosides to inhibit human immunodeficiency virus replication in cultured human macrophages.

Primary human monocyte-derived macrophages (MDM) were shown to have diminished deoxynucleoside kinase activities compared to T lymphoblasts, and a reduced ability to phosphorylate dideoxynucleosides with anti-human immunodeficiency virus (HIV) activity. These drugs, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyadenosine (ddA), which are potent anti-HIV agents in CD4 lymphocytes, did not inhibit HIV replication in MDM, even at concentrations of 100 microM. This drug concentration of AZT is approximately 100-fold higher than the levels attained in the serum of treated patients and the levels required to inhibit HIV replication in lymphocytes. These observations may explain the failure of AZT therapy to clear viremia, consistent with the presence of a drug-resistant reservoir of infected cells in vivo. New therapeutic approaches to inhibit the replication of HIV in MDM may be needed.

Interferons and bacterial lipopolysaccharide protect macrophages from productive infection by human immunodeficiency virus in vitro.

To determine the effects of immunomodulatory agents upon HIV replication in macrophages, cultured monocyte-derived macrophages were treated with various substances and then infected with a macrophage-tropic strain of HIV-1. Pretreatment with rIFN-alpha, IFN-beta, and IFN-gamma, or bacterial LPS prevented viral replication in macrophages. In treated cultures, little or no infectious HIV or p24 core antigen was released into the supernatant, no virions were seen by electron microscopy, no viral RNA or DNA was detectable in the cell lysates, and no cytopathology (as determined by multinucleated giant cell formation) occurred. In contrast, pretreatment with a wide dose range of recombinant IL-1 beta, IL-2, IL-4, IL-6, M-CSF, TNF, or lymphotoxin failed to protect macrophages from productive infection by HIV. A consistent effect of granulocyte/macrophage-CSF on HIV replication in macrophages was not observed. In dose response studies, pretreatment with approximately 100 U/ml of IFN-alpha, approximately 10 U/ml of IFN-beta, or approximately 100 U/ml of IFN-gamma was sufficient to prevent virion release maximally and to prevent cytopathology completely. In kinetic studies, IFN-alpha, IFN-gamma, or LPS were added to the macrophage cultures either before or after infection with HIV. Even when added 3 d after infection with a multiplicity of 1 50% tissue-culture infectious dose per cell, all three treatments markedly reduced virion release, suggesting that these agents act at a point in the viral life cycle beyond the early events of virus binding, penetration, and uncoating. These data indicate that HIV replication in previously uninfected macrophages may be regulated by an inducible host cell mechanism. These findings may explain the restricted replication of HIV in macrophages in vivo and suggest an antiviral role for interferons in the therapy of HIV infection.

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.

Apoptosis as a mechanism of cell death in cultured T lymphoblasts acutely infected with HIV-1.

The mechanisms by which HIV-1 infection kills T lymphocytes are not clearly established. Apoptosis is an internally programmed cell death pathway that may regulate both T cell development and senescence, and that is characterized by cleavage of DNA at internucleosomal regions. The present experiments show that acute HIV-1 infection of MT2 lymphoblasts and activated normal peripheral blood mononuclear cells induces apoptosis. The addition of anti-gp120 neutralizing antibody, after HIV-1 infection of MT2 cells, permitted sustained high levels of viral replication, but blocked apoptosis and cell death. Apoptosis may account for the direct cytopathologic effects of HIV-1 in T cells.

The emerging role of CD40 ligand in HIV infection.

CD40 ligand (also called CD40L, CD154, or TNFSF5) is a membrane protein expressed mainly by activated CD4+ T cells, which interacts with its receptor, CD40, on a variety of cells. The crucial importance of the CD40L-CD40 system for many immune responses has been extensively described. This review focuses on the multiple roles that this system may play in HIV infection. In early HIV infection, CD40L expression contributes to the immunological control of viral replication by inducing HIV-suppressive chemokines and supporting the production of anti-HIV antibodies and cytotoxic T cells. However, by activating antigen-presenting cells, such as dendritic cells and macrophages, CD40L can also lead to increased CD4+ T cell activation, which promotes the replication of HIV in these lymphocytes. Later, with the development of AIDS, CD40L-expressing CD4+ T cells become selectively depleted, perhaps as a result of a gp120-induced signal through CD4 that down-regulates CD40L expression. This acquired CD40L deficiency may explain the similarity between the types of opportunistic infections that occur in AIDS and in congenital CD40L deficiency. Vaccines or other strategies that promote the growth of CD4+ T cells capable of expressing CD40L may help to sustain host immunity against HIV and prevent AIDS-defining opportunistic infections.

Significance of T cell apoptosis for macrophages in HIV infection.

Apoptosis is a major form of cell death in HIV infection. This review presents current ideas on the role of apoptosis in the development of AIDS. HIV may cause apoptosis either directly in individual CD4+ T cells through cellular infection and through the release of gp120 envelope protein, or indirectly by initiating systemic disturbances in the immune system. Furthermore, although apoptosis is often assumed to be a biological dead end, linear, unintegrated retroviral DNA survives apoptosis in avian leukosis virus systems. Macrophages avidly phagocytose apoptosing cells, and the viral DNA in apoptotic debris might spontaneously transfect macrophages and lead to the production of new virions. Such a hypothetical accessory infection pathway may explain why anti-HIV cytotoxic cells are unable to clear this virus from the body. Strategies directed against the "recycling" of the retroviral genomes present in apoptotic debris may ultimately have a role in the treatment of HIV infection.

The immunological potential of apoptotic debris produced by tumor cells and during HIV infection.

Apoptosis is a major cause of cell death in health and disease. In contrast to necrosis, apoptosis does not induce an inflammatory response and the cellular debris produced by apoptosis has been assumed to be biologically inert. This review challenges this assumption by suggesting that apoptotic debris (especially in the context of growing tumors or during HIV infection) may have immunological activities, mainly immunosuppressive but perhaps also immunostimulatory. In many cases, the surface of apoptotic cells differs from normal cells in that phosphatidylserine (PS) is aberrantly exposed on the external face of the cell membrane. Liposomes composed of PS may down-modulate macrophage anti-leishmanial activities, suppress macrophage TNF production, suppress lymphocyte proliferation, and increase macrophage proliferation. "Membrane shedding" has been described in certain malignancies where apoptosis may be occurring, and the shed tumor membrane vesicles have been shown to reduce MHC class II expression on macrophages and decrease lymphocyte responsiveness, perhaps because of their ganglioside content. Finally, the apoptotic debris from HIV-infected cells may bear on its surface viral proteins which contain immunosuppressive peptide sequences. This debris may also use viral envelope proteins to fuse into macrophages and thereby avoid phagocytosis and lysosomal destruction. These considerations suggest that the flux of apoptosing cells and debris through the immune system that occurs during tumor growth and HIV infection should not be assumed to be immunologically neutral. In particular, HIV-related apoptosis may have immunosuppressive effects in addition to the numerical depletion of lymphocytes.

Characterization of a macrophage-tropic HIV strain that does not alter macrophage cytokine production yet protects macrophages from superinfection by vesicular stomatitis virus.

Macrophages, unlike CD4+ T cells, can be productively infected by human immunodeficiency virus (HIV) without prior cellular activation. Cytopathic infection ensues without the induction of tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), or tissue factor genes. In detailed studies on TNF alpha, HIV infection did not affect the regulation of TNF alpha in response to bacterial lipopolysaccharide. In an effort to examine the interferon responsiveness of HIV-infected macrophages, the cells were challenged with vesicular stomatitis virus (VSV) with or without interferon pretreatment. Surprisingly, HIV-infected macrophages were completely resistant to VSV-induced lysis even in the absence of interferon; however, no interferon was detected in the supernatants of these infected cells. The resistance of HIV-infected macrophages to superinfection with VSV indicates a previously undescribed effect of HIV upon macrophage cellular metabolism.

Tumor necrosis factor production by human monocytes is a regulated event: induction of TNF-alpha-mediated cellular cytotoxicity by endotoxin.

Expression of cellular cytotoxicity by monocytes or macrophages has been conceived as an induced function secondary to collaboration in the immune response or to other agonists. However, a form of spontaneous cellular cytotoxicity by monocytes analyzed with unseparated human peripheral blood mononuclear cells (PBM) has been described by using the 6-hr 51Cr release from actinomycin D (ActD)-treated murine WEHI 164 cells, a target cell refractory to the cytotoxic effects of natural killer and cytolytic T cells. We observe that when cells are isolated under rigorously endotoxin-free conditions, there is no cytotoxicity. Inclusion of serum does not induce cellular cytotoxicity; however, cytotoxic activity is induced by the presence of as little as 1 pg/ml of bacterial lipopolysaccharide (LPS). PBM required 2 hr of preexposure to endotoxin in order to express full cytotoxic activity. We investigated the basis of the cytotoxicity of WEHI 164 cells and the effect of ActD. ActD-treated target cells are highly susceptible to the effects of TNF-alpha and TNF-beta (alpha-lymphotoxin), whereas untreated target cells were resistant. In contrast, ActD does not affect susceptibility to the cytotoxic effects of H2O2, and interleukin 1 is not cytotoxic to the target cells. With the use of a neutralizing monoclonal antibody specific for TNF-alpha, the cytotoxic activity induced by LPS greatly diminished and the amount of TNF-alpha neutralized is similar to that required for equivalent cytotoxicity. We conclude that monocytes present in human PBM are not "spontaneously" cytotoxic for ActD-treated WEHI 164 target cells, but that the reported cytotoxicity results from exposure to a level of endotoxin or endotoxin-like agonists to which the cells are exposed. The cytotoxicity is mediated mostly if not entirely by TNF-alpha, an established product of monocytes/macrophages. With the use of endotoxin-free conditions, PBM can be isolated in a cytotoxically latent state, suitable for analysis of the immunologic regulation of TNF-alpha-mediated monocyte cellular cytotoxicity.

Managing HIV. Part 3: Mechanisms of disease. 3.3 How HIV leads to opportunistic infections.

HIV-related immune deficiency differs from that due to other causes in its severity and inexorably progressive nature. It leads to multiple opportunistic infections in patients with advanced HIV infection, mostly with common endogenous and environmental organisms that usually pose little threat to human health.

Initial characterization of a lymphokine pathway for the immunologic induction of tumor necrosis factor-alpha release from human peripheral blood mononuclear cells.

Under endotoxin-free conditions, unstimulated human PBMC do not release TNF-alpha, as measured in a sensitive assay with 51Cr release in 6 h from actinomycin D-treated WEHI 164 cells. IFN-gamma alone at less than or equal to 10,000 U/ml is insufficient to elicit TNF-alpha release. Similarly, the lymphokine-rich supernatant of PBMC stimulated by allogeneic cells is also insufficient to induce TNF-alpha release in a short term assay. However, when PBMC are first primed with IFN-gamma for 48 h and then exposed to lymphokine supernatant for 6 h, effector cells within the PBMC population are triggered to express TNF-alpha-mediated cytotoxicity. All of the measured cytotoxicity is attributable to TNF-alpha because it could be abolished by a specific anti-TNF-alpha neutralizing mAb. Although IFN-gamma serves to prime PBMC in this assay system, it fails to trigger the release of TNF-alpha. Instead, a second lymphokine (provisionally termed "cytotoxicity triggering factor" (CTF) is required to induce TNF-alpha release from IFN-gamma-primed human PBMC. In kinetic studies, IFN-gamma priming was optimal when PBMC were exposed to IFN-gamma (150 U/ml) for 48 h. In contrast to the prolonged interval for priming, CTF need be present for 6 h or less for maximal induction of TNF-alpha-mediated cytotoxicity. In dose-response studies, IFN-gamma priming (48 h) required at least 4 U/ml and was complete with 20 to 100 U/ml. By using fully primed PBMC, the response to CTF followed a sigmoidal dose-response curve, which allowed the quantitation of CTF in half-maximal units. Activated Th lymphocytes constitute one cellular source for CTF. CTF is produced by cloned allorective T3+T4+T8-M1- Th cells after alloantigen stimulation, and also by nylon wool-purified T cells after stimulation with PMA and A23187 calcium ionophore. Unstimulated T cells do not release CTF. In physicochemical studies, CTF activity elutes from Sephadex G-100 as a major discrete peak of Mr 55 kDa and minor peaks of 14 kDa and greater than 150 kDa. On the basis of multiple criteria, CTF is distinguishable from several other cytokines: IFN-gamma, IL-1, IL-2, GM-CSF, MIF, CSF-1, TNF-alpha, and lymphotoxin (TNF-beta). We conclude that, by acting together, IFN-gamma and CTF provide a lymphokine pathway whereby Ag-responsive human Th cells induce the immunologic release of TNF-alpha from effector cells present in PBMC.

Characterization and growth in human macrophages of Mycobacterium avium complex strains isolated from the blood of patients with acquired immunodeficiency syndrome.

Strains of the Mycobacterium avium complex (MAC) yield opaque and transparent colonial variants when cultivated in vitro. The transparent variants are more virulent than the opaque for animals, but little is known about the respective roles of these colonial variants in humans. To assess which variant infects humans, various blood fractions from eight patients with MAC bacteremia were plated directly onto 7H10 agar. In cell fractionation studies, all the M. avium complex CFU were associated with leukocytes and none were found free in plasma. All colonies on the primary culture plate exhibited the transparent phenotype. However, during subculture in 7H9 broth or on Lowenstein-Jensen agar, opaque variants appeared in seven of eight strains. Isogenic pairs of transparent and opaque variants were prepared and used to infect in vitro human monocyte-derived macrophages from healthy seronegative individuals. Transparent variants invariably grew inside macrophages, but only one of seven opaque variants did so. These observations indicate that the bacteremia of M. avium complex in acquired immunodeficiency syndrome patients consists exclusively of the transparent variants, perhaps because these variants are able to multiply inside macrophages. In contrast, opaque variants appear after in vitro subculture and are controlled by human macrophages, consistent with their reduced virulence in animals.

The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes.

Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), are unusual among retroviruses in their ability to infect nondividing cells. The matrix proteins of several lentiviruses contain a short stretch of amino acids reminiscent of known nuclear localization signals. In HIV-1, this motif has been shown to function as a nuclear targeting sequence when conjugated to a heterologous protein, and to permit the active nuclear import of the HIV-1 preintegration complex in growth-arrested cells. In the present work, mutations were introduced in the matrix nuclear localization region of T-cell- and macrophage-tropic HIV-1 clones. The resulting viral mutants replicated with normal or even accelerated kinetics in dividing cells, including activated peripheral blood lymphocytes. However, in sharp contrast with wild-type virus, the mutants could not grow efficiently in terminally differentiated macrophages or establish a stable and inducible infection intermediate in unstimulated peripheral blood lymphocytes. Because macrophages represent a major viral reservoir in vivo, and because at any given time most T cells in the body are quiescent, these results strongly suggest that the karyophilic properties of the matrix protein are critical for the spread of the virus in HIV-infected individuals, and consequently for AIDS pathogenesis.