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Regulation of human immunodeficiency virus type 1 and cytokine gene expression in myeloid cells by NF-kappa B/Rel transcription factors

CD4+ macrophages in tissues such as lung, skin, and lymph nodes, promyelocytic cells in bone marrow, and peripheral blood monocytes serve as important targets and reservoirs for human immunodeficiency virus type 1 (HIV-1) replication. HIV-1-infected myeloid cells are often diminished in their ability to participate in chemotaxis, phagocytosis, and intracellular killing. HIV-1 infection of myeloid cells can lead to the expression of surface receptors associated with cellular activation and/or differentiation that increase the responsiveness of these cells to cytokines secreted by neighboring cells as well as to bacteria or other pathogens. Enhancement of HIV-1 replication is related in part to increased DNA-binding activity of cellular transcription factors such as NF-kappa B. NF-kappa B binds to the HIV-1 enhancer region of the long terminal repeat and contributes to the inducibility of HIV-1 gene expression in response to multiple activating agents. Phosphorylation and degradation of the cytoplasmic inhibitor I kappa B alpha are crucial regulatory events in the activation of NF-kappa B DNA-binding activity. Both N- and C-terminal residues of I kappa B alpha are required for inducer-mediated degradation. Chronic HIV-1 infection of myeloid cells leads to constitutive NF-kappa B DNA-binding activity and provides an intranuclear environment capable of perpetuating HIV-1 replication. Increased intracellular stores of latent NF-kappa B may also result in rapid inducibility of NF-kappa B-dependent cytokine gene expression. In response to secondary pathogenic infections or antigenic challenge, cytokine gene expression is rapidly induced, enhanced, and sustained over prolonged periods in HIV-1-infected myeloid cells compared with uninfected cells. Elevated levels of several inflammatory cytokines have been detected in the sera of HIV-1-infected individuals. Secretion of myeloid cell-derived cytokines may both increase virus production and contribute to AIDS-associated disorders.


INTRODUCTION
The human immunodeficiency virus (HIV-1) was first identified in 1983 (21,106,197) and is now recognized as the virus responsible for the progressive immune system degeneration that leads to the development of AIDS. As knowledge of HIV-1 and its interactions with the host immune system increases, it is apparent that a number of secondary factors such as bacterial or viral infections and other opportunistic infections influence the progression of asymptomatic individuals to AIDS. Although T cells are targeted for infection, CD4 ϩ cells of the myeloid lineage are also very important in HIV-1 disease. Promonocytes and macrophages serve as reservoirs of HIV-1 virions within various tissues such as the lung, skin, lymph nodes, and bone marrow. Physiological changes in HIV-1-infected myeloid cells including alterations in cytokine secretion, antigen presentation, and phagocytosis-in conjunction with secondary infections-may enhance HIV-1 replication, contribute to AIDS-associated disorders, and accelerate the progression of HIV-1 disease.
Efforts to control the HIV-1 pandemic focus on all aspects of the disease, including public awareness, education, development of therapies aimed at interrupting the viral replication cycle, enhancement of the host immune system, and vaccine development. More is known about this virus, perhaps, than any other pathogen, yet the most realistic goal in terms of therapeutic intervention may be a regimen of combination therapy designed to delay viral spread and/or disease progression. Understanding the factors which influence HIV-1 gene expression and replication will form the basis for the development of effective antiviral therapies.

COURSE OF HIV-1 INFECTION
HIV-1 infection is transmitted sexually, through intravenous contact with contaminated blood products, or across broken mucosal or epidermal epithelia. HIV-1 can also be transmitted vertically from infected mothers in utero or through lactation after birth (225). The typical course of HIV-1 disease begins with a primary infection that is followed in 50 to 70% of individuals by an acute viral syndrome characterized by a mononucleosis-like illness, generalized lymphadenopathy, intense plasma viremia, and a significant decrease in circulating CD4 ϩ T cells approximately 3 to 6 weeks after the initial infection (380). This is followed by extensive viral dissemination to surrounding tissues such as the lungs, bone marrow, and liver but particularly to the lymph nodes (281). At these sites, an immune response to HIV-1 that is responsible for the suppression of plasma viremia is mounted. As a result, the acute syndrome resolves, the number of circulating CD4 ϩ T cells recovers, and infection may enter a period of clinical latency that can last for 10 years or longer (280). During this time, HIV-1 persists in the lymph nodes, where it continues to replicate and infect circulating T cells (279). Recent developments indicate that during this asymptomatic or stationary phase, plasma viremia is maintained in a low steady state by a continuous, vigorous response of the immune system to the rapid replication of HIV-1 (56). Treatment of patients with potent inhibitors of HIV replication revealed that throughout the period of clinical latency, virus is rapidly cleared from the bloodstream. Furthermore, during this phase, CD4 ϩ lymphocytes are destroyed and replenished at a rate of ϳ2 ϫ 10 9 cells per day and new virions are produced and cleared from the circulation at a rate of ϳ1 ϫ 10 9 virions per day. Such a dynamic replication rate yields an extremely high mutation rate (142,402). The result, over a time course that encompasses years, is the net decline in the number of CD4 ϩ T cells and the gradual deterioration of multiple aspects of the immune response. In later stages of HIV-1 disease, virus is usually detectable in the peripheral blood (281) because of a breakdown in the ultrastructure of lymph nodes that permits virus to escape into the circulation (87). Once the circulating CD4 ϩ T-cell level drops below 200 cells per mm 3 , patients may become increasingly susceptible to life-threatening opportunistic infections and malignancies (87).

HIV-1 INFECTION OF MYELOID CELLS
CD4 ϩ cells of the myeloid lineage, including monocytes and macrophages, are important targets for HIV-1 infection in vitro and in vivo (143,(229)(230)(231)304). The peripheral blood monocyte represents an intermediate stage of maturation; terminally differentiated macrophages are found only after migration from the bloodstream into organs and tissues (236). Myeloid cells are susceptible to HIV-1 infection at multiple stages of differentiation (247). HIV-1-infected CD34 ϩ myeloid precursor cells are present in the bone marrow of a significant number of AIDS patients (304) and can be infected in vitro (98). Estimates of the proportion of circulating blood monocytes infected by HIV-1 range from less than 14% (368) to greater than 50% (144,146) as measured by PCR. HIV-1 infection of mononuclear phagocytes in vivo occurs predominantly in terminally differentiated macrophages in tissues such as the lungs, skin, brain, and lymph nodes (reviewed in reference 143). In situ hybridization for HIV-1 RNA in brain tissue of infected individuals shows a frequency of productively infected macrophages of 1 to 10% (173). Similarly, HIV-1 proteins or nucleic acids are detected in 10 to 50% of the alveolar macrophages isolated from bronchiolar lavage fluids (300,339). HIV-1-infected epidermal Langerhans cells, the dendritic antigen-presenting cells of the skin, are detected in up to 20% of seropositive patients (229,386). CD4 ϩ myeloid cells infected with HIV-1 are present in various other tissues including the placenta (Hofbauer cells) (9,199), liver (Kupffer cells) (50), and lymph nodes (follicular dendritic cells) (278,279).
Myeloid cells play an important role in HIV-1 disease, since they are probably the first cells infected during sexual transmission. Following infection by the epidermal route, HIV-1 can bind to cells such as epidermal Langerhans cells, macrophages, and tissue eosinophils. These cells are able to move from the epidermis into proximal lymphoid organs, through either the peripheral vascular system or the lymphatic system (225). Lymphatic drainage of HIV-1-infected cells may play a particularly important role in the infectious process. Following entry through afferent lymphatics, macrophages become part of the dendritic antigen-presenting population of cells in the lymph nodes. Presentation of viral antigens leads to an immune response against HIV-1; virus is simultaneously spread to uninfected cells by cell-cell transmission. The immediate result is a massive follicular hyperplasia seen early in the course of HIV-1 infection (149). Most remarkably, HIV-1infected macrophages in vitro are not subject to the same cytopathic effects as are T cells; in fact, populations of chronically infected myeloid cell lines can be maintained in culture for prolonged periods (229). Thus, HIV-1-infected myeloid cells, sequestered from recognition by the host immune system, represent a true reservoir for dissemination of HIV-1 to other macrophages, T cells, or target tissues.

MACROPHAGE TROPISM AND HOST PERMISSIVITY
Although most clinical isolates of HIV-1 are able to infect primary T lymphocytes or macrophages (94,178), various isolates that have differing tropisms for macrophages and myeloid cell lines have been identified (110,116). These isolates may exhibit altered kinetics of replication within these cell lines, and many do not result in a productive viral infection. The ability of HIV-1 isolates to infect a wide variety of cell lines appears to correlate with disease progression in the AIDS patients from whom these viruses were isolated. HIV-1 isolates from asymptomatic individuals grow slowly in phytohemagglutinin-stimulated mononuclear cells, lack the capacity to form syncytia (non-syncytium inducing [NSI]), and cannot be passaged into cell lines. In contrast, HIV-1 isolates from AIDS patients are fast replicating, syncytium inducing (SI), and Tcell tropic. In 95% of asymptomatic individuals, only NSI variants of HIV-1 are detected (347). NSI variants in general have the capacity to replicate in primary monocyte-derived macrophages. SI variants, characterized by a high rate of replication, emerge during the course of infection and are detected in half of the individuals with advanced disease. It has been proposed that SI variants are cleared effectively during the initial stages of HIV-1 infection by a noncompromised immune system. The emergence of SI variants during the later stages of disease progression following immune deterioration supports the idea that SI variants are more susceptible to immune surveillance whereas NSI variants may be less vulnerable to immune system destruction because of their persistence in macrophages (347). Furthermore, NSI variants can be detected throughout the course of HIV-1 infection, even at stages when SI variants predominate, thus demonstrating the role of macrophagetropic variants in viral persistence. Virus isolates derived from monocytes and then serially passaged in T lymphocytes have a reduced capacity to reinfect monocytes or macrophages. However, viruses isolated from monocytes show dual tropism for T cells and monocytes.
Tropism for various cell types is a function of both viral and cellular determinants. The envelope V3 loop region is a major determinant of macrophage tropism. One group has identified a 20-amino-acid sequence in the envelope V3 loop of gp120 from the macrophage-tropic Bal isolate that is sufficient to confer macrophage tropism on the HTLV-IIIB strain (145). There are profound phenotypic differences in the virions produced from monocytes and lymphocytes. In T cells, 35% of the total viral proteins produced are env gene products, while in monocytes, the predominant viral proteins are capsid proteins and env gene products constitute less than 10% (229). Electron microscopy of virus produced from infected T cells reveals the characteristic spikes protruding from the membrane that correspond to gp120; these spikes are absent in virus from infected monocytes (143).
Initial experiments indicated that cellular activation influenced the permissiveness of cells to HIV-1 infection, since quiescent T cells were not permissive to HIV-1 infection (371,(415)(416)(417). Resting peripheral blood monocytes also require activation and/or intracellular changes to be permissive to HIV-1 infection (reviewed in references 47, 196, and 395). Myeloid cells become ''activated'' as they differentiate from a circulating monocyte to a tissue macrophage as a result of exposure to antigenic agents, cytokines, or extracellular matrix proteins whose expression is increased during an immune response (253). Peripheral blood mononuclear cells (PBMCs) isolated from normal donors are 10 to 100 times more susceptible to HIV-1 infection in vitro following antigenic stimulation with either keyhole limpet hemocyanin or tetanus toxoid than are cells not exposed to antigens (220). Exposure of PBMCs to noninfectious but mitogenic human T-cell leukemia virus type 1 (HTLV-1) virions before, immediately following, or several days after HIV-1 infection in vitro results in increased HIV-1 production (417). In vitro, freshly isolated primary monocytes do not support productive HIV-1 infection (349,391,404); however, after growth factor-enhanced differentiation, monocyte-derived macrophages (MDMs) are susceptible to infection by HIV-1 (58,272,285,319). Following terminal myeloid differentiation, HIV-1 replication is less efficient (196,391). HIV-1 production by macrophage cell lines and primary macrophages can be increased by treatment with growth and/or differentiation factors that increase the proliferative state of the cell (e.g., granulocyte-macrophage colony-stimulating factor [GM-CSF] and macrophage colony-stimulating factor [M-CSF]) (58,97,116,169,179). HIV-1 production from primary macrophages can also be increased by factors such as gamma interferon that activate macrophage function in the absence of proliferation (179). A fraction of terminally differentiated macrophages do proliferate in vitro (54), and recent evidence suggests that an activation state corresponding to the G 1 /S phase of the cell cycle, but not mitosis, is necessary for productive infection (348). Surprisingly, MDMs express fewer CD4 receptors than do freshly isolated blood monocytes (272,390,391) but are more susceptible to infection. This apparent paradox substantiates evidence that monocytic differentiation induces intracellular physiological changes which in turn enhance HIV-1 replication but do not alter viral entry (268). For example, decreased intracellular nucleotide pools appear to be the rate-limiting step for reverse transcriptase activity in macrophages (269). Increased permissiveness in mature myeloid cells may correlate with changes in the activity of cellular transcription factors. In monocytes, the level of constitutive NF-Bbinding activity increases with the stage of differentiation (127) and is capable of activating HIV-1 gene expression (256). Transient transfections show that monocytic differentiation results in a 20-fold upregulation of basal transcription from the HIV-1 long terminal repeat (LTR) and enhancement of Tatmediated transcription (249). Recent studies demonstrate that intracellular levels of both RelA(p65) and IB␣ proteins increase with in vitro differentiation of primary monocytes to MDMs (58a).
Virally encoded factors may also aid in the establishment of productive infection. Both the HIV-1 Gag (matrix) and Vpr proteins form a nucleophilic preintegration complex with viral nucleic acid (44,137). The function of these virion-associated proteins is to transport the preintegration complex to the nucleus in the absence of mitosis (44,137). In addition, Vpr may induce differentiation (195) in the infected host cell and thus indirectly modulate virus replication. Host and viral determinants of tropism, as well as the involvement of Th1/Th2 cytokines in pathogenesis, are described in detail in several recent articles (61,239).

REGULATION OF HIV-1 TRANSCRIPTION
Transcription of the HIV-1 genome depends on the intracellular environment into which the virus integrates and is regulated by a complex interplay between viral regulatory proteins and cellular transcription factors that interact with the viral LTR region (reviewed in references 12 and 111). Sequences involved in viral gene expression are contained within U3 and R regions of the HIV-1 LTR and may be subdivided into the core promoter elements, the enhancer, the modulatory and negative regulatory elements, and the Tat responsive element (TAR) (Fig. 1). The DNA-binding activity of several transcription factors is modulated in response to cellular activation, differentiation, and the actions of cytokines and mitogens.

Tat-TAR RNA Interactions
The tat gene encodes a 15-kDa trans-activator protein that enhances LTR-derived gene expression and is required for high-level expression of all viral genes (reviewed in references 62, 88, 389, and 407). Tat has three functional domains: an amino-terminal amphipathic ␣-helix, a domain found in many transcriptional activators; a cysteine-rich domain which may be important for dimerization; and a basic domain, critical for nuclear localization and TAR RNA binding (108,186,337). TAR RNA is formed by the nascent RNA transcript derived from nucleotides ϩ1 and ϩ59; the core secondary stem-loop structure (nucleotides ϩ18 to ϩ44) is critical for Tat-TAR interactions and for Tat-mediated transactivation of the HIV-1 LTR. The bulge region in TAR (ϩ22 to ϩ24) serves as the primary binding site for Tat, and the loop sequences (ϩ30 to ϩ35) also contribute to this interaction (334,335,353). TAR RNA essentially serves as an anchor for Tat and certain cellular factors (TRP-185 and p68) (218,360) to facilitate interactions with promoter elements such as TATA, Sp1, and the HIV-1 enhancer (29). Deletion of NF-B-and Sp1-binding sites from the HIV-1 promoter abrogates Tat-mediated transactivation, suggesting that once brought into the vicinity of the promoter, Tat interacts with transcription factors bound at the NF-B/Sp1 region to stimulate transcription initiation and stabilize elongation complexes (28). Tat may be able to transactivate independently of TAR RNA, since TAR-defective viruses are replication competent but still require Tat. In each case, TAR maps to the NF-B sites within the HIV enhancer (18,221,376).
In addition to transcription initiation, Tat plays a role in elongation and transcription processivity (reviewed in reference 155). Following transcription initiation, paused polymerase complexes accumulate and are prematurely terminated at random locations (191,219). The IST (initiator of short transcripts) sequence (312, 358) is a DNA sequence (Ϫ5 to ϩ80) that overlaps with TAR and mediates the synthesis of short transcripts in the absence of Tat (2,165). Although the mechanism remains unclear, Tat in combination with a cellular coactivator can bind TAR (217,359), modify the transcription complex, release it from the promoter, and allow full-length viral transcription to take place (126).

TAR DNA Element
The TAR DNA element extends from ϩ1 to ϩ59 within the R region of the HIV-1 LTR and includes binding sites for a number of cellular factors (Fig. 1). There is a high-affinity binding site for upstream binding protein/leader-binding protein (UBP-1/LBP-1) located from nucleotides Ϫ16 to ϩ27 within the HIV-1 LTR (159). In vitro transcription assays indicate that purified UBP-1/LBP-1 also binds to a low-affinity site (nucleotides Ϫ38 to Ϫ16, not included in Fig. 1) and inhibits HIV-1 gene expression possibly by interfering with TFIID binding to the TATA element (159). Depending on the level of UBP-1/LBP-1 relative to potentially competing DNAbinding proteins, both positive and negative effects of this protein on HIV-1 gene expression are possible. Other cellular factors, such as upstream binding protein 2 (UBP-2) or CTF/ NF1, also bind to TAR DNA (109,154), but the effect of these factors on HIV-1 gene expression is not known.

Core Promoter
The core promoter of the HIV-1 LTR resembles that of many eukaryotic genes transcribed by RNA polymerase II (Fig. 1). The promoter contains a consensus TATA sequence (Ϫ27 to Ϫ23) and initiator elements (Ϫ2 to ϩ8 and ϩ32 to ϩ41) to which general transcription factors bind (reviewed in references 29 and 311). A complex array of factors interacting either directly or indirectly with the TATA region in combination with upstream DNA-binding factors is critical for regulation of HIV-1 gene expression. Immediately upstream of the TATA element are three GC-rich sequences (Ϫ78 to Ϫ46), to which the ubiquitous Sp1 transcription factor binds (153). DNase I footprinting studies with templates mutated at Sp1 demonstrate a loss in the number of proteins that bind to Sp1 flanking sequences, indicating that Sp1 may facilitate gene expression by stabilizing the binding of both general and upstream transcription factors (153).

Modulatory Elements
The region located between nucleotides Ϫ410 and Ϫ157 (Fig. 1) has a negative effect on the rate of RNA initiation in vivo (211) and in vitro (271) and was originally defined as the negative regulatory element. Within this distal enhancer element are binding sites for a number of positively (Myb) (315) and negatively (USF, COUP) (59,212) acting cellular transcription factors; however, the mechanism by which this region downregulates HIV-1 transcription is unknown. Two potential AP-1-binding sites are located in the HIV-1 LTR between Ϫ357 and Ϫ316 bp from the transcription start site (101). AP-1 is present in all cells and regulates the expression of immediate-early genes in response to mitogenic stimuli including serum, growth factors, and phorbol esters. The region within the HIV-1 LTR that binds NF-AT (between nucleotides Ϫ254 and Ϫ216) is partially responsible for upregulation of HIV-1 gene expression in response to T-cell activation by agents such as ionomycin, phorbol esters, and phytohemagglutinin (363). NF-AT is a cytoplasmic T-cell-specific factor which combines with members of the AP-1 and possibly NF-B/Rel proteins to activate transcription (265). LEF-1 (TCF-1␣) is a T-cell-specific factor which binds between nucleotides Ϫ139 and Ϫ124 of the HIV-1 LTR (384,401). Within the context of the TcR␣ promoter, LEF-1 bends DNA, facilitating the formation of nucleoprotein complexes between upstream factors and the basal transcription machinery (121). In transient transfections, LEF-1 activates transcription from the HIV-1 LTR alone and in synergy with Tat by a similar DNA-bending mechanism (120a).

HIV-1 Enhancer
The most widely studied element in the modulatory region of the HIV-1 LTR is the enhancer region, which consists of two 10-bp conserved sequences (Ϫ109 to Ϫ79) known as NF-B motifs. Nabel and Baltimore (256) first established the role of NF-B in controlling transcription from the HIV-1 LTR by demonstrating a direct correlation between increases in NF-B DNA-binding activity and HIV-1 LTR-directed transcriptional activity during T-cell activation. Mutation of NF-B motifs in HIV-1 LTR chloramphenicol acetyltransferase reporter constructs results in a marked decrease in gene expression following transfection into lymphoid cells, in both the presence and absence of the tat gene (256). Transient-transfection studies, with combinations of the HIV-1 LTR and HIV-1 enhancer sequences linked to reporter genes, demonstrate that induction of NF-B DNA-binding activity in T cells and monocytes leads to increased HIV-1 LTR-directed gene expression (reviewed in reference 128). The NF-B sites act in synergy with NF-AT sites in phorbol myristate acetate (PMA)-stimulated T cells (75,182,260,371) to increase the rate of viral replication in cells that respond to mitogenic stimulation. Among the elements identified as positively acting factors in basal HIV-1 transcription, TATA, Sp1, and NF-B elements are the most influential in primary monocytes while NF-B and NF-AT have strong effects in activated T cells (249).
The transcription factor AP-2 is present in unstimulated Jurkat cells and binds to the motif 5Ј-CCGCTGGGGA-3Ј between the two NF-B motifs within the HIV enhancer (406). AP-2 may have a positive effect on HIV transcription in unstimulated T cells since mutations within the AP-2 site reduce HIV LTR directed transcription fivefold (288). AP-2 binds in a mutually exclusive manner with NF-B proteins and does not interfere with the PMA or tumor necrosis factor alpha (TNF-␣) responsiveness of the NF-B element (288).

NF-B/Rel FAMILY OF TRANSCRIPTION FACTORS
NF-B is a ubiquitous transcription factor that participates in various biological processes including immunological re-sponsiveness, lymphoid cell differentiation, growth control, and dorsal-ventral axis determination in Drosophila melanogaster (reviewed in reference 17, 128, 129, 205, and 253). NF-B exists in the cytoplasm in latent form as a complex consisting of a dimer of DNA-binding subunits bound to an inhibitor, IB. NF-B-regulated genes encode proteins involved in the rapid response to pathogens or stress, including the acute-phase proteins, cytokines, and cellular adhesion molecules (17). NF-B also plays a critical role in T-cell activation. In activated monocytes and macrophages, genes such as those encoding granulocyte colony-stimulating factor (G-CSF), M-CSF, and GM-CSF, the inflammatory cytokines beta interferon [IFN-␤], TNF-␣, interleukin-1 (IL-1) and IL-6, receptors for tissue factor and IL-2 ␣-chain, the chemotactic protein MCP-1/JE, and nitric oxide synthase are highly induced as a result of regulatory control by NF-B (253). The production of these immunomodulatory cytokines is an essential part of the host immune response to bacterial and viral pathogens.

NF-B/Rel DNA-Binding Subunits
Each of the five DNA-binding members of the Rel family (p50, p52, RelA, c-Rel, and RelB) shares a region of homology termed the NF-B/Rel/dorsal (NRD) region, which spans approximately 300 amino acids at the amino terminus of each protein (36,205). The domain structures of the NF-B/IB family members are illustrated schematically in Fig. 2. The NRD region is responsible for DNA binding (184,223), dimerization (210), nuclear localization, and interaction with the inhibitory subunit(s), IB (26,409). The crystal structure of p50 homodimers bound to DNA reveals that each DNA-binding subunit surrounds DNA by folding into a pattern that resembles immunoglobulin-like modules (120,250). The NRD DNA-binding region is divided into two domains separated by a flexible hinge: the amino-terminal portion contains a recognition loop which interacts within the major groove of DNA, and the carboxyl-terminal region functions as a dimerization interface (120,250).
Two of the DNA-binding subunits are synthesized as inactive cytoplasmic precursor proteins (Fig. 2). The product of the nfkb1 gene is a 105-kDa protein which gives rise to a 50-kDa DNA-binding subunit, p50 (36,166,237). The nfkb2-encoded p100 precursor is very similar to p105; its mature DNA-binding form is p52 (262). Each precursor protein has two functional domains: the carboxy-terminal inhibitory domain, containing seven ankyrin repeats, and the amino-terminal NRD (134,206,318). A glycine-rich region divides the two functional domains and allows sufficient structural flexibility for amino and carboxy termini to physically interact (139). This physical interaction masks the nuclear localization sequence within the NRD region, preventing nuclear translocation and DNA binding. The processed DNA-binding subunits which are composed almost entirely of the NRD region, arise following ubiquitin-dependent proteolytic degradation of the carboxy termini up to a site adjacent to the glycine-rich hinge (84,277). The other NF-B/ Rel DNA-binding proteins are not proteolytically processed from precursors but remain cytoplasmic through interactions with inhibitory molecules. c-Rel is an 85-kDa protein, which in addition to the NRD region, contains a unique 300-amino-acid carboxy-terminal domain and is highly related to RelA(p65) (46,148). The carboxy termini of c-Rel and RelA contain two distinct activation domains similar to those in the viral transactivator VP16 (20,129,289,343). Finally, the RelB subunit contains an elongated amino-terminal segment but lacks transactivating potential (338).
The physiological role of the two DNA-binding subunits in vivo has been characterized in mice homozygously deficient in NF-B p50 and RelB subunits (356,403). Targeted disruption of the relb locus yields mice with multiple immunological abnormalities including dysfunction of the hematopoietic system and inflammatory response; however, there are no apparent defects in lymphoid cell development (403). Mice deficient in the p50 subunit show no developmental abnormalities but have impaired B-cell-mediated (specific) and nonspecific immune responses (356).

IB Regulatory Proteins
The IB family of proteins is defined by its ability to interact with NF-B/Rel subunits (Fig. 2) and by the presence of between five and seven repeats of a 33-amino-acid sequence termed the ankyrin motif (reviewed in reference 24). The ankyrin motif may mediate the sequestration of NF-B in the cytoplasm, possibly in association with cytoskeletal proteins (35). IB␣ (132) is the most extensively studied protein in this family, but other members include IB␤ (379), IB␥ (147,206), and the product of the proto-oncogene bcl-3 (134,270). The full-length precursors p105 and p100 also have demonstrated inhibitory capabilities, not only over their own amino termini but also through interaction with c-Rel and RelA (318). There is specificity in the interaction between the inhibitory subunits and the DNA-binding members; IB␣ and IB␤ exclusively inhibit dimers containing RelA and c-Rel (66,132,164), whereas IB␥ and Bcl-3 preferentially interact with p50and/or p52-containing dimers (103,163,206,266,409).

Regulation of NF-B DNA-Binding Activity
In most cell types, NF-B is present in an inactive cytosolic form that can be induced following exposure to a wide variety of activating agents including cytokines, T-cell mitogens, bacterial products, and viruses (Fig. 3). Activation of NF-B requires the disruption of IB-DNA-binding subunit interactions. Several lines of evidence indicate that phosphorylation of IB is an important posttranslational modification involved in NF-B activation. In vitro, direct phosphorylation of IB␣ FIG. 2. Schematic representation of the NF-B/Rel and IB family of proteins. The different mammalian NF-B/Rel and IB proteins are organized as DNA-binding proteins, nonbinding proteins, and inhibitors. The name of each protein is indicated below each structure. Arrows indicate the proteolytic cleavage of the non-DNA-binding p105 and p100 proteins into the DNA-binding p50 and p52 proteins, respectively. A second arrow in the p105 structure indicates the alternative splicing of the p105 mRNA to produce the inhibitory molecule IB␥. Symbols and abbreviations: , DNA-binding portion of the Rel homology (RH) domain; , dimerization portion of the RH domain; , PKA phosphorylation site present in the RH domain; NLS, nuclear localization signal; G, glycine-rich hinge region present in p105 and p100; P and S/P, proline-rich and serine-and proline-rich regions, respectively; , transactivation domains present in p65, p65⌬, c-Rel, and RelB; , ankyrin repeats present in p105, p100, and the inhibitor proteins; , protein degradation signal PEST present in IB␣ and IB␤. occurs with protein kinase C (PKC), PKA, heme-regulated eukaryotic initiation factor 2 kinase, cyclic AMP-responsive kinase, and the double-stranded RNA (dsRNA)-dependent kinase (PKR) (34,139,183,205,216). In cells, both Raf and Src kinases have been implicated in the induction of NF-Bbinding activity (92,174,200). In addition, kinase inhibitors can block PMA-induced NF-B activation (381). IB degradation also coincides directly with the appearance of nuclear NF-B and is necessary for the induction of NF-B DNAbinding activity (138). Events responsible for IB degradation are highly inducible, and stimulation of cells may lead to a modification of IB (such as phosphorylation) that targets the inhibitor for rapid degradation. Recent work by Palombella et al. demonstrates that TNF-␣-induced IB␣ degradation is blocked by inhibitors of ubiquitin-dependent proteasomes (277). Proteolytic processing of the precursors p105 and p100 into their active p50 and p52 forms is an additional level at which NF-B DNA-binding activity may be controlled. For example, p105 and p100 exist complexed to other Rel family members such as RelA, c-Rel, and proteolytically released p50 (233,259,318,373). Proteolytic cleavage of the carboxyl terminal of p105 activates DNA-binding p50-p50, p50-c-Rel, and p50-RelA dimers (233,259,318). This mechanism, however, is not likely to result in a rapid appearance of NF-B in the nucleus. Recent experiments in vivo have shown that p105 is phosphorylated and its carboxy terminus is degraded by the ubiquitindependent proteasome pathway (277). Therefore, two types of NF-B complexes can exist in the cytoplasm: those retained by IB␣ and those retained by NF-B precursors (Fig. 2). Each target may respond independently to different types of environmental stimuli or differential signals.
Once NF-B DNA-binding activity is activated, numerous target genes are selectively regulated by the transcriptional activation potential of different homo-and heterodimer combinations. As well, variations in the NF-B consensus sequence FIG. 3. Biochemistry of NF-B activation. The NF-B/Rel transcription factor exists in two latent cytoplasmic forms: (i) complexed to an inhibitor IB and (ii) in association with NF-B precursors p100 or p105. NF-B DNA-binding activity is induced by a variety of agents and pathogens, some of which interact with specific receptors on the cell surface. Second-messenger signaling molecules generated by these agents lead to the phosphorylation followed by polyubiquitination (Ub) and subsequent degradation of IB␣ in the 26S multisubunit proteasome complex (277). IB degradation liberates dimeric combinations of NF-B DNA-binding subunits, which rapidly translocate to the nucleus. Active NF-B dimers are also generated following proteolytic degradation of the carboxy termini of the p105 or p100 precursor proteins. Target genes are selectively regulated by the distinct transcriptional activation potential of different subunit combinations, variations in NF-B consensus sequence, promoter context, and synergism with other transcription factors (17,205). NF-B-regulated transcription is restricted as newly synthesized IB interacts with cytoplasmic NF-B subunits, restoring the latent cytoplasmic complex; nuclear IB␣ may directly dissociate NF-B-DNA complexes. The transcriptional upregulation of nfkb1, nfkb2, c-rel, bcl-3, and Ikba (MAD-3) genes in response to NF-B-activating stimuli restores intracellular pools of latent NF-B. VOL. 59,1995 REGULATION OF HIV-1 AND CYTOKINE EXPRESSION BY NF-B 487 (5Ј-GGGANNYYCC-3Ј) to which the subunits bind (185,205,388) and cooperativity between different transcription factor families and NF-B/Rel contribute to the specificity of gene activation (17,205). NF-B-mediated transcription is downregulated by de novo synthesis of IB␣ which interacts with nuclear or cytoplasmic NF-B subunits in transit to the nucleus (26). Increased levels of IB␣ may also dissociate protein-DNA complexes and restore the cytoplasmic pool of latent NF-B/IB complexes (14,414). Furthermore, transcriptional upregulation of nfkb1, nfkb2, c-rel, bcl-3, and Ikba (MAD-3) genes by NF-B provides an autoregulatory mechanism to restore intracellular pools of latent NF-B (36, 45, 232, 237) (Fig. 3). Disruption of IB␣ autoregulation with IB␣ antisense RNA results in malignant transformation and altered NF-B-regulated gene activity, indicating that IB␣ may represent a potential growth suppressor activity (22).

IB␣ Phosphorylation and Degradation
IB␣ has a half-life of 1 to 2 h when complexed with NF-B but is rapidly degraded following inducer-mediated stimulation of cells (317,352,372). The short half-life of IB␣ may be due to the presence of a C-terminal domain rich in proline, glutamic acid, serine, and threonine, called the PEST domain (132,324). Activating agents, such as dsRNA, phorbol esters, TNF-␣, IL-1, and lipopolysaccharide (LPS), accelerate the degradation of cytosolic IB␣, thereby promoting the release and nuclear translocation of NF-B/Rel dimers (24-26, 129, 205). Nuclear NF-B/Rel dimers transactivate target gene expression, including transcriptional upregulation of the MAD-3 (IB␣) gene, thereby establishing an autoregulatory loop in which newly synthesized IB␣ restores the cytoplasmic pool of latent NF-B (43,77,193,372).
Phosphorylation and subsequent degradation of IB␣ appear to be critical processes involved in NF-B activation (Fig.  3). Following inducer-mediated stimulation, IB␣ becomes hyperphosphorylated, detectable in immunoblots as a slowly migrating form that is sensitive to phosphatase treatment (25,43). Hyperphosphorylation does not impair the ability of IB␣ to associate with NF-B but is believed to represent a signal for subsequent degradation (6,71,93,204,383) via the ubiquitinproteasome degradation pathway (277).
An important question with regard to the regulation of NF-B transcriptional activity concerns the signaling pathways that stimulate IB␣ phosphorylation and degradation. It re-mains unclear whether different stimuli trigger multiple independent signaling pathways or whether signaling events converge upon a common host kinase activity that stimulates phosphorylation and subsequent degradation of IB␣. A number of potential kinases which are capable of phosphorylating IB␣ in vitro have been identified. dsRNA-dependent protein kinase (PKR) phosphorylates IB␣ in vitro (183), and antisense RNA complementary to PKR blocks NF-B activation induced by dsRNA (216). The role of PKR appears to be restricted to dsRNA stimulation, since antisense PKR RNA does not block TNF-␣-induced NF-B activity. TNF-␣ stimulation appears to involve at least three kinases: Raf-1, the isoform of PKC, and casein kinase II (CKII). Both PKC-and CKII are activated by TNF-␣, whereas Raf-1 activity is not stimulated by TNF-␣ (70,297,405). However, Raf-1 dominant positive mutants induce NF-B DNA-binding activity. Conversely, dominant negative mutants of Raf-1 or PKC-block TNF-␣-induced NF-B activation (69,92,200). Although Raf-1 has been shown to interact with IB␣ in experiments with the yeast two-hybrid system (200), it is not known whether Raf-1 or PKC-is directly involved in phosphorylating IB␣ or, rather, plays a role in the signal transduction cascade leading to IB␣ phosphorylation.
To further characterize the signaling events that contribute to IB␣ phosphorylation and degradation, we isolated a kinase activity from Jurkat T cells that specifically interacts with IB␣ in an affinity chromatography step and phosphorylates IB␣ with high specificity in vitro. Biochemical and immunological cross-reactivity identified the kinase activity as CKII (202). Point mutation analysis revealed that a double mutation of residues Thr-291 and Ser-283 eliminated phosphorylation of IB␣ by the CKII in vitro and reduced phosphorylation about 10-fold in vivo, although IB␣ degradation was unaffected. Brown et al. demonstrated that Ser-32 and Ser-36, as well as a portion of the C-terminal end, were both required for inducermediated degradation (41,42). Interestingly, both Ser-32 and Ser-36 residues also represent natural CKII sites (Fig. 4).
These results indicate that CKII or an enzyme with the same phosphorylation sites represents the IB␣ kinase.
CKII is a highly conserved and ubiquitous serine/threoninespecific kinase composed of two larger catalytic subunits (␣ and/or ␣Ј; relative molecular mass, 35 to 44 kDa) and two smaller ␤ regulatory subunits (relative molecular mass, 25 to 28 kDa) (227,297). Immunocytochemical studies localize CKII to FIG. 4. Schematic representation of human IB␣. Human IB␣ contains five internal ankyrin repeats (SWI6/ANK) that bind to NF-B molecules and mask the nuclear localization sequence (26). The N-terminal region of IB␣ is expanded above the schematic to show the one-letter amino acid sequence between amino acids 25 and 45. Within this region of IB␣ are two phosphorylation sites (Ser-32 and Ser-36, indicated by triangles), shown previously to play a role in inducer-mediated degradation (42). Both sites represent potential phosphorylation sites by CKII (CKII consensus S/T-X-X-E/D). A region rich in proline, serine, threonine, and glutamic acid (the PEST domain) spans amino acids 264 to 317; the C-terminal region of IB␣ between amino acids 251 and 317 is expanded below the schematic to show the one-letter amino acid sequence. The PEST domain contains several CKII phosphorylation sites clustered within the highly acidic domain (thick line) at S-283, T-291, and T-299 (underlined) (23,202). Phosphorylation of residues T-291 and T-299 also generates new potential CKII phosphorylation sites at residues S-288, T-296, and S-293 (denoted by lines and asterisks above the sequence). A potential PKC site located between the ankyrin repeats and the PEST domain is also shown.
Recently, CKII␣ transgenic mice were created that developed lymphoproliferative disorders resembling the fatal lymphoproliferative syndrome associated with the protozoan parasite Theileria parva in cattle. Interestingly, theileriosis in cattle is characterized by the overexpression of CKII. In the murine transgenic model, CKII␣-expressing mice displayed a stochastic potential to develop lymphoma; coexpression of a c-myc transgene in addition to CKII␣ resulted in neonatal leukemia. This study showed that CKII can serve as an oncogene, and its dysregulated expression is capable of transforming lymphocytes in cooperation with c-myc (354).
A highly acidic region of IB␣ composed of 20 amino acids (Ser-283 to Asp-302) contains six potential sites of phosphorylation by CKII (23,82,202). The consensus CKII sites (S/T-X-X-E/D) are formed by an N-terminal serine or threonine separated by two residues from a C-terminal acidic amino acid (aspartic acid or glutamic acid). Interestingly, the acidic amino acid at position 4 of the consensus sequence may be contributed by any phosphorylated amino acid (227,297). Thus, in IB␣, Ser-283, Thr-291, and Thr-299 represent natural sites of phosphorylation by CKII and phosphorylation at Thr-291 and Thr-299 creates the potential for sequential CKII phosphorylation at Ser-288 and Thr-296, respectively. Finally, phosphorylation of Thr-296 generates a potential CKII phosphorylation site at Ser-293 (Fig. 4). Deletion of the C-terminal PEST domain or, more precisely, the CKII cluster between amino acids 283 and 302, results in an IB␣ molecule that is resistant to inducer-mediated degradation (23,202). Biochemical characterization of the domain structure of IB␣ demonstrates that IB␣ contains a highly structured central domain that is resistant to proteolysis and flexible N-and C-terminal extensions that are sensitive to proteolytic digestion (151). Also, the Cterminal region, but not the N-terminal region, is protected from proteolysis when IB␣ is bound to RelA(p65), suggesting that this region of the protein directly interfaces with Re-lA(p65) and was thus masked in the IB␣-RelA complex.
It is likely that both the N-terminal and C-terminal domains are recognized by the protease machinery after inducer-mediated phosphorylation (23,41,42,323). Alternatively, the PEST domain alone may be recognized but in uninduced cells the C-terminal region is masked from recognition by the proteasome because of the conformation of IB␣ or its association with other proteins. Simultaneous phosphorylation of N-terminal and C-terminal sites may increase the acidic nature of the regions and force IB␣ to adopt a new conformation in which the PEST domain is exposed to the proteasome (Fig. 5).
Two major classes of proteases are known to operate outside of the lysosome, the calpains (Ca 2ϩ -dependent cysteine proteases) and the proteasome. Central to the proteasome machinery is the ATP-dependent 26S multisubunit proteasome, which can operate in a ubiquitin-dependent or -independent fashion (292). The proteases involved in the degradation of IB␣ have not yet been identified; however, two observations implicate the proteasomes: (i) ATP is required for IB␣ degradation upon LPS stimulation (150); and (ii) with the exception of calpain inhibitor I (204), only proteasome inhibitors have been shown to prevent IB␣ degradation induced by TNF-␣ (93,277,383). Interestingly, PSI and MG115 proteasome inhibitors prevent IB␣ degradation but not IB␣ hyperphosphorylation, illustrating that these two events are independent (277,383).
A unique function of IB␣ is the capacity to dissociate NF-B-DNA complexes, an activity that is mediated by the ankyrin repeats as well as the PEST domain. In view of the predominantly cytoplasmic localization of IB␣, the biological significance of NF-B-DNA complex dissociation by IB␣ is not yet understood, although IB␣ has previously been identified in the nucleus (60). Furthermore, in vitro transcription studies with purified NF-B proteins demonstrate that addition of recombinant IB␣ to the transcription reactions inhibits NF-B-dependent transcription (203,414). These experiments suggest that a novel nuclear role for newly synthesized IB␣ may be to directly inhibit NF-B-dependent gene expres- This idea is further supported by the observation that following induction, de novo synthesized IB␣ protein transiently appears in the nucleus and negatively regulates NF-B-dependent transcription (14). IB␤ was recently cloned and characterized (379). Structurally, IB␤ contains six ankyrin repeat regions and a putative C-terminal PEST domain; interestingly, several potential CKII sites are conserved in IB␤ in the C-terminal PEST domain. A primary biological difference between IB␣ and IB␤ is their response to different inducers of NF-B activity. IB␤ is rapidly degraded upon LPS or IL-1 stimulation, but neither TNF-␣ nor phorbol ester stimulation induces IB␤ degradation. Conversely, IB␣ degradation is induced by all four stimuli. These results suggest that unlike IB␣, IB␤ is used not for regulating rapid responses but for responding to persistent signals that result in a more permanent activation of gene expression (379). Additional studies will define more precisely the complex associations between IB␣ and NF-B proteins, as well as the relationship of these interactions with the newly cloned IB␤.

PHYSIOLOGICAL CHANGES IN HIV-1-INFECTED MYELOID CELLS
The primary cause of immune system dysfunction in HIV-1-infected individuals is depletion of the CD4 ϩ helper T-cell population. However, the impaired function of other subsets of immune effector cells, such as myeloid cells, may also contribute to immune dysfunction by increasing susceptibility to secondary pathogenic infections. The primary function of mature myeloid cells or macrophages is to clear the blood, lymph, and tissues of microbial pathogens and other foreign particles by phagocytosis. Effective killing of microorganisms requires several important steps: migration of monocytes to the appropriate site, particle recognition and attachment, and particle ingestion by phagocytosis. Following phagocytosis, most organisms are killed by oxidative mechanisms (1).

Functional Changes
Several studies document defective chemotaxis of phagocytic cells from HIV-1-infected individuals (264,301,367). Interestingly, defective chemotaxis is also seen in monocytes from seronegative donors after in vitro exposure to gp41 or gp120 (399). Monocytes from AIDS patients do not appear to be impaired for nonspecific (C3-receptor-mediated) or immune system-specific (Fc-receptor-mediated) phagocytosis (27,83,118,264,301,332). Estevez et al. (83) reported that phagocytosis of Candida pseudotropicalis is efficient whereas intracellular killing (lytic activity) is reduced. Similarly, intracellular killing by superoxide generation is reported to be either normal (255,264) or deficient (39,252) in cells isolated from AIDS patients. In contrast, in vitro models of HIV-1infected promonocytic cells display an amplification of the oxidative burst response (167). Altered oxidative burst may be related to differences in the disease stage of patients under study, since Roux-Lombard et al. demonstrated a decline in the oxidative burst response with advancing disease (332). In addition, monocytes and macrophages isolated from HIV-1infected individuals at various disease stages show decreased ability to mediate tumor cell lysis (327).
Monocytes serve as accessory cells in the immune system. Antigen presentation in the context of a major histocompatibility complex class II molecule (HLA-DR in humans) on the macrophage cell surface is required for CD4 ϩ T-lymphocyte activation. Normal (130,228) or decreased (136,333) HLA-DR expression, correlating with decreased ability to stimulate T cells (95,168,357), is reported to occur in chronically infected myeloid cells. However, treatment of monocytes in vitro with either free HIV-1 or purified gp120 leads to differentiation and increased HLA-DR expression (7,399). These studies support the idea that a biphasic response of host mononuclear cells begins with an increase in HLA-DR expression followed by a decline during chronic infection (136,182,295). The downregulation of antigen-presenting complexes may be a mechanism by which infected cells escape immune surveillance and promote viral persistence.

Cytokine Gene Expression
Secretion of immunoregulatory cytokines by macrophages is essential for lymphocyte activation, antibody production, and cytotoxic cellular responses. Cytokine gene expression studies of PBMCs from HIV-1-infected individuals generate conflicting results. For example, spontaneous and LPS-induced TNF-␣, IL-1, and IL-6 secretion is not significantly different in PBMCs isolated from AIDS patients and those isolated from uninfected donors (291,396). In contrast, alveolar macrophages (but not monocytes) isolated from patients with early-and late-stage disease express increased levels of LPS-stimulated TNF-␣, IL-1␤, and IL-6 mRNA (364). Also, levels of basal and stimulated TNF-␣ secretion by PBMCs from HIV-1-infected individuals increase with disease progression (327,408). Cytokine release from HIV-1-infected myeloid cells may contribute to the elevated levels of TNF-␣ (172,188,194), IL-1 (224), IL-6 (32, 40), transforming growth factor ␤ (TGF-␤) (8), and IFN-␥ (104), present in the sera of AIDS patients with latestage disease. Elevated IFN-␣/␤ activity is also present in the sera of AIDS patients with late-stage disease and serves as a marker for poor prognosis (320). Paradoxically, in PBMCs isolated from HIV-1-infected individuals, IFN-␣ production becomes increasingly impaired as the disease progresses (396). A similar defect in IFN-␣ production occurs in HIV-1-infected PBMCs treated with dsRNA, Newcastle disease virus, or herpes simplex virus (HSV); IFN mRNA expression is reduced at the transcriptional level (115). Cells isolated from patients at different stages of disease and with different degrees of immunodeficiency may account for reported variations in cytokine secretion from primary cells.
Recent work demonstrates that when PBMCs isolated from HIV-infected individuals are stimulated in vitro with Staphylococcus aureus, they produce fivefold-less IL-12 than do PBMCs from healthy donors (53). Furthermore, PBMCs infected with HIV in vitro also have a reduced ability to synthesize IL-12. Since IL-12 is involved in the stimulation of natural killer (NK) cell activity and the differentiation of Th1 cells, defective IL-12 production may contribute to immunosuppression in the HIV-infected patient (reviewed in reference 385).
Constitutive expression of IL-1␤, TNF-␣ and M-CSF mRNA is not observed in PLB-985, THP-1, and U937 cell lines chronically infected by HIV-1. Nonetheless, increased cytokine mRNA levels are seen after treatment with LPS or Sendai virus compared with those in uninfected controls (63,64,244,411). No difference in mRNA stability is detected between HIV-1-infected and uninfected cell lines, suggesting that cytokine genes may be upregulated at the transcriptional level (328). In chronically infected U1 cells stimulated with Epstein-Barr virus-or cytomegalovirus (CMV)-derived antigens, enhanced monokine secretion correlates directly with enhanced HIV-1 replication (55). Both normal and enhanced cytokine gene expression is reported in PBMCs infected with HIV-1 in Transcriptional upregulation of cytokine genes may involve signal transduction pathways activated by gp120-CD4 interactions at the cell surface. TNF-␣, IL-1, and IL-6 mRNA, as well as metabolites of arachidonic acid, are produced from normal PBMCs exposed to either purified gp120 (119,235,243,398) or inactivated HIV-1 (258). Stimulation of the CD4 antigen induces the lymphoid cell-specific tyrosine kinase Lck, involved in T-cell activation (393). Induction of the Lck signaling cascade or a similar pathway in monocytes may also lead to cytokine gene activation.
The mechanism by which cytokine secretion is upregulated in response to secondary antigenic challenge may not be HIV-1 specific, since viral infection in other myeloid cell systems also gives rise to a potentiated cytokine response. Murine CMV infection of a macrophage cell line is insufficient to induce IL-1 secretion, but following stimulation with LPS, IL-1 secretion is enhanced more than 100-fold (198). Potentiated TNF-␣ mRNA expression is also observed with influenza virus-infected macrophages challenged with LPS (124,257). These observations indicate that changes in cellular physiology associated with virus infection may give rise to altered cytokine gene transcription.
Physiological changes associated with cellular differentiation may cause myeloid cells to be highly responsive to antigenic stimulation. For example, when PBMCs are fractionated into nonadherent (immature) and adherent (mature) populations and then stimulated with Sendai virus, LPS, or PMA, approximately 200-fold-higher levels of TNF-␣ are produced from the mature adherent cells than from the nonadherent cells (3). Dudding et al. (78) report that human CMV infection of the ML-3 promyelomonocytic cell line does not induce cytokine or inflammatory response gene transcription. However, PMA-induced differentiation of ML-3 cells followed by CMV infection leads to enhanced transcription, translation, and stability of IL-1␤, TNF-␣, MAD-9, and c-fms mRNA (78).

Myeloid Cell Differentiation
HIV-1 infection of promonocytic U937 cells induces phenotypic changes associated with cellular differentiation and viral expression (112,207,284,285,295). Chronic HIV-1 infection of PLB-985 cells leads to morphological changes consistent with irreversible differentiation along the monocytic pathway and mRNA expression of myeloid cell-specific surface markers (CD14 and c-fms) (331). Acutely but not chronically HIV-1infected THP-1 monocytes also display changes indicative of monocytic maturation (244). In other studies, the promyelocytic cell lines PLB-985 and HL-60 do not spontaneously differentiate. Despite infection by HIV-1 over 20 days, each cell line maintains its capacity for either monocytic or granulocytic differentiation in response to differentiation-inducing agents (49,298). In addition, primary monocytes isolated from HIV-1-infected donors fail to undergo normal in vitro maturation after 7 days in culture (10).
Changes in the expression of surface receptors associated with myeloid differentiation or activation may be one mechanism whereby differentiation-induced signals can lead to a ''priming effect'' in HIV-1-infected cells. Upregulation of CD14 is reported to occur in HIV-1-infected promonocytic cells (331) and may lead to increased responsiveness to LPS and LPS-induced HIV-1 gene expression (19). Similarly, HIV-1infected monocytic cell lines show increased TNF receptor (48), IL-2 receptor (7), and c-Fms (CSF-1 receptor) (331) expression, which could lead to increased cytokine and growth factor responsiveness.

NF-B DNA-Binding Activity
HIV-1 infection in myeloid cells may also induce alterations in signal transduction pathways which modulate cellular transcription factors. NF-B-binding activity, expression of an HIV-1 LTR reporter gene, and monocytic differentiation are coordinately inducible in immature myeloid cells (HL-60 and U937) (127). In the more mature P388, THP-1, and P45-1.8 cells and in primary macrophages, NF-B-binding activity and expression of an HIV-1 LTR reporter gene are constitutively high and not further inducible, indicating that NF-B-binding activity is induced during the promyelocytic-to-monocytic transition (127). However, examination of the nuclear NF-B levels in a wide variety of promonocytic and monocytic cell lines, as well as in primary blood monocytes, does not always yield a correlation between maturation state and constitutive, nuclear NF-B (17,100). The NF-B-regulated Ikba (MAD-3) gene is highly induced in macrophages upon adherence to their substratum (132). NF-B-binding activity is also activated following binding of ␤1 integrins to cellular adhesion molecules (209), suggesting that NF-B is easily activated upon adherence or physical manipulation; this could lead to variations in constitutive NF-B DNA-binding activity between various cell lines and primary cells.
Productive viral infection also causes sustained NF-B DNA-binding activity in HIV-1-infected monocytic cell lines (16,286,314,329,331,375). A direct temporal correlation exists between HIV-1 infection and the appearance of NF-B DNA-binding activity in the monomyeloblastic cell line PLB-985 (330). Sustained NF-B DNA-binding activity may be dependent on the maintenance of a virus-mediated activation signal. Bachelerie et al. demonstrate constitutive NF-B-binding activity in the chronically HIV-1-infected promonocytic cell line U937 (16). Increased transcription and processing of the p105 precursor gives rise to increased intracellular pools of NF-B p50 in chronically infected U937 cells (286). In HIV-1-infected cells, p105 processing may be mediated by the virusencoded protease (321).
HIV-1 infection of the monocytic THP-1 cell line results in two populations of chronically infected cells: those with productive and those with restricted virus expression (314). Nuclear extracts from productively infected cells contain increased NF-B p50-RelA heterodimer-binding activity, whereas extracts from cells expressing low levels of virus (restricted expression) display only p50 homodimer-binding activity. Similarly, p50-RelA heterodimer-binding activity is constitutively present in chronically HIV-1-infected (and more differentiated) PLB-IIIB cells but is not present or inducible in the immature parental PLB-985 myelomonoblastic cell line (329)(330)(331). Increased NF-B p50-RelA-binding activity is observed in HIV-1-infected PBMCs and macrophages but not in the promonocytic cell line U937, suggesting that a certain level of myeloid maturation is required prior to induction of NF-B DNA-binding activity (375).
The activity of specific lysosomal proteases expressed in myelomonocytic cells, including primary monocytes, HL-60, and certain clones of U937 and THP-1 cells, results in the carboxylterminal cleavage of the RelA subunit of NF-B, giving rise to VOL. 59, 1995 REGULATION OF HIV-1 AND CYTOKINE EXPRESSION BY NF-B 491 protein-DNA complexes without transcriptional activity that functionally resemble p50 homodimers (102). Chronically infected cells exhibiting ''restricted'' viral replication express the protease, whereas clones which are ''productively'' infected do not. Furthermore, the activity of the proteases is downregulated after exposure to agents that induce monocytic but not granulocytic differentiation. Cleavage of RelA is thought to occur only in vitro during cell extract preparation; however, protease expression may correlate with the expression of other genes responsible for the replicative phenotype of HIV in myeloid cells (102).

COFACTORS IN HIV-1 DISEASE PROGRESSION
Many pathogenic agents upregulate HIV-1 transcription by inducing NF-B DNA-binding activity (326). In this respect, secondary opportunistic infections which result in immune system activation may play an important role in the progression of AIDS. Furthermore, pathogens encountered by an HIV-1-infected individual during a normal immune response can increase the susceptibility of monocytes and T cells to infection by HIV-1.
AIDS patients are subject to multiple pathogenic opportunistic infections, such as those caused by Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium (336). As a sexually transmitted and blood-borne agent, HIV-1 is often found in the presence of other pathogens, including Treponema pallidum, Neisseria gonorrhoeae, CMV, HSV-1 and HSV-2, human herpesvirus 6, hepatitis B virus, and human papillomavirus. The human herpesviruses are among the most common viral pathogens associated with HIV-1 infection. Many individuals infected with HIV-1 are also infected with amoebae and other parasites that cause malaria (reviewed in reference 260). The response of the infected host to these organisms could indirectly favor HIV-1 replication and heighten progression to AIDS.

Viral Cofactors
There are several mechanisms by which viruses can increase HIV-1 gene expression. First, at a molecular level, DNA viruses commonly found in AIDS patients can encode transactivator proteins capable of increasing expression of the HIV-1 LTR. These include the herpesviruses, e.g., CMV (65,261), HSV (89,117,248), Epstein-Barr virus (162), and human herpesvirus 6 (215), as well as adenoviruses (171), papovaviruses (117), and hepatitis B virus (387). The CMV and HSV trans-activator proteins, involved in increased expression of the HIV-1 LTR, are encoded by immediate-early gene products (275,287,378). The effects of these trans-activators on LTRdriven expression are shown to be mediated by promoter regions, some of which are distinct from the HIV-1 enhancer (89,287). Second, several viruses or viral gene products (e.g., hepatitis B virus X and HTLV-1 Tax) can activate NF-B DNA-binding activity (reviewed in reference 17), which directly enhances HIV-1 LTR-regulated transcription. One common point at which the infection cycle of these viruses may overlap is in generation of dsRNA during viral replication, which may in turn activate PKR (reviewed in reference 341). PKR is induced by interferon and is activated by binding to dsRNA molecules generated during viral infection. Recent work implicates PKR in the activation of NF-B DNA-binding activity in vitro and in vivo. PKR can phosphorylate IB␣ and activate NF-B DNAbinding activity in cell extracts (183). In PKR-depleted HeLa cells, NF-B DNA-binding activity is not activated by dsRNA [poly(I)-poly(C)] (216). Increased PKR protein and activity is a possible mechanism by which constitutive activation of NF-B may be perpetuated in chronically infected cells. Furthermore, PKR may induce NF-B by two potential mechanisms: phosphorylation-induced degradation of IB and/or inhibition of de novo IB protein synthesis at the translational level. Studies are under way to dissect the role of PKR in NF-B activation and enhancement of HIV-1 infection.
Third, viruses play an indirect role in increasing virus spread and susceptibility of cells to infection. For example, some strains of human herpesvirus 6 upregulate CD4 surface expression on CD8 ϩ T cells, expanding the repertoire of cells permissive to HIV-1 infection (214). Herpesviruses also upregulate Fc receptors on myeloid cells, thus providing an alternate route of HIV-1 entry into uninfected cells (226). Viral dissemination may also be enhanced by attraction of HIV-1-infected monocytes to sites of secondary infection (e.g., CMV in the central nervous system [CNS]) (260). Finally, HIV-1 upregulation may occur indirectly in virally infected cells through the production of inflammatory cytokines (see below) (293).

Bacterial Products
LPS is a major component of the outer membrane of most gram-negative bacteria and is used to simulate certain effects of bacterial infection in cell culture models. LPS binds to a 55-kDa cell surface protein, CD14, expressed mainly on monocytes, macrophages, and B cells but to a lesser extent on epithelial cells. Interestingly, CD14 expression is upregulated in chronically HIV-1-infected PLB-985 cells as an marker of monocytic differentiation (331). Exposure to LPS leads to the induction of genes associated with cellular activation and differentiation, i.e., the light-chain gene in pre-B cells and cytokine genes in myeloid cells. LPS is able to rapidly stimulate NF-B-binding activity in both of these cell types (reviewed in reference 253). LPS induces transcription from the HIV-1 LTR in monocytes but not T cells (19,310), depending on the differentiation state of the cells. For example, LPS stimulates the HIV-1 enhancer in dividing, freshly isolated monocytes and U937 promonocytic cells (30) but downregulates HIV-1 expression in PMA-stimulated, nondividing U937 cells and macrophages. The reduction in HIV-1 LTR-directed gene expression is mediated through an element distinct from NF-B enhancer sites (30).

Physical Stress
Physical factors causing cellular stress, such as UV light and heat, enhance HIV-1 replication during de novo infection and in chronically infected cells (370). A transcriptional mechanism, mediated by stress-induced activation of NF-B, is implicated in the process, since UV light and heat both induce expression of HIV-1 LTR-driven reporter constructs in an NF-B-dependent manner (392). Temperatures of approximately 41ЊC synergize with IL-6 in the enhancement of HIV-1 expression in chronically infected promonocytic and T cells (369). Furthermore, deletion of NF-B sites within the HIV-1 enhancer abrogates heat shock induction of HIV-1 LTR-directed gene expression (369).
Emotional stress can impair several immunological responses in humans and animals and is usually accompanied by altered levels of neurohormones or CNS-related peptides in the blood (125). Myeloid cells express surface receptors for several of these CNS-derived proteins, which result in impaired macrophage cytotoxic functions (133). However, establishing a correlation between in vitro studies and the effects of psychological stress to the in vivo progression of HIV disease 492 ROULSTON ET AL. MICROBIOL. REV.
is exceedingly difficult. One study showed no correlation between CD4 cell counts and degree of emotional stress in seropositive individuals over a 1-year period (290). Similarly, malnutrition is associated with immunological impairment (51). Malnutrition occurs frequently during HIV infection and is enhanced with disease progression. Indicators of malnutrition such as low albumin and transferrin levels in serum correlate with poor clinical outcome in AIDS patients, independent of CD4 lymphocyte counts (374).

CYTOKINE MODULATION OF HIV-1 REPLICATION
Inflammatory mediators and cytokines are secreted by activated myeloid and lymphoid cells during normal immune responsiveness to pathogenic infections. Many cytokines released by activated lymphoid and myeloid cells stimulate viral replication (Table 1), contribute to HIV-1 pathogenesis, and hasten progression to AIDS (reviewed in references 47, 85, 304, and 305). Cytokines stimulate the production of a variety of additional cytokines in an autocrine and/or paracrine manner. The microenvironment of the lymph node provides an environment where B cells, T cells, and myeloid cells are in close contact; hence, complex cytokine networks involving paracrine and autocrine effects may be important in such tissues with regard to clinical latency and disease progression. Increased cytokine expression occurs in lymph nodes proximal to areas of virus replication (80). Furthermore, activated B lymphocytes, isolated from the lymph nodes of AIDS patients, are able to stimulate HIV-1 expression in autologous T cells by secretion of IL-6 and TNF-␣ (377). In addition, coculture of monocytes from infected donors with allogeneic T cells and mitogens results in monocytic differentiation and HIV-1 expression (346); cell-to-cell contact and cytokine gene expression is required to stimulate HIV-1 replication (345). Therefore, the effects of a given cytokine on HIV-1 replication are dependent on the mixture of soluble mediators present within the immediate environment of infected cells. Although the stimulatory and/or inhibitory effects of many cytokines are studied in vitro, a direct correlation with cytokine effects in vivo is difficult to establish.

Activating Cytokines
Several cytokines exert stimulatory effects on HIV-1 replication and HIV-1 LTR-driven transcription (Table 1). TNF-␣, also known as cachectin, was first described as an endotoxininduced factor that was cytotoxic for certain tumor cells (31). TNF-␣ is produced primarily by macrophages, NK cells, and T cells and plays a critical role in the acute-phase response to bacterial pathogens. TNF-␤ (lymphotoxin) is closely related to TNF-␣ (30% amino acid homology) but is produced exclusively by lymphocytes (394). Both molecules bind with high affinity to the same two receptors, TNFR-␣ (75 kDa) and TNFR-␤ (55 kDa), expressed on virtually all cell types (68,208). Consistent with a common receptor, TNF-␣ and TNF-␤ exhibit similar biological activities including induction of B-cell and myeloid cell differentiation, procoagulant activity, bone resorption, and the induction of the cytokines IL-1, IL-6, and GM-CSF (5). Overproduction of TNF-␣ and TNF-␤ in vivo is implicated in cachexia (progressive wasting), septic shock, and autoimmune disorders (273,382).
The inductive effect of TNF-␣/␤ on HIV-1 expression is well documented for T lymphocytes and mononuclear phagocytes. When supernatants from phytohemagglutinin-stimulated PB-MCs or LPS-stimulated macrophages are added to chronically infected T-cell lines, upregulation of HIV-1 replication is observed (55,97). Most of the inductive activity from these supernatants originates from TNF-␣ (55,96). TNF-␣/␤ can also enhance viral replication when added exogenously in vitro or when produced endogenously during virus spread in culture (55, 96, 180, 187, 306; reviewed in reference 242). TNF-␣ triggers HIV-1 activation by inducing NF-B (79,274). The signal transduction pathway leading to NF-B DNA-binding activity involves signaling through the 55-kDa TNF-␣ receptor via sphingomyelinase and ceramide (350). In this respect, TNF-␣ can rescue virus replication in cell lines infected with a mutant clone deleted in the Sp1 region of the HIV-1 LTR (282). TNF-␣/␤ is therefore an important mediator of HIV-1 replication, and elevated levels in blood serum seen in advanced disease may contribute to the severe weight loss common in AIDS patients (355). IL-1 is a pleiotropic cytokine consisting of two distinct but related proteins, IL-1␣ and IL-1␤. IL-1 is produced mainly by activated macrophages but also by fibroblasts, T and B lymphocytes, and brain astrocytes and microglia (5). Its biological activities are diverse and include growth and differentiation of T and B cells and induction of prostaglandin E 2 synthesis. IL-1 contributes to symptoms such as fever, sleep, and anorexia, seen in individuals exposed to pathogenic stimuli (72). IL-1 also stimulates HIV-1 at the transcriptional level by inducing NF-B DNA-binding activity in T cells (180) and in chronically infected promonocytes (307).
IL-2 was originally described as a lymphokine produced by antigen-or mitogen-stimulated T cells. Its primary function is to cause the proliferation and activation of CD4 ϩ T lymphocytes, although it also stimulates macrophages, B cells, and NK cells (13). In contrast to its role in T-cell activation, IL-2 does not influence HIV-1 replication in T-cell lines (55,135). However, enhanced replication of HIV-1 occurs in PBMCs in patients undergoing IL-2 cancer therapy (158). Transcriptional mechanisms may be involved since IL-2 stimulates NF-B DNA-binding activity in primary monocytes but not in T cells (37). IL-2 may also stimulate HIV-1 replication indirectly in vivo via the production of TNF-␣ and IL-6 (242).
IL-3 and the CSFs GM-CSF and M-CSF upregulate HIV-1 expression in primary macrophages and several myeloid cell lines (97,114,116,179,349). These growth factors are primarily responsible for hematopoiesis in the bone marrow. They are produced by T cells, B cells, macrophages, and endothelial cells in response to cytokine or inflammatory stimuli (13). In fact, myelomonocytic progenitor cells (which are permissive to HIV-1 infection) are highly sensitive to these growth factors, suggesting that the CSFs may affect HIV-1 replication in cells at multiple stages of myeloid cell differentiation (304). The increased expression of HIV-1 following stimulation of macrophages with GM-CSF does not act through the NF-B-binding region of the HIV-1 LTR; the region responsible for GM-CSF activation in these cells is located slightly upstream of and partially overlaps the NF-B binding regions (415).
IL-6 was first characterized as a B-cell stimulatory factor that was strongly induced in virus-infected fibroblasts (5). It is secreted by macrophages, T cells, B cells, endothelial cells, brain astrocytes, and bone marrow stromal cells. Similar to the cytokines IL-1 and TNF, IL-6 is multifunctional and is produced as an acute-phase response factor (13). Its biological activities include induction of T-cell, B-cell, macrophage, and neural cell differentiation and hematopoietic stem cell growth (5). IL-6 induces HIV-1 expression and replication in both acutely infected MDMs and a chronically infected monocytic cell line (302). IL-6 does not seem to affect viral replication in T cells (302), even though it is known to induce T-cell activation. Of interest is the fact that IL-6 induces HIV-1 expression at a posttranscriptional level, most probably by enhancing translation of viral proteins (302). However, IL-6 can also synergize with TNF-␣ and IL-1 to enhance viral transcription (302,307).

Bifunctional Cytokines
A level of complexity exists in terms of the regulatory effects of cytokines on HIV-1 replication. In some cases, either stimulatory or inhibitory activities seem to be a function of cell type and differentiation state (Table 1). TGF-␤ is produced primarily by platelets and bone marrow stromal cells (222). TGF-␤ has an extremely wide range of biological activities which vary depending on cell type, state of differentiation, and the presence of other cytokines. With few exceptions, all cells have surface receptors for and respond to TGF-␤. Although TGF-␤ has stimulatory effects on growth in some situations, the most common effects are inhibition of proliferation in T cells, B cells, and hematopoietic progenitors. TGF-␤ is also suppressive for the killing activity of NK cells and macrophages (reviewed in reference 222). With respect to HIV-1 replication, TGF-␤ has both stimulatory and inhibitory effects. In primary T-cell or MDM cultures and in promonocytic U937 cells, TGF-␤ treatment prior to infection has a predominantly positive effect on viral replication (192,294,308). However, if either primary cells or cell lines are infected prior to cytokine addition, TGF-␤ attenuates PMA-, TNF-␣-, or IL-6-stimulated HIV-1 infection (308).
IFN-␥ is produced by T lymphocytes and NK cells in response to antigenic or mitogenic stimuli. IFN-␥ induces expression of major histocompatibility complex class II expression on epithelial and mononuclear cells, allowing them to become active antigen-presenting cells. IFN-␥ also acts as a very potent stimulus of macrophage cytocidal activity against tumors (13). One study reported that IFN-␥ enhances the formation of syncytia in lymphocytes and PBMCs (397). Similar to the effects of TGF-␤, pretreatment of cells with IFN-␥ prior to infection results in upregulation of HIV-1 production whereas postinfection treatment with IFN-␥ decreases virus expression (179). Decreased virus expression in macrophages may result from the accumulation of virus particles in intracytoplasmic vacuoles (33). These results suggest that the state of differentiation or activation can also influence the effects of IFN-␥ on HIV-1 replication.
IL-4 is a pleiotropic cytokine that is released primarily by activated lymphocytes. IL-4 enhances the generation of cytotoxic T lymphocytes, induces macrophage cytocidal activity, and enhances cell adhesion molecule expression on endothelial cells. In B cells, IL-4 plays a role in growth, differentiation, and immunoglobulin isotype switching (13,283). IL-4 either induces or suppresses HIV-1 replication in primary MDMs as a function of maturational stage. In freshly isolated monocytes, IL-4 increases both viral replication and cell proliferation (160,349). In differentiated macrophages, IL-4 has a suppressive effect on HIV-1 gene expression (267,349). IL-4 seems to have no effect on HIV-1 replication or HIV-1 LTR-directed chloramphenicol acetyltransferase activity in T cells (96,274). Differences in culture conditions, strain of virus, differentiation state of cells, and time of cytokine addition relative to HIV-1 exposure may account for differences in modulatory effects seen in various studies.

Inhibitory Cytokines
The type 1 IFNs possess a broad spectrum of antiviral, immunoregulatory, and antiproliferative activities (reviewed in reference 340). Type 1 IFNs are produced mainly in leukocytes (IFN-␣) and fibroblasts (IFN-␤) in response to viral infection. A single gene encodes IFN-␤, whereas over 20 subtypes of IFN-␣ exist, each encoded by a distinct gene. IFN-␣ and IFN-␤ bind to a common ubiquitously expressed cell surface receptor, leading to the induction of specific genes, the interferon-stimulated genes, which are responsible for cellular antiviral and immunoregulatory activities (340). Two of the best-characterized interferon-stimulated genes encode 2Ј,5Ј-oligoadenylate synthetase and PKR (340). 2Ј,5Ј-Oligoadenylate synthetase exerts antiviral activity by activating an RNase that degrades viral RNA, whereas PKR phosphorylates the initiation factor eukaryotic initiation factor 2␣ thus preventing efficient translation of viral proteins (340).
Both IFN-␣ and IFN-␤ exert similar anti-HIV-1 effects on replication in monocytes and macrophages and appear to act at  (113,114). Both inhibition of HIV-1 LTR-directed transcription and increased mRNA degradation are responsible for the IFN-mediated inhibition of HIV-1 (113). Studies of acute HIV-1 infection of PBMCs demonstrate that IFN inhibits replication at a posttranslational level (141,366,410), since the synthesis of viral proteins is unaffected. IFN inhibits terminal events in the replication cycle, including viral assembly and budding, resulting in the formation of defective viral particles attached to the host cell membrane (366). In T cells, IFN interrupts the HIV-1 life cycle through transient reductions in viral mRNA synthesis and through inhibition of virus assembly and release (113,412). IFN-␣ also blocks an early event following viral entry, preventing the reverse transcription of the viral genome in acutely infected CEM T cells (362). In vitro, both IFN-␣ and zidovudine (AZT) are synergistic in their ability to slow the spread of HIV-1 replication in promonocytic cells (76). Clinically, IFN-␣ is used to treat Kaposi's sarcoma (KS) (190), and current clinical trials are exploring the potential benefit of the combined use of IFN-␣ and AZT or other nucleoside analogs (303). Two additional cytokines, IL-10 and IL-13, also specifically inhibit HIV-1 replication in MDMs. Both cytokines are members of the IL-4 superfamily, released by activated Th2 lymphocytes, and negatively regulate the activities of antigen-stimulated Th1 cells (IL-10) and monocytes (IL-13) (1, 241). IL-10 is thought to inhibit at the level of virus assembly (175). The mechanism by which IL-13 inhibits HIV-1 is still not clear, although no effect on viral entry, reverse transcriptase, or viral release has been reported (246).

Neurological Disorders
As many as 80% of all AIDS patients with advanced disease have neurological abnormalities, and approximately half of these suffer symptoms associated with CNS disorders (234). The most common disorder in HIV-1-infected individuals is encephalopathy, a fatal illness causing severe dementia. Events leading to encephalopathy are unclear, but infiltration by monocytes and macrophages is a consistent finding in the brains and CNS of AIDS patients (173). In AIDS dementia, macrophages and microglial cells of the CNS are the predominant cell types that are infected and produce HIV-1. These cells also produce cytokines such as IL-1, TNF-␣, TGF-␤, G-CSF, GM-CSF, prostaglandin E 2 , and IL-6. Many of these cytokines are chemotactic for other monocytes and cause death of oligodendrocytes and/or destruction of myelin in vitro (322,400). In a study of 38 patients, elevated levels of cytokines such as IL-1␤ and IL-6 were present in the cerebrospinal fluid of about half of the HIV-1-infected individuals studied (105). Therefore, many of the clinical and histological effects of HIV-1 infection in the CNS may be an indirect effect of cytokines and other soluble mediators secreted by resident macrophages and microglial cells.

Hematopoietic Disorders
Viral infection of CD34 ϩ myeloid precursor cells may be related to a variety of hematological abnormalities (cytopenias) seen in adults and children with AIDS (reviewed in reference 73). Hematopoietic stem cells can also act as reservoirs of HIV-1 within the bone marrow, which may enable the virus to spread to other cells, thus accelerating hematopoietic failure (73,225). Anemia is the most common cytopenia associated with HIV-1 infection, and the condition increases in severity with disease progression (418). Approximately, 10 to 20% of patients with AIDS-related complex and 35 to 75% of those with full-blown AIDS have neutropenia, granulocytopenia, or monocytopenia and also exhibit abnormalities of the bone marrow (73,342). Hematological abnormalities during the course of HIV-1 disease are largely due to ineffective hematopoiesis, which may be the result of HIV-1 infection of bone marrow stromal cells. HIV-1 infection of bone marrow stromal cells, particularly cells of the monocyte lineage, induces alterations in the production of growth factors and cytokines that adversely affect hematopoiesis (240). For example, levels of IFN secretion early in HIV-1 infection are lower than those seen in other viral infections. Later in the disease, IFN levels in serum may be elevated to myelosuppressive levels, not inhibitory to HIV-1 replication (57,99,325). IFN-␣, in particular, can inhibit bone marrow progenitor cell development (107). Concentrations of IL-1, TNF-␣, IFN-␣, and TGF-␤ in blood serum increase with progression of HIV-1 infection, and each of these cytokines can have detrimental effects on hematopoiesis (15,161,244,320). Both IL-1 and TNF-␣ induce the secretion of growth factors such as CSFs from stromal cells, which can stimulate HIV-1 replication. These cytokines are also responsible for T-cell activation and subsequent production of IFN-␥ that can in turn suppress hematopoiesis (38,90,316).

AIDS-Associated Malignancies
KS is the most common malignancy in AIDS patients, followed by B-cell lymphoma (122). Although HIV-1 does not infect either KS-derived spindle cells or B lymphocytes in vivo, several cytokines directly influence the proliferation and differentiation of these cells in vitro. These cytokines include basic fibroblast growth factor, IL-1␤, IL-6, GM-CSF, and platelet-derived growth factor for KS-derived cells (81) and IL-6, IL-4, and IL-10 for B lymphocytes (122). Recently, DNA encoding a new herpesvirus was isolated from over 90% of KS tissues from AIDS patients (52). Elevated levels of cytokines may arise in KS-derived cells as a result of infection with a virus such as a herpesvirus. As mentioned previously, elevated levels of IL-1, IL-6, and TNF are present in the sera of AIDS patients, particularly in the advanced stages of disease (40,188,194). These observations suggest that increased circulating cytokine levels may contribute to the development of AIDSassociated malignancies. VOL. 59, 1995 REGULATION OF HIV-1 AND CYTOKINE EXPRESSION BY NF-B

POTENTIAL ANTI-HIV-1 THERAPIES
NF-B is an important transcription factor in all hematopoietic cells and plays a central role in the host immune response to a wide variety of pathogens. A cysteine deficiency is reported in AIDS patients (74), and since cysteine has an antioxidant effect, its decrease in immune cells of HIV-1-infected individuals might create a prooxidant state, leading to the activation of NF-B (74,344). Although the antioxidants butylated hydroxyanisole and N-acetyl-L-cysteine inhibit TNF-or PMA-induced NF-B activity in HIV-1-infected cells, limited effects on HIV-1 replication are observed (4). Furthermore, these antioxidants have a suppressive effect on PBMC immune function in vitro (4). Although a rationale may exist for inhibition of NF-B during the early latent phase of infection, impeding hematopoietic cell function at later stages of disease may enhance immunodeficiency and increase the risk of secondary pathogenic infections.
In the face of profound immunosuppression, such as that induced by HIV-1, it is logical to propose the administration of immunostimulants. These agents, however, may induce proliferation of resting CD4 ϩ cells and enhance viral spread, thus provoking an effect opposite to the one they were designed to avoid (225). A more beneficial approach may be the use of immunosuppressants such as cyclosporin A (CsA), particularly in early stages of disease before the onset of HIV-1-induced immunodeficiency (86). CsA is commonly used to prevent tissue rejection in patients undergoing organ transplantation (365). The drug suppresses T-cell activation by blocking the activation of NF-AT (67). In vitro, CsA inhibits HIV-1 expression and cell growth in chronically infected cells (157). However, in vivo, the effects of CsA treatment in HIV-1-infected individuals leads to either clinical improvement (11) or deterioration (296), possibly as a function of the stage at which CsA treatment is commenced. A marked delay in HIV-1 disease progression and AIDS development is reported to occur in transplant patients receiving CsA treatment from the onset of infection (351). In addition to blocking cellular activation, CsA may affect other stages of the viral life cycle. CsA blocks HIV-1 virion binding to CD4 (170) and is thought to interfere with replication by preventing interactions between cyclophilins and the HIV-1 Gag protein (213). Immunosuppressants and antiinflammatory drugs may also interfere with the NF-B pathway. For example, aspirin (sodium salicylate) (176) and rapamycin (189) inhibit NF-B activation by interfering with the degradation of IB␣. Furthermore, dexamethasone, when bound to endogenous glucocorticoid receptor, can inhibit the ability of p50, RelA, and c-Rel subunits to bind DNA (313). Thus, immunosuppressants and anti-inflammatory drugs may provide therapeutic intervention at multiple levels in the treatment of HIV-1 infection.

CONCLUDING REMARKS
This review summarizes studies characterizing the mechanisms in which chronic HIV-1 infection of myeloid cells can lead to physiological changes that perpetuate HIV-1 replication and contribute indirectly to AIDS-associated disorders and symptoms (Fig. 6). HIV-1-infected myeloid cells are often diminished in the ability to participate in chemotaxis, phagocytosis, and intracellular killing. Chronic HIV-1 infection of promonocytic cells leads to constitutive NF-B DNA-binding activity and increased intracellular stores of latent NF-B. Phosphorylation and degradation of the cytoplasmic inhibitor IB␣ are crucial regulatory events in the activation of NF-B DNA-binding activity. Both N-and C-terminal residues of IB␣ are required for inducer-mediated degradation. Changes in the regulation of NF-B provide an intranuclear environment capable of maintaining a high level of HIV-1 LTR activity and NF-B-dependent cytokine gene expression. HIV-1 infection of myeloid cells can also lead to the expression of surface receptors associated with cellular activation and/or differentiation and increase the responsiveness of these cells to cytokines secreted by neighboring cells as well as bacteria or other pathogens. In response to secondary pathogenic infections, cytokine gene expression is rapidly induced and levels may be sustained over prolonged periods in HIV-1-infected myeloid cells compared with uninfected cells. Altered cytokine responsiveness of HIV-1-infected myeloid cells may contribute to the elevated levels of several inflammatory cytokines present in the sera of HIV-1-infected individuals.
Secondary bacterial or viral infections in HIV-1-infected individuals may result in unscheduled cytokine production by infected monocytic cells and may generate a potentiated inflammatory response. Secretion of myeloid cell-derived cytokines in lymph nodes or in the circulation of infected individuals may increase viral production and spread and may contribute to AIDS-associated pathogenesis (Fig. 6). Future directives for therapeutic intervention may therefore be designed to immunocompromise asymptomatic individuals as a means of controlling HIV-1 replication.