Replication of human immunodeficiency virus in monocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) potentiates viral production yet enhances the antiviral effect mediated by 3'-azido-2'3'-dideoxythymidine (AZT) and other dideoxynucleoside congeners of thymidine.

We have investigated the influence of granulocyte-macrophage CSF (GM-CSF) on the replication of HIV-1 in cells of monocyte/macrophage (M/M) lineage, and its effect on the anti-HIV activity of several 2'3'-dideoxynucleoside congeners of thymidine in these cells in vitro. We found that replication of both HTLV-IIIBa-L (a monocytotropic strain of HIV-1) and HTLV-IIIB (a lymphocytotropic strain) is markedly enhanced in M/M, but not in lymphocytes exposed to GM-CSF in culture. Moreover, GM-CSF reduced the dose of HIV required to obtain productive infection in M/M. Even in the face of this increased infection, GM-CSF also enhanced the net anti-HIV activity of 3'-azido-2'3'-dideoxythymidine (AZT) and several related congeners: 2'3'-dideoxythymidine (ddT), 2'3'-dideoxy-2'3'-didehydrothymidine (D4T), and 3'-azido-2'3'-dideoxyuridine (AZddU). Inhibition of viral replication in GM-CSF-exposed M/M was achieved with concentrations of AZT and related drugs, which were 10-100 times lower than those inhibitory for HIV-1 in monocytes in the absence of GM-CSF. Other dideoxynucleosides not related to AZT showed unchanged or decreased anti-HIV activity in GM-CSF-exposed M/M. To investigate the possible biochemical basis for these effects, we evaluated the metabolism of several drugs in M/M exposed to GM-CSF. We observed in these cells markedly increased levels of both parent and mono-, di-, and triphosphate anabolites of AZT and D4T compared with M/M not exposed to GM-CSF. By contrast, only limited increases of endogenous competing 2'-deoxynucleoside-5'-triphosphate pools were observed after GM-CSF exposure. Thus, the ratio of AZT-5'-triphosphate/2'-deoxythymidine-5'-triphosphate and 2'3'-dideoxy-2'3'-didehydrothymidine-5'-triphosphate/2'-deoxythymi dine- 5'-triphosphate is several-fold higher in GM-CSF-exposed M/M, and this may account for the enhanced activity of such drugs in these cells. Taken together, these findings suggest that GM-CSF increases HIV-1 replication in M/M, while at the same time enhancing the anti-HIV activity of AZT and related congeners in these cells. These results may have implications in exploring new therapeutic strategies in patients with severe HIV infection.

LFA-3, CD44, and CD45: physiologic triggers of human monocyte TNF and IL-1 release.

The monocyte-derived cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), are central regulators of the immune response, but the physiologic stimuli for their release remain largely undefined. Engagement of three monocyte glycoproteins, LFA-3, CD44, and CD45, by specific monoclonal antibodies immobilized on plastic induced TNF-alpha and IL-1 beta release. In addition, TNF-alpha was released when monocyte LFA-3 bound immobilized, purified CD2, which is its physiologic receptor. Thus, a receptor-ligand interaction that mediates cell-cell adhesion can transmit the necessary signals for the release of monokines.

Tyrosine phosphorylation of DNA binding proteins by multiple cytokines.

Interferon-alpha (IFN-alpha) and IFN-gamma regulate gene expression by tyrosine phosphorylation of several transcription factors that have the 91-kilodalton (p91) protein of interferon-stimulated gene factor-3 (ISGF-3) as a common component. Interferon-activated protein complexes bind enhancers present in the promoters of early response genes such as the high-affinity Fc gamma receptor gene (Fc gamma RI). Treatment of human peripheral blood monocytes or basophils with interleukin-3 (IL-3), IL-5, IL-10, or granulocyte-macrophage colony-stimulating factor (GM-CSF) activated DNA binding proteins that recognized the IFN-gamma response region (GRR) located in the promoter of the Fc gamma RI gene. Although tyrosine phosphorylation was required for the assembly of each of these GRR binding complexes, only those formed as a result of treatment with IFN-gamma or IL-10 contained p91. Instead, complexes activated by IL-3 or GM-CSF contained a tyrosine-phosphorylated protein of 80 kilodaltons. Induction of Fc gamma RI RNA occurred only with IFN-gamma and IL-10, whereas pretreatment of cells with GM-CSF or IL-3 inhibited IFN-gamma induction of Fc gamma RI RNA. Thus, several cytokines other than interferons can activate putative transcription factors by tyrosine phosphorylation.

Relationship of glucocorticoid suppression of arachidonic acid metabolism to alteration of neutrophil function.

The role of arachidonic acid metabolism in glucocorticoid-induced suppression of neutrophil function was investigated. In vivo treatment of cattle with dexamethasone decreased the production of lipoxygenase products of arachidonic acid metabolism (leukotriene B4 [LTB4] and H-5-hydroxy-6,8,11,14-eicosatetraenoic acid [5-HETE]) in neutrophils. We previously reported that in vivo dexamethasone treatment resulted in alteration of in vitro neutrophil oxidative metabolism, iodination, antibody-dependent cell-mediated cytotoxicity (ADCC), and random migration. To determine if the decrease in production of LTB4 and 5-HETE were responsible for the changes in neutrophil function, neutrophils were treated in vitro with inhibitors of the lipoxygenase enzyme (BW755c and nordihydroguaiaretic acid), and their function was evaluated. A decrease in neutrophil oxidative metabolism and iodination was observed, but there was no effect on neutrophil random migration or ADCC. The results suggest that in vivo treatment with glucocorticoids inhibit neutrophil oxidative metabolism and iodination by decreasing the formation of lipoxygenase products of arachidonic acid metabolism but alter neutrophil random migration and ADCC by a mechanism independent of arachidonic acid metabolism.

Enhanced luminol-dependent chemiluminescence of stimulated rat alveolar macrophages by pretreatment with alveolar lining material.

Previous reports indicate that the in vitro bactericidal activity of rat alveolar macrophages (AM) is dependent on the lipid fraction (ALM-L) of the alveolar lining material (ALM). The present study demonstrates that luminol-dependent chemiluminescence of stimulated rat AM is increased when rat AM are preincubated in the ALM or in the ALM-L. Evidence is presented that oxidation of the unsaturated lipids is responsible for the increase. In addition to pretreatment with the ALM, cells were also pretreated in commercial preparations of several lipids found to be present in the ALM. Preincubation in these lipids produced a significant increase in the luminol-dependent chemiluminescence response. However, when a saturated lipid, dipalmitoyl phosphatidylcholine, was used no increase was found. Pretreatment in ALM did not increase the nitro blue tetrazolium dye reduction by the AM, nor was the phagocytosis of latex beads by the AM altered by the addition of the ALM.

Role for arachidonic acid metabolism and protein synthesis in recombinant bovine interferon-gamma-induced activation of bovine neutrophils.

Bovine neutrophils were preincubated with recombinant bovine interferon-gamma (rboIFN-gamma), and the molecular events leading to enhanced antibody-dependent (ADCC) and antibody-independent (AINC) neutrophil-mediated cytotoxicity and reduced random migration under agarose were investigated. Addition of alpha-amanitin, puromycin, or cycloheximide (RNA and protein synthesis inhibitors) during preincubation and assaying prevented the rboIFN-gamma-induced AINC enhancement and migration inhibition, but did not influence the enhancement of ADCC. Treatment of neutrophils with rboIFN-gamma consistently increased the synthesis of a 60 and a 94-95 kilodalton protein and of leukotriene B4 and 5-HETE. Neutrophils isolated from dexamethasone-treated cattle showed similar protein synthetic activity after rboIFN-gamma treatment, but these cells failed to show enhanced AINC. Neutrophils from dexamethasone-treated cattle have been shown to have depressed arachidonic acid metabolism. Addition of lipoxygenase inhibitors (NDGA and BW755c) to neutrophils from nondexamethasone-treated cattle following preincubation with rboIFN-gamma (i.e., after protein synthesis had occurred) eliminated the enhancement of AINC. Thus it was concluded that the metabolic events required for neutrophil activation by rboIFN-gamma are different for the three functions measured. The enhanced AINC response required both arachidonic acid metabolism and protein synthesis, inhibition of random migration required only protein synthesis, and enhancement of ADCC required neither.

Effects of human immunodeficiency virus and colony-stimulating factors on the production of interleukin 6 and tumor necrosis factor alpha by monocyte/macrophages.

Patients infected with human immunodeficiency virus (HIV) frequently have increased production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), and these cytokines may in turn contribute to the disease pathogenesis. It has been hypothesized that secretion of these cytokines by HIV-exposed mononuclear cells or HIV-infected monocyte/macrophages (M/Ms) is the principal source of their overproduction in HIV-infected patients, and the present study was undertaken to explore this issue. We observed that in the absence of endotoxin or cytokines, M/Ms productively infected by HIV do not produce detectable IL-6 or TNF-alpha. However, granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that enhances HIV replication in M/Ms and is frequently used to propagate monocytotropic strains of HIV, can induce the relatively long-term production of IL-6 (up to 47 U/ml) and TNF-alpha (up to 47 pg/ml) by M/Ms, even in the absence of HIV. Also, HIV induced production of a relatively small (< or = 9 U/ml) quantity of IL-6 in M/Ms stimulated with macrophage-colony stimulating factor (M-CSF). Finally, while highly concentrated HIV induced production of both cytokines by either M/Ms or peripheral blood mononuclear cells (PBMCs), this production was almost completely eliminated when care was taken to avoid contamination of HIV by endotoxin. These data suggest that the excess IL-6 and TNF-alpha in HIV-infected patients does not simply result from their production by HIV-infected M/Ms and that alternative mechanisms are involved in this process.

Re-evaluation of the involvement of the adhesion molecules ICAM-1/LFA-1 in syncytia formation of HIV-1-infected subclones of a CEM T-cell leukemic line.

The role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and LFA-1 in human immunodeficiency virus type 1 (HIV-1)-induced cell fusion was investigated in subclones of a T-cell leukemic cell line (CEM) with differing abilities to form syncytia. Addition of monoclonal antibodies 84H10 directed against ICAM-1 and MHM23 directed against the common beta subunit of LFA-1 (CD18) resulted in greater than 50% suppression of syncytia formation in cultures of these clones infected with cell-free virus. Two subclones, 2G5-144-84 and 2G5-1, were deficient in their ability to form syncytia and expressed reduced amounts of LFA-1 compared with the parental line. The expression of ICAM-1 but not LFA-1 was upregulated on the clones following treatment with interferon-gamma (IFN gamma); however, this did not overcome the delay in syncytia formation observed in these cells. The syncytia-positive subclones 1B11-39 and 17D-9 expressed high levels of LFA-1. Basal expression of ICAM-1 was upregulated on these cells by treatment with tumor necrosis factor-alpha (TNF alpha), which also accelerated and enhanced syncytia formation. However, anti-ICAM-1 and anti-LFA-1 (CD18) antibodies did not reverse the TNF alpha-induced enhancement of syncytia formation of HIV-1-infected clones 1B11-39 and 17D-9. Under conditions of low viral expression, adhesion molecules may contribute to syncytia formation if adequate levels of both receptor and ligand in the ICAM-1/LFA-1 complex are expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

IFN-alpha and IFN-gamma can affect both monocytes and tumor cells to modulate monocyte-mediated cytotoxicity.

IFN-alpha and IFN-gamma were found to enhance monocyte-mediated activity by acting on both tumor cells and monocytes. The addition of IFN-alpha or IFN-gamma enhanced the monocyte-mediated cytotoxicity of the human melanoma cell line, A375, as well as the human colon carcinoma cell line, HT-29. However, IFN-alpha generally induced more monocyte-mediated lysis of the A375 cells, whereas IFN-gamma induced more monocyte-mediated lysis of the HT-29 cells. These differences are, in part, due to the direct effects of the IFN on the tumor cells. Pretreatment of A375 cells with either IFN-alpha or IFN-gamma significantly enhanced their susceptibility to lysis by untreated monocytes. However, only IFN-gamma pretreatment of HT-29 cells enhanced the lysis of these cells by untreated monocytes. Differences were also observed in the activation of monocytes by IFN-alpha vs IFN-gamma with respect to their ability to induce soluble cytotoxic factors. We found that the addition of IFN-gamma and tumor cells (either the A375 or HT-29 cells) to monocytes induced TNF release, whereas IFN-alpha or IFN-gamma alone or IFN-alpha and tumor cells had no effect. Despite its presence, TNF did not appear to play a major role in the killing of either tumor cell line. However, inhibitors of H2O2-myeloperoxidase system suppressed both IFN-alpha- and IFN-gamma-induced cytotoxicity of the HT-29 cells. Thus IFN-alpha and IFN-gamma can induce both similar and distinct mechanisms of cytotoxicity in monocytes. In addition, both IFN types can increase the susceptibility of tumor cells to lysis by untreated monocytes, although sensitivity to IFN-alpha vs IFN-gamma may vary with different tumor cell lines. These differences observed between IFN-alpha and IFN-gamma on monocytes and tumor cells could have important implications for the clinical use of these cytokines.

Stimulation of human monocytes via CD45, CD44, and LFA-3 triggers macrophage-colony-stimulating factor production. Synergism with lipopolysaccharide and IL-1 beta.

Stimulation of human monocytes with immobilized mAb directed against the CD45, CD44, or LFA-3 Ag induced the production of macrophage-CSF (M-CSF). M-CSF-specific transcripts appeared at 3 h poststimulation, were further increased by 12 h, and were still detectable at 24 h. M-CSF gene expression was accompanied by the induction of small but detectable amounts of M-CSF protein. LPS and IL-1 beta, but not IL-6 or TNF-alpha, dramatically augmented the ability of anti-CD45, anti-CD44, and anti-LFA-3 antibodies to induce M-CSF secretion but failed to stimulate M-CSF secretion in the absence of antibody. M-CSF activity in the culture supernatants was first detectable at 8 h of culture, peaked at day 2, and declined thereafter. Purified F(ab)2 fragments of anti-CD45 antibody were also effective in inducing M-CSF message and secretion, indicating that the Fc gamma RII is not involved in this response. Stimulation of cells with antibodies to the monocyte surface Ag MAC-1, LFA-1, and ICAM-1 did not result in M-CSF secretion. Moreover, LPS and IL-1 beta failed to synergize with these Ag in inducing M-CSF production. Together, these results indicate that stimulation of monocytes via the cell surface Ag CD45, CD44, and LFA-3 can trigger M-CSF production. However, second signals that can be provided by IL-1 beta or LPS are required to regulate optimal M-CSF protein secretion.

Cytokine-induced enhancement of ICAM-1 expression results in increased vulnerability of tumor cells to monocyte-mediated lysis.

Pretreatment of the human melanoma cell line, A375, and the human colon carcinoma cell line, HT-29, with certain cytokines was found to increase the vulnerability of these cells to monocyte-mediated killing. This activity was found to correlate with increased expression of intercellular adhesion molecule-1 (ICAM-1) on the tumor cells and was blocked by anti-ICAM-1 antibodies. Both IFN-gamma and TNF induced large increases in the ICAM-1 expression on both cell lines and increased the susceptibility of the tumor cells to monocyte-mediated killing. IFN-alpha and IL-1 beta, however, induced only small increases in ICAM-1 expression and enhanced the lysis of the A375 cells but not the HT-29 cells by monocytes. These differences may be the result of a higher basal expression of ICAM-1 found on the A375 cells when compared with the HT-29 cells. These data indicate that regulation of ICAM-1 expression on tumor cells can alter the vulnerability of these cells to lysis by monocytes.

Enhancement of monocyte-mediated tumoricidal activity by multiple interferon-alpha species.

Twenty-one interferon (IFN)-alpha species were evaluated for their ability to enhance monocyte-mediated lysis of the human melanoma cell line, A375. A wide variation in the potency of the different species in inducing monocyte tumoricidal action was observed. In addition, many IFN-alpha species were found to induce as much or more tumoricidal activity than recombinant IFN-gamma. The degree of monocyte activation induced by the various species generally correlated with their antiviral activity. Those which were better at inducing monocyte tumoricidal action also gave the highest antiviral specific activities. Studies were conducted to determine if the relative potency of the IFN-alpha species could be changed by altering certain parameters of the cytotoxicity assay. All IFN-alpha species tested required only 30 min in culture with the monocytes to induce activation. There were no changes in the relative potency of the species when cytotoxicity was measured at different times, nor when the effector:target ratio was altered. Competitive binding studies revealed that those IFN-alpha species which induced little activity in the bioassays were also generally poor in their ability to bind the IFN-alpha receptor on human monocytes, while the IFN-alpha species which induced relatively more activity in the bioassays were better able to bind to the IFN-alpha receptor. These data indicate that there are dramatic differences in activities among the IFN-alpha species which may, in part, be explained by different binding affinities. In addition, the differences observed among the IFN-alpha species demonstrate the need for further functional and structural characterization of the individual IFN-alpha species which could lead to a more effective clinical application of IFN-alpha.

Amino terminus is essential to the structural integrity of recombinant human interferon-gamma.

Species lacking either 8 or 10 residues at the amino terminus of recombinant human interferon-gamma (Hu-IFN-gamma) were generated by limited digestion with Staphylococcus aureus V8 protease. A crude digest, consisting predominantly of these species, were completely inactive in inducing antiviral activity and the expression of HLA-DR antigens on HL-60 cells. The NH2-terminal deletion fragments were separated from residual intact IFN-gamma and from smaller polypeptides by reverse phase high performance liquid chromatography (HPLC) at pH 2.2. Intact IFN-gamma, purified by HPLC and subsequently refolded by dilution in 0.1 M sodium phosphate buffer (pH 7.5, 0.1% bovine serum albumin) was similar to untreated IFN-gamma in terms of binding to its cell surface receptor and in inducing antiviral activity and the expression of HLA-DR molecules. Conversely, biological activity was not detected in purified fragments 8-139 and 10-139. Examination of fragments 8-139 and 10-139 by far-UV circular dichroism revealed that cleavage of 8-10 residues at the amino terminus accompanied a dramatic change in secondary structure (6% alpha-helical and 36% beta-sheet content) as compared to untreated or HPLC-purified IFN-gamma (66% alpha-helix and 0% beta-sheet content). In summary, these results indicate that the amino terminus contributes to the structural integrity of the IFN-gamma molecule.

Seismic evaluation of existing liquid low level waste system

The existing liquid low level waste (LLLW) ystem of the Oak Ridge National Laboratory is used to collect, newtralize, concentrate, and store the radiactive and toxic waste from various sources and the Laboratory. The waste solutions are discharged from source facilities to individual collection tanks, transferred by underground piping to an evaporator facility for concentration, and pumped through the underground piping storage in underground tanks. The existing LLLW system was installed in the 1950s with several system additions up to the present. The worst-case accident postulated is an earthquake of sufficient magnitude to rupture tha tanks and/or piping so as to damage the containment integrity to thesurrounding soil and environment, The objective of an analysis of the system to provide a level of confidence in the seismic resistance of the LLWsystem to withstand the postulated earthquake (AU)