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Human herpesvirus 6 (HHV-6)-associated dysfunction of blood monocytes
V7ru.sResearch, 29 (1993) 79-90 0 1993 Elsevier Science Publishers B.V.
Virus
All rights reserved 016%1702/93/$06.00
Research
VIRUS 00894
Human herpesvirus 6 (HHV-6) -associated dysfunction of blood monocytes Eileen M. Burd and Donald R. Carrigan Department of Pathology, The Medical College of Wmconsin, Milwaukee, WI 53226, USA
(Received 12 November 1992; revision received and accepted 3 February 1993)
Summary
HHV-6 is a recently described member of the herpesvirns family. HHVd-associated marrow failure and interstitial pneumonitis where macrophages are the primary infected cell type have been described in marrow transplant patients (Carrigan, 1991; Drobyski et al., 1993). In recent studies we have shown that exposure of normal human marrow to HHV-6,, (a type A strain) or several type B strains resulted in suppression of growth factor induced outgrowth of macrophages by > 90% (Burd and Carrigan, 1993). Additional experiments using HHV-6,, to characterize the effects of the virus on peripheral blood monocytes showed that the respiratory burst capacity of these cells as determined by luminol-enhanced chemiluminescence using phorbol myristate acetate as a trigger was decreased by 83% f 13% in a series of 5 experiments. The decreased respiratory burst was evident as early as 15 min after exposure to virus. Experiments in which cells were separated on a fluorescence activated cell sorter prior to respiratory burst assay showed that the response was mediated solely by peripheral blood monocytes. The respiratory burst response of virus-exposed cells to opsonized zymosan was not affected, indicating that the virus may selectively interfere with the protein kinase C pathway of cellular activation. Ultracentrifugation of stock material to remove infectious virus showed that the suppressive factor was associated with the supernatant fraction. These findings suggest that HHV-6 infection may be associated
Correspondence
to: E.M. Burd, Department kee, WI 53226, USA.
of Pathology, The Medical College of Wisconsin, Milwau-
with a defect in one of the major monocyte activation pathways, and this could be of importance with respect to persistent infection by HHV-6 in immune compromised patients.
Human herpesvirus 6; Respiratory suppression
burst; Monocyte; Signal transduction;
Immune
Introduction HHV-6 was first isolated in 1986 from patients with lymphoproliferative disorders (Salahuddin, 1986). Subsequently it has been determined that HHV-6 infection is common and occurs early in life as evidenced by the high percentage (60-90%) of individuals over the age of two years who are seropositive (Pellet 1992). The most clearly established clinical manifestations of primary HHV-6 infection are exanthem subitum (roseola) and an acute febrile illness without rash in young children (Pellet et al., 1992; Pruksanonda et al., 1992). Serious complications are not rare and include encephalitis, fatal hepatitis, and fatal hemophagocytic syndrome (Pruksanonda et al., 1992; Asano et al., 1990; Huang et al., 1990). HHV-6 infection has recently been implicated as a major cause of febrile convulsions in children (Segondy et al., 19921, and acute HHV-6 infections account for between 10 and 20% of all febrile children seen in pediatric emergency rooms (Pruksanonda et al., 1992). HHV-6 shares with the other members of the herpesvirus family the propensity to establish latent infections that are subject to frequent reactivations. In immune compromised patients such reactivations often progress to persistent, active infections associated with considerable morbidity and mortality. A diverse range of pathogenic mechanisms appear to be operative in these patients. In bone marrow transplant (BMT) patients persistent HHV-6 infection of the lung causes an interstitial pneumonitis most probably mediated by immunopathological mechanisms since the infection is restricted to macrophages and lymphocytes with no apparent involvement of lung parenchymal cells (Carrigan et al., 1991). A second, and apparently more common and dangerous, manifestation of HHV-6 infection in BMT patients is marrow suppression and marrow failure (Drobyski et al., 1993). In vitro evidence suggests that these marrow-suppressive effects of HHV-6 are likely to be mediated by a combination of alpha-IFN induction by the virus within the infected marrow and the release of a cytokine-like activity from infected cells that blocks maturation of marrow stem cells and stromal elements (Knox and Carrigan, 1992a), probably by interfering with the responses of marrow cells to growth factors (Burd and Carrigan, 1993). Persistent HHV-6 infections in renal allografts show a strong correlation with rejection of the organ (Okuno et al., 1990), suggesting that HHV-6 shares with cytomegalovirus the ability to enhance im-
81
munologic responses against a tissue, perhaps by up-regulating major histocompatibility complex antigen expression (Apperly and Goldman, 1988). Another important aspect of the pathogenic potential of HHV-6 may be related to its ability to interfere with the specific and non-specific defensive responses of the infected host. Central to this potential mechanism is the tropism of HHV-6 for CD4 + T-lymphocytes (Takahashi et al., 1989), and its ability to down-regulate the expression of CD3 from the surface of T-lymphocytes (Lusso et al., 1988). Mature macrophages also provide a target for HHV-6 in the lungs of BMT patients with HHV-6 pneumonitis (Carrigan et al., 1991), in macrophage cultures derived from peripheral blood mononuclear cells (PBMC) (Carrigan, 1992; Knox and Carrigan, 1992b) and in normal human bone marrow cultures (Knox and Carrigan, 1992a). Monocyte infection by HHV-6 has been described (Kondo et al., 19911, but little is known about the effects of the infection on the antimicrobial and other functions of the monocyte. Other viruses such as HIV and CMV which infect leukocytes are known to have serious immunosuppressive effects in infected individuals. In vitro, CMV infection of monocytes has been shown to result in severe dysfunction of these cells with suppression of a variety of activities including phagocytosis, oxidative metabolism, production of inflammatory mediators and immunoregulation (Rouse and Horohov, 1986). By analogy to these and other macrophage-tropic viruses, HHV-6 infection is likely to cause similar immunosuppressive effects and may amplify the immunocompromised status in already compromised patients. Exploration of the effects of HHV-6 infection on monocyte functions may provide important information about the potential for HHV-6 to directly cause disease or to synergize with other pathogens to transform a mild illness into a life-threatening infection. Specifically, the work presented here describes the effect of exposure to HHV-6 on the respiratory burst capacity of peripheral blood monocytes as detected in a chemiluminescence assay.
Materials and Methods
Two different strains of HHV-6, HHV-6,, and HHV-6,,,, were used in these studies (provided by Dr. D. Ablashi of the National Cancer Institute, Bethesda, MD). These are prototype A and B types of HHV-6, respectively (Ablashi et al., 1991). HHV-6,, was grown in the HSB-2 human leukemia cell line and HHV-6,,, was propagated in the Molt-3 T-leukemia cell line. Virus stocks were prepared by homogenization and clarification by centrifugation of infected cell cultures showing greater than 50% cytopathic effect. ‘Mock’ homogenates of identically prepared uninfected cells were used as controls in all experiments. To determine the role of infectious virus, the stocks were ultracentrifuged at 150,000 X g for 2 h at 4°C to eliminate the infectious virus particles. Elimination of
82
virus was confirmed in each experiment by back-titration appropriate cell line. Chemiluminescence
of the sup~matant
in the
assay
A luminol-enhanced chemiluminescent assay was modified from the procedure described by Prendergast and Proctor (1981). All samples were assayed in triplicate by repetitive scintillation counting using a Packard Tri-Carb Model ZOOOCAliquid scintillation counter (Packard Instrument Co., Downer’s Grove, IL). Results were expressed as counts per minute (cpm) using the full tritium counting channel. All samples were counted at 5- to lo-min intervals after the addition of inducer for at least one hour. Comparisons among samples were based upon the m~mum cpm value measured for each sample at any time during the counting period. PBMC were separated from whole heparinized blood by Ficoll-Hypaque density centrifugation and were exposed to virus, mock control, or culture medium at a concentration of 0.5 ml of virus or control material per lo6 cells for 15 min. The cells were then pelleted, resuspended in Basal Medium Eagle (BME: Sigma Chemical Co., St, Louis, MO) and aliquoted into dark-adapted, sterile, siliconized, glass scintillation vials at a concentration of lo6 cells in 100 ~1 BME per vial. An 8 X lo-’ M working solution of luminol (Sigma Chemical Co, St. Louis, MO) in distilled water, was prepared from a 5 X lo-’ M stock solution in dimethylsulfoxide (DMSO) and was added to each vial (100 pi/vial). Phorbol myristate acetate (PMA: Sigma Chemical Co., St. Louis, MO), 5 @g/ml in BME diluted from a 5 mg/ml DMSO stock solution was used as a soluble stimulus. Zymosan (Sigma Chemical Co., St. Louis, MO) was opsonized by incubation in autologous serum at 37°C for 30 min, washed, and resuspended in BME at a concentration of 5 mg/ml for use as a particulate stimulus. PMA or opsonized zymosan (OZ) was added (100 &&ial) to the vials in the scintillation counter. To ensure that the chemiluminescence response corresponded to the production of oxygen metabolites, superoxide dismutase (SOD) from human e~hro~es (Sigma Chemical Co., St. Louis, MO), 46.5 units in 100 ~1 BME was added directly to control vials at the beginning of the assay along with one of the stimuli. Addition of exogenous SOD inhibited peak chemiluminescence by an average of 99.6% in assays using PMA as the stimulus and by 81% in assays using OZ as the stimulus, confirming that superoxide generation is responsible for the majority of the luminescence response seen/under these conditions. Proof that it was the monocytes in the mononuclear cell preparation that were responsible for the respiratory burst being assayed was obtained by testing whole mononuclear cell preparations for their ability to produce a respiratory burst in comparison with mononuclear cell preparations from which the monocytes have been removed by cell separation using a Coulter EPICS 5 flow cytometer. The separated cells were determined to be free of monocytes by a two-cofor flow cytometric assay using monoclonal antibodies specific for CD45 and CD14. The monocyte-free samples showed responses similar to those of the luminol background control while unseparated PBMC preparations in the same assay gave a
83 TABLE 1 Donor
Experiment no.
% suppression of PMA-induced respiratory burst by:
H~-6zz,
HHV-6,,
99 88
84 not done
87 90
60 not done
86
68
typical respiratory burst response. These results demonstrate that monocytes were responsible for producing the respiratory burst in this system. Controls of the respiratory burst assay system using BME, PBMC, OZ, or PMA by themselves generated no detectable chemiluminescence.
Results
Effect of HHV-6 on phorbol ester-stimulated monocyte respiratory burst HHV-6 had a markedly suppressive effect on PMA-stimulated respiratory burst after as little as 15 min of exposure to the virus material. In each of 5 experiments using PBMC from three different donors, HHV-6,, suppressed chemiluminescence by at least 86% and as much as 99% compared to mock-infected controls as measured by peak CPM values in the assay (Table 1). Exposure of PBMC to HHV-6,, also reduced the chemiluminescence response. The suppression with HHV-6,, ranged from 60-84% in 3 experiments (Table 1). A representative pattern of PMA-stimulated chemiluminescence observed with HHV-6,, and HHV-6 z29 in one experiment is presented in Fig. 1. Of special note is the persistence of the burst suppression throughout the assay by HHV-6,,, whereas the inhibition by HHV-6,,, was more transient. This increased suppression by the type A HHV-6 was consistently observed and corresponded to a similar greater suppression of bone marrow stem cell responses to growth factors by the A type of virus (Burd and Carrigan, 1993). Effect of HHV-6 on opsonized Zymosan-stimulated respiratory burst The marked suppression of phorbol ester (PMA)-stimulated monocyte respiratory burst made it of interest to determine whether the suppression extended to OZ stimulated respiratory bursts. In contrast to PMA stimulation, which is mediated through direct binding of the phorbol ester to protein kinase C (PKC) (Tauber, 1987), OZ stimulates phagocyte respiratory bursts through a membrane receptor system coupled to a PKC-independent, phospholipase A2 (PL-A2) and
e-e HliMm X-X MockHHV4&bfdt-3) CI
Me&m
60
’ t
Resting PMA
4 li b i3 411I9 :, Time (min)
Fig. 1. Chemiluminescence response of PBMC after in vitro exposure for 15 min to materials derived from HHV-6-infected cell cultures. Panels A and B show results with HHV-6,s and HHV-6,, respectively. The cells were assayed for chemiluminescence response following PMA stimulation. ‘Mock virus’ was culture medium derived from uninfected cells grown in parallel with the virus-infected culture. ‘Medium’ refers to a sample run with plain culture medium not exposed to cells. Data are presented as the mean value of triplicate assays. Bars indicate the standard error of the mean.
calmodulin-associated signal transduction pathway (Maridonneau-Parini et al., 1986; Rossi, 1986). The results obtained indicated that respiratory bursts stimulated by OZ were not suppressed in cells exposed to either HHV-6,, (Fig. 2a) or (Fig. 2b). Microscopic examination of the mixture of virus-treated HEW-6,, PBMCs and 02, at the end of the assay did not demonstrate phagocytosis of the OZ particles. However, many 02 particles were seen associated with the membranes of the mononuclear cells, indicating that surface contact alone was sufficient to initiate a respiratory burst. Therefore, a clear distinction was seen between monocyte respiratory burst activity mediated directly through PKC and that mediated through a membrane receptor linked signal transduction pathway.
Effect burst
of ultracentrifuged
(virus-free) stock preparations
on monocyte respiratory
The apparent distinction between phorbol ester-stimulated (PKC dependent, membrane receptor-independent) and OZ-stimulated (PKC-independent, membrane receptor-dependent) monocyte respiratory bursts with respect to sensitivity to HHV-6-mediated suppression, when coupled with the very low inputs of infectious virus used in these experiments, suggested that an HHV-6-associated
85
H w
HHv6,
X-X Mock HHVX.= owl w
HHV6,9
X-X Mock HHVdm w
(Mdt-3)
M.&m
Medium
Fig. 2. Zymosan-induced respiratory burst response of PBMC after 15 min of exposure to HHV-6o, (a) or HHV-6x,, (b). ‘Mock virus’ was culture medium derived from uninfected cells grown in parallel with the virus-infected culture. ‘Medium’ refers to a sample run with plain culture medium not exposed to cells. Data are presented as the mean value of triplicate assays. Bars indicate the standard error of the mean.
soluble mediator might be involved. Therefore, the experiments were repeated with medium from infected cell cultures from which infectious virus had been eliminated by ultracentrifugation (Fig. 3). These preparations suppressed PMA stimulated monocyte respiratory burst responses to a degree similar to that seen in the studies above using stocks which contained infectious virus. Virus-free HHV-6,s preparations suppressed PMA-stimulated respiratory burst by 92% and virus-free HHV-6 z29 similarly suppressed respiratory burst by 85%. This finding strongly supported the idea that a virus-associated soluble factor or factors were responsible for the respiratory burst suppression observed.
Discussion Phagocytic leukocytes generate reactive oxygen intermediates (ROI) in response to a wide variety of soluble and particulate stimuli in a complex metabolic process known as respiratory burst. ROIs are important for host defense against microorganisms and some types of cancer cells. In addition, virus-infected cells and virus-containing cell debris are rapidly cleared and inactivated by the mononuclear phagocytes (monocytes, macrophages and possibly dendritic cells) via phagocytosis
t
PMA
6
12
18
24
30
36
42
Time (min)
48
54
60
‘-t! ’ ! ! ! ! ! ! ! ! 6
PMA
12
18
24
30
36
42
48
54
60
Time (min)
Fig. 3. PMA-induced respiratory burst of PBMC after a 1.5mitt exposure to virus-free (ultracentrifuged at 150,000 X g for 2 h at 4°C) medium from HHV-6,, (a) or HHV-6 z29 (b) infected cell cultures. ‘Mock virus’ was culture medium derived from uninfected cells grown in parallel with the virus-infected culture and subjected to an identical ultracentifugation procedure. ‘Medium’ refers to a sample run with plain culture medium not exposed to cells. Data are presented as the mean value of duplicate assays. Bars indicate data ranges.
and destruction by hydrolytic enzymes and ROIs (Morahan et al., 1985). Some viruses have the ability to interfere with the normal functions of phagocytic cells. This has two major consequences for the host. First, evasion of normal antiviral host defenses gives the virus a survival advantage. Second, disabling of cells involved in specific and non-specific immune functions may lead to secondary immune suppression and subsequent opportunistic infections in the host. Data from the present studies show that two strains of HHV-6, HHV-6,, (prototype type A strain) and HHV-6 zz9 (prototype type B strain) (Ablashi et al., 1991), can suppress the respiratory burst response of normal peripheral blood monocytes to PMA stimulation. The suppression was at least 60% in a series of experiments. Results were reproducible with PBMC from three different donors. The suppressive effect was detectable within 15 min of exposure to culture medium from virus-infected cultures. Lack of a respiratory burst response in preparations from which monocytes were removed by size separation using a fluorescence-activated cell sorter confirmed that the observed respiratory burst was produced by the monocytes in the original preparation. Addition of SOD to a positive control reaction vial resulted in elimination of most of the respiratory burst response, indicating that the response was a result of the production of superoxide.
87
The control ‘mock virus’ materials derived from both the HSB-2 and Molt-3 cells significantly influenced the monocyte respiratory burst in these experiments. An enhancement of the burst was seen with respect to the PMA-induced reaction, while a suppression was noted in the OZ induced burst (Figs. 1 and 2). The mechanisms of these effects are unknown. However, both of these cell lines were derived from T-lymphocytic leukemias, and secreted products of T-lymphocytes can influence the respiratory bursts of monocytes (Yuo et al., 1992). In both systems HHV-6 infection acted so as to reduce the ‘mock’ cell effects on the monocyte respiratory burst. In other words, the virus suppressed the burst in the PMA-induced system, and functionally enhanced the burst in the OZ system, relative to the mock-infected control material. However, in several experiments (data not shown) the PMA-induced monocyte respiratory burst was suppressed below that of the unmanipulated control (‘medium’ sample in Figs. 1 and 21, demonstrating the ability of the virus to suppress the native respiratory burst of these cells. Thus, the main conclusion from these studies was that the virus can suppress the monocyte burst, but the complexity of these systems must be kept in mind with respect to the mechanisms involved. Details of the mechanism by which HHV-6 suppresses PMA-induced respiratory burst are not known. Disabling of monocytes by direct infection and cell damage is one possible mechanism. However, the suppression of respiratory burst does not require the presence of infectious virus since the suppression was reproduced using materials from which infectious virus had been removed by ultracentrifugation. Thus the suppression appears to be mediated by a virally encoded soluble factor or a host cell factor induced by the infecting virus. This, in turn, suggests that the suppression is likely to be associated with alterations in one or more of the signal transduction pathways that are involved in the regulation of the respiratory burst. Regulation of the leukocyte respiratory burst is complex. Two major pathways have been identified, although they share several components in common. These pathways are reviewed in detail in Rossi (19861 and Tauber (1987). The phorbol ester-triggered, PKC-dependent pathway can be summarized as follows: (1) phorbol ester binds to and activates PKC, (2) activated PKC phosphorylates and activates the NADPH oxidase, and (3) NADPH oxidase catalyzes the respiratory burst. In contrast, the pathway utilized by OZ consists of the following steps: (1) OZ binds to a membrane receptor (probably an F, receptor) which activates phospholipase C, (2) phospholipase C hydrolyzes phosphatidylinositol biphosphate to form inositol triphosphate (IP,) and diacylglycerol (DAG), (3) DAG can then activate PKC, but this is not necessary for the subsequent respiratory burst, (4) IP, triggers an increase in intracellular Ca+*, (5) increased Ca+* activates phospholipase A2 and binds to and activates calmodulin, (6) calmodulin activates a calmodulin-dependent kinase (CDK), (7) PL-A2 catalyzes the production of arachidonic acid (AA), (8) CDK and AA separately activate NADPH oxidase which produces the respiratory burst. The respiratory burst response to PMA was suppressed after exposure of the cells to HHV-6 in comparison to the mock-infected control materials, while the
88
OZ-induced respiratory burst response remained intact or was even enhanced. The simplicity of the signal transduction pathway involved in the PMA-induced respiratory burst (PMA to PKC to NADPH oxidase) strongly suggests that PKC may be the target of the HHV-6-associated soluble factor. That the NADPH oxidase system remains intact after exposure to the HHV-6-associated factor was demonstrated by the resistance of the OZ-induced response to HHV-6-mediated suppression. Inhibition of PKC by an HHV-6-derived soluble factor would be consistent with the suppression of the responses of bone marrow precursors to granulocyte/macrophage-colony stimulating factor (GM-CSF) (Burd and Carrigan, 1993) since responses to this growth factor are dependent upon PKC (Farrar et al., 1990). Also, exposure of THP-1 promonocytic cells to medium from HHV-6,s infected cell cultures dramatically alters the level of PKC activity present in the cells (Knox and Carrigan, unpublished observation). However, with respect to the results presented here a note of caution must be sounded. The description of the PKC-associated respiratory burst presented above is over-simplified. Recent studies (Carrigan, manuscript in preparation) have demonstrated that the phorbol ester-induced leukocyte respiratory burst is totally dependent upon the continuous activation of PKC but that it also contains components that are sensitive to inhibition by calmodulin antagonists and tyrosine kinase inhibitors. Thus, more detailed biochemical studies will be necessary to completely define the cellular target of the HHV-6-associated soluble factor. These findings have important implications with respect to the pathogenic potential of HHV-6 in both immunologically normal individuals and in patients with immunodeficiencies. Interference with monocytic respiratory burst activity could potentially abrogate host defenses against bacterial, fungal and other viral pathogens. In this way HHV-6 could theoretically synergize with other causes of immunodeficiency, such as the iatrogenic immunosuppression of bone marrow transplantation or the acquired immunodeficiency associated with infection by HIV.
Acknowledgements This work was supported by Grant EDT-19 from the American Cancer Society. We thank Ms. Konstance K. Knox for many helpful discussions and for her insights concerning cellular signal transduction pathways.
References Ablashi, D.V., Balachandran, N., Josephs, S.F., Hung, C.L., Krueger, G.R.F., Kramarsky, B., Salahuddin, S.Z. and Gallo, R.C. (1991) Genomic polymorphism, growth properties, and immunologic variations in human herpesvirus- isolates. Virology 184, 545-552. Apperley, J.F. and Goldman, J.M. (1988) Cytomegalovirus: biology, clinical features and methods for diagnosis. Bone Marrow Transplantation 3, 253-264.
89 Asano, Y., Yoshikawa, T., Suga, S., Yazaki, T., Kondo, K. and Yamanishi, K. (1990) Fatal fulminant hepatitis in an infant with human herpesvirus- infection. Lancet 335, 862-863. Burd, E.M., Knox, K. and Carrigan, D.R. (1993) Human herpesvirus six mediated suppression of growth factor induced macrophage maturation in human bone marrow cultures. Blood, 81, 16451650. Carrigan, D.R., Drobyski, W.R., Russler, S.K., Tapper, M.A., Knox, K.K. and Ash, R.C. (1991) Interstitial pneumonitis associated with human herpesvirus- infection after marrow transplantation. Lancet 338, 147-149. Carrigan, D.R. (1992) Human herpesvirus six and bone marrow transplantation. In: D.V. Ablashi, G.R.F. Krueger and S.Z. Salahuddin (Eds), Biology, Epidemiology, Molecular Biology and Clinical Pathology of HHV-6. Elsevier, Amsterdam, pp. 281-301. Drobyski, W.R., Dunne, W.M., Burd, E.M., Knox, K.K., Ash, R.C., Horowitz, M.M., Flomenberg, N. and Carrigan D.R. (1993) Human herpesvirus(HHV-6) in allogeneic bone marrow transplant recipients: I. Evidence of a marrow suppressive role for HHV-6 in vivo. J. Infect. Dis., 167, 735-739. Farrar, W.L., Ferris, DK and Linnekin, D. (1990) Haemopoietic growth factor regulation of protein kinases and genes associated with cell proliferation. In Molecular Control of Haemopoiesis. CIBA Foundation Symposia 148. John Wiley, New York, pp. 127-144. Huang, L.M., Courtier, A.M., Lowry, J.W., Buckley, N.H., White, K.T. and Hoegerman, SF. (1990) Human herpesvirus- associated with fatal haemophagocytic syndrome. Lancet 336, 60-61. Knox, K.K. and Carrigan, D.R. (1992a) In vitro suppression of bone marrow progenitor cell differentiation by human herpesvirus 6 infection. J. Inf. Dis. 165, 925-929. Knox, K.K. and Carrigan, D.R. (1992b) HHV-6 kills human peripheral blood monocyte derived macrophages by inducing programmed cell death (apoptosis). International herpesvirus workshop, Edinburgh, Scotland, August 1992. Kondo, K., Kondo, T., Okuno, T., Takahashi, M. and Yamanishi, K. (1991) Latent human herpesvirus 6 infection of human monocytes/macrophages. J. Gen. Virol. 72, 1401-1408. Lusso, P., Markham, P.D., Tschachler, E., di Marzo Veronese, F., Salahuddin, S.Z., Ablashi, D.V., Pahwa, S., Krohn, K. and Gallo, R.C. (1988) In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6). J. Exp. Med. 167, 1658-1670. Maridonneau-Parini I., Tringale S.M. and Tauber AI. (1986) Identification of distinct activation pathways of the human neutrophil NADPH oxidase. J. Immunol. 137, 2925-2929. Morahan, P.S., Connor, J.R. and Leary, K.R. (19851 Viruses and the versatile macrophage. Br. Med. Bull. 41, 15-21. Okuno, T., Higashi, K., Shiraki, K., Yamanishi, K., Takahashi, M., Kokado, Y., Ishibashi, M., Takahar, S., Sonoda, T., Tanaka, K., Baba, K., Yabucchi, H. and Kurata, T. (1990) Human herpesvirus 6 infection in renal transplantation. Transplantation 49, 519-522. Pellet, P.E., Black, J.B. and Yamamoto, M. (1992) Human herpesvirus 6: the virus and the search for its role as a human pathogen. Adv. Virus Res. 41, 1-51. Prendergast E. and Proctor R. (1981) Simple procedure for measuring neutrophil chemiluminescence. J. Clin. Micro. 13, 390-392. Pruksananonda, P., Hall, C.B., Insel, R.A., McIntyre, K., Pellett, P.E., Long, C.E., Schnabel, K.C., Pincus, P.H., Stamey, F.R., Dambaugh, T.R. and Stewart J.A. (1992) Primary human herpesvirus 6 infection in young children. N. Engl. J. Med. 326, 1445-1450. Rossi, F. (1986) The 0, forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim. Biophys. Acta 853, 65-89. Rouse, B.T. and Horohov, D.W. (1986) Immunosuppression in viral infections. Rev. Inf. Dis. 8, 850-873. Salahuddin, S.Z., Ablashi, D.V., Markham, P.D., Josephs, S.F., Sturzenegger, S., Kaplan, M., Halligan, G., Biberfeld, P., Wong-Stall, F., Kramarsky, B. and Gallo, R.C. (1986) Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders. Science 234, 596-601. Segondy, M., Astruc, J., Atoui, N., Echenne, B., Robert, C. and Agut, H. (1992) Herpesvirus 6 infection in young children. N. Engl. J. Med. 327, 1099-1100. Takahashi, K., Sonoda, S., Higashi, K., Kondo, T., Takahashi, H., Takahashi, M. and Yamanishi, K. (1989) Predominant CD4 T-lymphocyte tropism of human herpesvirus 6-related virus. J. Virol. 63, 3161-3163.
90
Tauber, A.I. (1987) Protein kinase C and the activation of the human neutrophil NADPH-oxidase. Blood 69, 711-720. Yuo, A., Kitagawa, K., Motoyoshi, K., Azuma, E., Saito, M. and Takaku, F. (1992) Rapid priming of human monocytes by human hematopoietic growth factors: granulocyte-macrophage colony stimulating factor (CSF), macrophage (CSF), and interleukin-3 selectively enhance superoxide release triggered by receptor-mediated agonists. Blood 79, 1553-1557.
Virus
All rights reserved 016%1702/93/$06.00
Research
VIRUS 00894
Human herpesvirus 6 (HHV-6) -associated dysfunction of blood monocytes Eileen M. Burd and Donald R. Carrigan Department of Pathology, The Medical College of Wmconsin, Milwaukee, WI 53226, USA
(Received 12 November 1992; revision received and accepted 3 February 1993)
Summary
HHV-6 is a recently described member of the herpesvirns family. HHVd-associated marrow failure and interstitial pneumonitis where macrophages are the primary infected cell type have been described in marrow transplant patients (Carrigan, 1991; Drobyski et al., 1993). In recent studies we have shown that exposure of normal human marrow to HHV-6,, (a type A strain) or several type B strains resulted in suppression of growth factor induced outgrowth of macrophages by > 90% (Burd and Carrigan, 1993). Additional experiments using HHV-6,, to characterize the effects of the virus on peripheral blood monocytes showed that the respiratory burst capacity of these cells as determined by luminol-enhanced chemiluminescence using phorbol myristate acetate as a trigger was decreased by 83% f 13% in a series of 5 experiments. The decreased respiratory burst was evident as early as 15 min after exposure to virus. Experiments in which cells were separated on a fluorescence activated cell sorter prior to respiratory burst assay showed that the response was mediated solely by peripheral blood monocytes. The respiratory burst response of virus-exposed cells to opsonized zymosan was not affected, indicating that the virus may selectively interfere with the protein kinase C pathway of cellular activation. Ultracentrifugation of stock material to remove infectious virus showed that the suppressive factor was associated with the supernatant fraction. These findings suggest that HHV-6 infection may be associated
Correspondence
to: E.M. Burd, Department kee, WI 53226, USA.
of Pathology, The Medical College of Wisconsin, Milwau-
with a defect in one of the major monocyte activation pathways, and this could be of importance with respect to persistent infection by HHV-6 in immune compromised patients.
Human herpesvirus 6; Respiratory suppression
burst; Monocyte; Signal transduction;
Immune
Introduction HHV-6 was first isolated in 1986 from patients with lymphoproliferative disorders (Salahuddin, 1986). Subsequently it has been determined that HHV-6 infection is common and occurs early in life as evidenced by the high percentage (60-90%) of individuals over the age of two years who are seropositive (Pellet 1992). The most clearly established clinical manifestations of primary HHV-6 infection are exanthem subitum (roseola) and an acute febrile illness without rash in young children (Pellet et al., 1992; Pruksanonda et al., 1992). Serious complications are not rare and include encephalitis, fatal hepatitis, and fatal hemophagocytic syndrome (Pruksanonda et al., 1992; Asano et al., 1990; Huang et al., 1990). HHV-6 infection has recently been implicated as a major cause of febrile convulsions in children (Segondy et al., 19921, and acute HHV-6 infections account for between 10 and 20% of all febrile children seen in pediatric emergency rooms (Pruksanonda et al., 1992). HHV-6 shares with the other members of the herpesvirus family the propensity to establish latent infections that are subject to frequent reactivations. In immune compromised patients such reactivations often progress to persistent, active infections associated with considerable morbidity and mortality. A diverse range of pathogenic mechanisms appear to be operative in these patients. In bone marrow transplant (BMT) patients persistent HHV-6 infection of the lung causes an interstitial pneumonitis most probably mediated by immunopathological mechanisms since the infection is restricted to macrophages and lymphocytes with no apparent involvement of lung parenchymal cells (Carrigan et al., 1991). A second, and apparently more common and dangerous, manifestation of HHV-6 infection in BMT patients is marrow suppression and marrow failure (Drobyski et al., 1993). In vitro evidence suggests that these marrow-suppressive effects of HHV-6 are likely to be mediated by a combination of alpha-IFN induction by the virus within the infected marrow and the release of a cytokine-like activity from infected cells that blocks maturation of marrow stem cells and stromal elements (Knox and Carrigan, 1992a), probably by interfering with the responses of marrow cells to growth factors (Burd and Carrigan, 1993). Persistent HHV-6 infections in renal allografts show a strong correlation with rejection of the organ (Okuno et al., 1990), suggesting that HHV-6 shares with cytomegalovirus the ability to enhance im-
81
munologic responses against a tissue, perhaps by up-regulating major histocompatibility complex antigen expression (Apperly and Goldman, 1988). Another important aspect of the pathogenic potential of HHV-6 may be related to its ability to interfere with the specific and non-specific defensive responses of the infected host. Central to this potential mechanism is the tropism of HHV-6 for CD4 + T-lymphocytes (Takahashi et al., 1989), and its ability to down-regulate the expression of CD3 from the surface of T-lymphocytes (Lusso et al., 1988). Mature macrophages also provide a target for HHV-6 in the lungs of BMT patients with HHV-6 pneumonitis (Carrigan et al., 1991), in macrophage cultures derived from peripheral blood mononuclear cells (PBMC) (Carrigan, 1992; Knox and Carrigan, 1992b) and in normal human bone marrow cultures (Knox and Carrigan, 1992a). Monocyte infection by HHV-6 has been described (Kondo et al., 19911, but little is known about the effects of the infection on the antimicrobial and other functions of the monocyte. Other viruses such as HIV and CMV which infect leukocytes are known to have serious immunosuppressive effects in infected individuals. In vitro, CMV infection of monocytes has been shown to result in severe dysfunction of these cells with suppression of a variety of activities including phagocytosis, oxidative metabolism, production of inflammatory mediators and immunoregulation (Rouse and Horohov, 1986). By analogy to these and other macrophage-tropic viruses, HHV-6 infection is likely to cause similar immunosuppressive effects and may amplify the immunocompromised status in already compromised patients. Exploration of the effects of HHV-6 infection on monocyte functions may provide important information about the potential for HHV-6 to directly cause disease or to synergize with other pathogens to transform a mild illness into a life-threatening infection. Specifically, the work presented here describes the effect of exposure to HHV-6 on the respiratory burst capacity of peripheral blood monocytes as detected in a chemiluminescence assay.
Materials and Methods
Two different strains of HHV-6, HHV-6,, and HHV-6,,,, were used in these studies (provided by Dr. D. Ablashi of the National Cancer Institute, Bethesda, MD). These are prototype A and B types of HHV-6, respectively (Ablashi et al., 1991). HHV-6,, was grown in the HSB-2 human leukemia cell line and HHV-6,,, was propagated in the Molt-3 T-leukemia cell line. Virus stocks were prepared by homogenization and clarification by centrifugation of infected cell cultures showing greater than 50% cytopathic effect. ‘Mock’ homogenates of identically prepared uninfected cells were used as controls in all experiments. To determine the role of infectious virus, the stocks were ultracentrifuged at 150,000 X g for 2 h at 4°C to eliminate the infectious virus particles. Elimination of
82
virus was confirmed in each experiment by back-titration appropriate cell line. Chemiluminescence
of the sup~matant
in the
assay
A luminol-enhanced chemiluminescent assay was modified from the procedure described by Prendergast and Proctor (1981). All samples were assayed in triplicate by repetitive scintillation counting using a Packard Tri-Carb Model ZOOOCAliquid scintillation counter (Packard Instrument Co., Downer’s Grove, IL). Results were expressed as counts per minute (cpm) using the full tritium counting channel. All samples were counted at 5- to lo-min intervals after the addition of inducer for at least one hour. Comparisons among samples were based upon the m~mum cpm value measured for each sample at any time during the counting period. PBMC were separated from whole heparinized blood by Ficoll-Hypaque density centrifugation and were exposed to virus, mock control, or culture medium at a concentration of 0.5 ml of virus or control material per lo6 cells for 15 min. The cells were then pelleted, resuspended in Basal Medium Eagle (BME: Sigma Chemical Co., St, Louis, MO) and aliquoted into dark-adapted, sterile, siliconized, glass scintillation vials at a concentration of lo6 cells in 100 ~1 BME per vial. An 8 X lo-’ M working solution of luminol (Sigma Chemical Co, St. Louis, MO) in distilled water, was prepared from a 5 X lo-’ M stock solution in dimethylsulfoxide (DMSO) and was added to each vial (100 pi/vial). Phorbol myristate acetate (PMA: Sigma Chemical Co., St. Louis, MO), 5 @g/ml in BME diluted from a 5 mg/ml DMSO stock solution was used as a soluble stimulus. Zymosan (Sigma Chemical Co., St. Louis, MO) was opsonized by incubation in autologous serum at 37°C for 30 min, washed, and resuspended in BME at a concentration of 5 mg/ml for use as a particulate stimulus. PMA or opsonized zymosan (OZ) was added (100 &&ial) to the vials in the scintillation counter. To ensure that the chemiluminescence response corresponded to the production of oxygen metabolites, superoxide dismutase (SOD) from human e~hro~es (Sigma Chemical Co., St. Louis, MO), 46.5 units in 100 ~1 BME was added directly to control vials at the beginning of the assay along with one of the stimuli. Addition of exogenous SOD inhibited peak chemiluminescence by an average of 99.6% in assays using PMA as the stimulus and by 81% in assays using OZ as the stimulus, confirming that superoxide generation is responsible for the majority of the luminescence response seen/under these conditions. Proof that it was the monocytes in the mononuclear cell preparation that were responsible for the respiratory burst being assayed was obtained by testing whole mononuclear cell preparations for their ability to produce a respiratory burst in comparison with mononuclear cell preparations from which the monocytes have been removed by cell separation using a Coulter EPICS 5 flow cytometer. The separated cells were determined to be free of monocytes by a two-cofor flow cytometric assay using monoclonal antibodies specific for CD45 and CD14. The monocyte-free samples showed responses similar to those of the luminol background control while unseparated PBMC preparations in the same assay gave a
83 TABLE 1 Donor
Experiment no.
% suppression of PMA-induced respiratory burst by:
H~-6zz,
HHV-6,,
99 88
84 not done
87 90
60 not done
86
68
typical respiratory burst response. These results demonstrate that monocytes were responsible for producing the respiratory burst in this system. Controls of the respiratory burst assay system using BME, PBMC, OZ, or PMA by themselves generated no detectable chemiluminescence.
Results
Effect of HHV-6 on phorbol ester-stimulated monocyte respiratory burst HHV-6 had a markedly suppressive effect on PMA-stimulated respiratory burst after as little as 15 min of exposure to the virus material. In each of 5 experiments using PBMC from three different donors, HHV-6,, suppressed chemiluminescence by at least 86% and as much as 99% compared to mock-infected controls as measured by peak CPM values in the assay (Table 1). Exposure of PBMC to HHV-6,, also reduced the chemiluminescence response. The suppression with HHV-6,, ranged from 60-84% in 3 experiments (Table 1). A representative pattern of PMA-stimulated chemiluminescence observed with HHV-6,, and HHV-6 z29 in one experiment is presented in Fig. 1. Of special note is the persistence of the burst suppression throughout the assay by HHV-6,,, whereas the inhibition by HHV-6,,, was more transient. This increased suppression by the type A HHV-6 was consistently observed and corresponded to a similar greater suppression of bone marrow stem cell responses to growth factors by the A type of virus (Burd and Carrigan, 1993). Effect of HHV-6 on opsonized Zymosan-stimulated respiratory burst The marked suppression of phorbol ester (PMA)-stimulated monocyte respiratory burst made it of interest to determine whether the suppression extended to OZ stimulated respiratory bursts. In contrast to PMA stimulation, which is mediated through direct binding of the phorbol ester to protein kinase C (PKC) (Tauber, 1987), OZ stimulates phagocyte respiratory bursts through a membrane receptor system coupled to a PKC-independent, phospholipase A2 (PL-A2) and
e-e HliMm X-X MockHHV4&bfdt-3) CI
Me&m
60
’ t
Resting PMA
4 li b i3 411I9 :, Time (min)
Fig. 1. Chemiluminescence response of PBMC after in vitro exposure for 15 min to materials derived from HHV-6-infected cell cultures. Panels A and B show results with HHV-6,s and HHV-6,, respectively. The cells were assayed for chemiluminescence response following PMA stimulation. ‘Mock virus’ was culture medium derived from uninfected cells grown in parallel with the virus-infected culture. ‘Medium’ refers to a sample run with plain culture medium not exposed to cells. Data are presented as the mean value of triplicate assays. Bars indicate the standard error of the mean.
calmodulin-associated signal transduction pathway (Maridonneau-Parini et al., 1986; Rossi, 1986). The results obtained indicated that respiratory bursts stimulated by OZ were not suppressed in cells exposed to either HHV-6,, (Fig. 2a) or (Fig. 2b). Microscopic examination of the mixture of virus-treated HEW-6,, PBMCs and 02, at the end of the assay did not demonstrate phagocytosis of the OZ particles. However, many 02 particles were seen associated with the membranes of the mononuclear cells, indicating that surface contact alone was sufficient to initiate a respiratory burst. Therefore, a clear distinction was seen between monocyte respiratory burst activity mediated directly through PKC and that mediated through a membrane receptor linked signal transduction pathway.
Effect burst
of ultracentrifuged
(virus-free) stock preparations
on monocyte respiratory
The apparent distinction between phorbol ester-stimulated (PKC dependent, membrane receptor-independent) and OZ-stimulated (PKC-independent, membrane receptor-dependent) monocyte respiratory bursts with respect to sensitivity to HHV-6-mediated suppression, when coupled with the very low inputs of infectious virus used in these experiments, suggested that an HHV-6-associated
85
H w
HHv6,
X-X Mock HHVX.= owl w
HHV6,9
X-X Mock HHVdm w
(Mdt-3)
M.&m
Medium
Fig. 2. Zymosan-induced respiratory burst response of PBMC after 15 min of exposure to HHV-6o, (a) or HHV-6x,, (b). ‘Mock virus’ was culture medium derived from uninfected cells grown in parallel with the virus-infected culture. ‘Medium’ refers to a sample run with plain culture medium not exposed to cells. Data are presented as the mean value of triplicate assays. Bars indicate the standard error of the mean.
soluble mediator might be involved. Therefore, the experiments were repeated with medium from infected cell cultures from which infectious virus had been eliminated by ultracentrifugation (Fig. 3). These preparations suppressed PMA stimulated monocyte respiratory burst responses to a degree similar to that seen in the studies above using stocks which contained infectious virus. Virus-free HHV-6,s preparations suppressed PMA-stimulated respiratory burst by 92% and virus-free HHV-6 z29 similarly suppressed respiratory burst by 85%. This finding strongly supported the idea that a virus-associated soluble factor or factors were responsible for the respiratory burst suppression observed.
Discussion Phagocytic leukocytes generate reactive oxygen intermediates (ROI) in response to a wide variety of soluble and particulate stimuli in a complex metabolic process known as respiratory burst. ROIs are important for host defense against microorganisms and some types of cancer cells. In addition, virus-infected cells and virus-containing cell debris are rapidly cleared and inactivated by the mononuclear phagocytes (monocytes, macrophages and possibly dendritic cells) via phagocytosis
t
PMA
6
12
18
24
30
36
42
Time (min)
48
54
60
‘-t! ’ ! ! ! ! ! ! ! ! 6
PMA
12
18
24
30
36
42
48
54
60
Time (min)
Fig. 3. PMA-induced respiratory burst of PBMC after a 1.5mitt exposure to virus-free (ultracentrifuged at 150,000 X g for 2 h at 4°C) medium from HHV-6,, (a) or HHV-6 z29 (b) infected cell cultures. ‘Mock virus’ was culture medium derived from uninfected cells grown in parallel with the virus-infected culture and subjected to an identical ultracentifugation procedure. ‘Medium’ refers to a sample run with plain culture medium not exposed to cells. Data are presented as the mean value of duplicate assays. Bars indicate data ranges.
and destruction by hydrolytic enzymes and ROIs (Morahan et al., 1985). Some viruses have the ability to interfere with the normal functions of phagocytic cells. This has two major consequences for the host. First, evasion of normal antiviral host defenses gives the virus a survival advantage. Second, disabling of cells involved in specific and non-specific immune functions may lead to secondary immune suppression and subsequent opportunistic infections in the host. Data from the present studies show that two strains of HHV-6, HHV-6,, (prototype type A strain) and HHV-6 zz9 (prototype type B strain) (Ablashi et al., 1991), can suppress the respiratory burst response of normal peripheral blood monocytes to PMA stimulation. The suppression was at least 60% in a series of experiments. Results were reproducible with PBMC from three different donors. The suppressive effect was detectable within 15 min of exposure to culture medium from virus-infected cultures. Lack of a respiratory burst response in preparations from which monocytes were removed by size separation using a fluorescence-activated cell sorter confirmed that the observed respiratory burst was produced by the monocytes in the original preparation. Addition of SOD to a positive control reaction vial resulted in elimination of most of the respiratory burst response, indicating that the response was a result of the production of superoxide.
87
The control ‘mock virus’ materials derived from both the HSB-2 and Molt-3 cells significantly influenced the monocyte respiratory burst in these experiments. An enhancement of the burst was seen with respect to the PMA-induced reaction, while a suppression was noted in the OZ induced burst (Figs. 1 and 2). The mechanisms of these effects are unknown. However, both of these cell lines were derived from T-lymphocytic leukemias, and secreted products of T-lymphocytes can influence the respiratory bursts of monocytes (Yuo et al., 1992). In both systems HHV-6 infection acted so as to reduce the ‘mock’ cell effects on the monocyte respiratory burst. In other words, the virus suppressed the burst in the PMA-induced system, and functionally enhanced the burst in the OZ system, relative to the mock-infected control material. However, in several experiments (data not shown) the PMA-induced monocyte respiratory burst was suppressed below that of the unmanipulated control (‘medium’ sample in Figs. 1 and 21, demonstrating the ability of the virus to suppress the native respiratory burst of these cells. Thus, the main conclusion from these studies was that the virus can suppress the monocyte burst, but the complexity of these systems must be kept in mind with respect to the mechanisms involved. Details of the mechanism by which HHV-6 suppresses PMA-induced respiratory burst are not known. Disabling of monocytes by direct infection and cell damage is one possible mechanism. However, the suppression of respiratory burst does not require the presence of infectious virus since the suppression was reproduced using materials from which infectious virus had been removed by ultracentrifugation. Thus the suppression appears to be mediated by a virally encoded soluble factor or a host cell factor induced by the infecting virus. This, in turn, suggests that the suppression is likely to be associated with alterations in one or more of the signal transduction pathways that are involved in the regulation of the respiratory burst. Regulation of the leukocyte respiratory burst is complex. Two major pathways have been identified, although they share several components in common. These pathways are reviewed in detail in Rossi (19861 and Tauber (1987). The phorbol ester-triggered, PKC-dependent pathway can be summarized as follows: (1) phorbol ester binds to and activates PKC, (2) activated PKC phosphorylates and activates the NADPH oxidase, and (3) NADPH oxidase catalyzes the respiratory burst. In contrast, the pathway utilized by OZ consists of the following steps: (1) OZ binds to a membrane receptor (probably an F, receptor) which activates phospholipase C, (2) phospholipase C hydrolyzes phosphatidylinositol biphosphate to form inositol triphosphate (IP,) and diacylglycerol (DAG), (3) DAG can then activate PKC, but this is not necessary for the subsequent respiratory burst, (4) IP, triggers an increase in intracellular Ca+*, (5) increased Ca+* activates phospholipase A2 and binds to and activates calmodulin, (6) calmodulin activates a calmodulin-dependent kinase (CDK), (7) PL-A2 catalyzes the production of arachidonic acid (AA), (8) CDK and AA separately activate NADPH oxidase which produces the respiratory burst. The respiratory burst response to PMA was suppressed after exposure of the cells to HHV-6 in comparison to the mock-infected control materials, while the
88
OZ-induced respiratory burst response remained intact or was even enhanced. The simplicity of the signal transduction pathway involved in the PMA-induced respiratory burst (PMA to PKC to NADPH oxidase) strongly suggests that PKC may be the target of the HHV-6-associated soluble factor. That the NADPH oxidase system remains intact after exposure to the HHV-6-associated factor was demonstrated by the resistance of the OZ-induced response to HHV-6-mediated suppression. Inhibition of PKC by an HHV-6-derived soluble factor would be consistent with the suppression of the responses of bone marrow precursors to granulocyte/macrophage-colony stimulating factor (GM-CSF) (Burd and Carrigan, 1993) since responses to this growth factor are dependent upon PKC (Farrar et al., 1990). Also, exposure of THP-1 promonocytic cells to medium from HHV-6,s infected cell cultures dramatically alters the level of PKC activity present in the cells (Knox and Carrigan, unpublished observation). However, with respect to the results presented here a note of caution must be sounded. The description of the PKC-associated respiratory burst presented above is over-simplified. Recent studies (Carrigan, manuscript in preparation) have demonstrated that the phorbol ester-induced leukocyte respiratory burst is totally dependent upon the continuous activation of PKC but that it also contains components that are sensitive to inhibition by calmodulin antagonists and tyrosine kinase inhibitors. Thus, more detailed biochemical studies will be necessary to completely define the cellular target of the HHV-6-associated soluble factor. These findings have important implications with respect to the pathogenic potential of HHV-6 in both immunologically normal individuals and in patients with immunodeficiencies. Interference with monocytic respiratory burst activity could potentially abrogate host defenses against bacterial, fungal and other viral pathogens. In this way HHV-6 could theoretically synergize with other causes of immunodeficiency, such as the iatrogenic immunosuppression of bone marrow transplantation or the acquired immunodeficiency associated with infection by HIV.
Acknowledgements This work was supported by Grant EDT-19 from the American Cancer Society. We thank Ms. Konstance K. Knox for many helpful discussions and for her insights concerning cellular signal transduction pathways.
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Tauber, A.I. (1987) Protein kinase C and the activation of the human neutrophil NADPH-oxidase. Blood 69, 711-720. Yuo, A., Kitagawa, K., Motoyoshi, K., Azuma, E., Saito, M. and Takaku, F. (1992) Rapid priming of human monocytes by human hematopoietic growth factors: granulocyte-macrophage colony stimulating factor (CSF), macrophage (CSF), and interleukin-3 selectively enhance superoxide release triggered by receptor-mediated agonists. Blood 79, 1553-1557.