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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Hawiger, J. Articles by Ballard, D. W. Search for Related Content PubMed PubMed Citation Articles by Hawiger, J. Articles by Ballard, D. W. Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 94 No. 5 (September 1), 1999: pp. 1711-1716 IB Kinase Complex Is an Intracellular Target for Endotoxic Lipopolysaccharide in Human Monocytic Cells By Jacek Hawiger, Ruth Ann Veach, Xue-Yan Liu, Sheila Timmons, and Dean W. Ballard From the Department of Microbiology and Immunology and Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, TN.     ABSTRACT TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Endotoxic lipopolysaccharide (LPS) is a proinflammatory agonist produced by gram-negative bacteria and a contributor to the majority of the 400,000 septic shock cases recorded annually in US hospitals. The primary target cells for LPS are monocytes and macrophages. Their response consists of massive production of proinflammatory cytokines, reactive oxygen- and nitrogen-intermediates, procoagulants, and cell adhesion molecules. In turn, expression of these LPS-responsive factors contributes to collapse of the circulatory system, to disseminated intravascular coagulation, and to a 30% mortality rate. A common intracellular mechanism responsible for the expression of septic shock genes in monocytes and macrophages involves the activation of NF-B. This transcription factor is regulated by a family of structurally related inhibitors including IB, IB, and IB, which trap NF-B in the cytoplasm. In this report, the investigators show that LPS derived from different gram-negative bacteria activates cytokine-responsive IB kinases containing catalytic subunits termed IKK (IKK1) and IKK (IKK2). The kinetics of IKK and IKK activation in LPS-stimulated human monocytic cells differ from that recorded on their stimulation with tumor necrosis factor-, thereby implying a distinct activation mechanism. LPS-activated IKK complexes phosphorylate all 3 inhibitors of NF-B: IB, IB, and IB. Moreover, LPS activates IKK preferentially, relative to IKK. Thus, IKK complex constitutes the main intracellular target for LPS-induced NF-B signaling to the nucleus in human monocytic cells to activate genes responsible for septic shock. © 1999 by The American Society of Hematology.     INTRODUCTION TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES PROINFLAMMATORY STIMULI signal to the nucleus via transcription factor NF-B, which activates a set of genes responsive to inflammatory, immune, and oxidant stress. These stimuli encompass biologic agents such as endotoxic lipopolysaccharide (LPS) and bacterial superantigens (eg, toxic shock syndrome toxin and streptococcal pyrogenic exotoxin), which are responsible for the majority of septic shock cases. In addition, intracellular bacteria (eg, Mycobacterium tuberculosis), viruses (eg, human immunodeficiency virus, human T-cell lymphotropic virus, and cytomegalovirus), cytokines (tumor necrosis factor- [TNF-], interlukin-1 [IL-1]), and lipid peroxides induce nuclear import of NF-B.1-3 In response to these agents, the cytoplasmic ankyrin motif-rich inhibitors of NF-B, IB, IB, and IB are phosphorylated, ubiquitinated, and then degraded by adenosine triphosphate (ATP)-dependent 26S proteosomes.4-9 Thus, phosphorylation-dependent proteolysis of IB proteins releases NF-B for subsequent import to the nucleus. In turn, NF-B stimulates transcription of a number of genes containing cognate B sites in their enhancer/promoter regions. These NF-B-responsive genes encode cytokines (TNF-, IL-1, IL-6, IL-8, and IL-12), procoagulant molecules (tissue factor and plasminogen activator inhibitor 1), signal transducers (inducible nitric oxide synthase and cyclooxygenase 2), cell adhesion molecules (endothelial selectin, intracellular cell adhesion molecule-1, and vascular cell adhesion molecule-1), and growth factors (granulocyte-colony-stimulating factor).3,6,10 The products of these NF-B-regulated genes expressed in monocytes, macrophages, and vascular endothelial cells contribute to the development of septic shock and disseminated intravascular coagulation, leading to multiple organ failure and death.1,3 These findings highlight NF-B as a major intracellular mediator of the systemic inflammatory response syndrome known as sepsis and septic shock.11 The fact that multiple proinflammatory stimuli, LPS, TNF-, and IL-1, involved in induction and mediation of septic shock syndrome, evoke activation of NF-B indicates that the signals generated by these stimuli and their cognate receptors converge at the common step of NF-B activation.6 This step appears to be specific phosphorylation of inhibitory proteins, such as IB, by their kinase(s). A primary target for IB kinases is IB. This inhibitor is characterized by a signal response domain in the amino-terminal segment, ankyrin repeats in the midsection, and a PEST domain in the carboxyl terminal segment.12 The signal response domain contains two serines at positions 32 and 36, which are targeted for specific phosphorylation by IB kinases.5,13 The PEST domain bears serines phosphorylated by casein kinase II involved in constitutive turnover of IB.14 The IB kinase that catalyzes inducible phosphorylation of serines 32 and 36 in response to cytokines was initially reported by Chen et al15 as part of a 700-kD complex requiring ubiquitin for full activity. Subsequently, two serine/threonine kinases termed IB kinase  (IKK or IKK1) and IB kinase  (IKK or IKK2) of molecular weight 85 kD and 87 kD, respectively, were identified.16-19 Both of these kinases are homologous to a previously reported conserved helix-loop-helix ubiquitous kinase with a unique structure containing a kinase domain linked to a leucine zipper domain and a helix-loop-helix domain. Both IKK and IKK phosphorylate serines 32 and 36 in IB, suggesting functional homology as well.19 Although it is well known that primary target cells for LPS are monocytes and macrophages,3,20 the mechanism by which LPS induces NF-B in this cellular setting remained unknown. Therefore, we hypothesized that the recently discovered IB kinases constitute an intracellular target for LPS. Our results provide evidence that IB kinase in human monocytic THP-1 cells is targeted by LPS, thereby assigning this multiunit kinase complex a primary role as the signal transducer responsible for NF-B mobilization in response to septic-shock inducers.     MATERIALS AND METHODS TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Reagents.   Rabbit polyclonal antibodies were generated by immunizing rabbits with synthetic peptides corresponding to amino acids 1-28 (NH2-terminal peptide) and 731-744 (COOH-terminal peptide) of human IKK and a synthetic peptide corresponding to amino acids 743-756 of human IKK. The peptides were covalently coupled to keyhole limpet hemocyanin before injection together with complete Freund adjuvant. The antibody lgG fraction of each antiserum was purified by absorption to and elution from Protein A Sepharose (Zymed, San Francisco, CA). IB-specific (amino acids 289-317) antipeptide rabbit antibodies were prepared as described previously.9 Glutathione S-transferase (GST)-IB (1-54), IB (1-44), and IB (1-61) fusion proteins (wild type and IB mutant S32A and S36A) were prepared as described.17,19,21 LPS preparations were derived from: (1) Escherichia coli 0127:B8 (extracted by Boivin method; DIFCO, Detroit, MI), (2) E coli 0127:B8 (prepared by phenol extraction and gel filtration chromatography), (3) Pseudomonas aeruginosa serotype 10 (chromatographically purified by gel filtration), and (4) Salmonella minnesota (prepared by phenol extraction and gel filtration chromatography). The latter three LPS preparations were obtained from Sigma Chemical Company (St. Louis, MO). Cell culture.   Human monocytic THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum containing no detectable LPS (<0.006 ng/mL in Limulus Amebocyte Gel Clot as determined by the manufacturer, Atlanta Biologicals, Norcross, GA), 2 mmol/L L-glutamine, and antibiotics as described.4 Where indicated, 5 mL of THP-1 cells (106/mL) were stimulated with E coli LPS O127:B8 (DIFCO) or TNF- with the potency of 32,000 U per µg (Mallinckrodt, St Louis, MO) at concentrations and times specified in the text. Immunoprecipitation and immunoblotting.   Cytoplasmic extracts were prepared from THP-1 cells by detergent lysis (0.1 % Nonidet P40 in 10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 300 mmol/L sucrose) in the presence of phosphatase inhibitors (12.5 mmol/L -glycerophosphate, 2 mmol/L NaF, 100 µmol/L Na3VO4) and protease inhibitors (0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mg/mL pefabloc SC, 50 µg/mL antipain, 1 mg/mL aprotinin, 20 µg/mL chymostatin, 5 µg/mL E64, 1 µg/mL leupeptin, 1 µg/mL pepstatin, and 20 µg/mL phosphoramidon). Lysates were adjusted to a final concentration of 50 mmol/L HEPES (pH 7), 250 mmol/L NaCl, and 5 mmol/L EDTA before addition of specific IKK or IKK antibody IgG (20 µg added to reaction mixture). Specific IB antipeptide antibody was used as described previously.9 Immunoprecipitations were typically performed in 400 µL reaction mixtures containing 100 to 500 µg of total cytoplasmic protein and 10 µL of protein A-Sepharose beads (Zymed). In some immunoprecipitation experiments, protein A-Sepharose beads were precoated with specific antibodies and washed once with ELB buffer (50 mmol/L HEPES, 250 mmol/L NaCl, 5 mmol/L EDTA, and 0.1% Nonidet P-40).5 Immunoprecipitates were washed three times with ELB buffer in the presence of phosphatase inhibitors, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidine difluoride membranes. Immunoreactive polypeptides were detected with anti-IKK antibodies and an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). IKK assay.   IB kinase activity in immunoprecipitates obtained with anti-IKK and anti-IKK antibodies in cytoplasmic lysate of THP-1 cells was measured as described previously.17, 22 Briefly, cytoplasmic extracts from 5 × 106 THP-1 cells were prepared and immunoprecipitated as described above. They were immunoprecipitated with anti-IKK antibody IgG (10 µg added to 100 to 500 µg of total protein), unless specified otherwise. Immunoprecipitates were washed thrice with ELB buffer and once with kinase buffer (10 mmol/L HEPES pH 7.4, 1 mmol/L MnCl2, 5 mmol/L MgCl2, 12.5 mmol/L -glycerophosphate, 50 µmol/L Na3VO4, 2 mmol/L NaF, 0.5 mmol/L dithiothrietol, and 10 µmol/L ATP). Immunoprecipitates were resuspended in kinase buffer (20 µL) containing 1 µg IB (residues 1-54), GST fusion protein, and 5µCi of 32P--ATP (ICN cat no. 38101x, 4,500 Ci/mmol). The kinase reaction was done at 30°C for 30 minutes and was stopped by addition of Laemmli buffer containing 2.5% -mercaptoethanol. After a 5-minute incubation at 100°C, the mixture was spun down and the supernatant applied to SDS-PAGE (10%). Dried gels were applied to Biomax film (Eastman Kodak, Rochester, NY). In some experiments, dried gels were analyzed in Fuji FLA 2000 Fluorescent Image Analyzer (Fuji Medical Systems, Stamford, CT). Electrophoretic mobility shift assay (EMSA).   To measure the nuclear import of NF-B in THP-1 cells, EMSA was performed as described with a radiolabeled B probe.4,9     RESULTS TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Expression of endogenous IKK and IKK in human monocytic THP-1 cells.   At least 2 kinases specific for IB and IB were recently identified in Hela and 293 EBNA cells and named IKK and IKK.16-19 To monitor their expression in human monocytic cells, we prepared rabbit polyclonal antibodies directed against peptides derived from the NH2-terminal segment (residues 1-28) and the COOH-terminal segment (residues 731-744) of IKK and the COOH-terminal segment of IKK (residues 743-756). The antibodies were monospecific for homologous peptides in an enzyme-linked immunosorbent assay using peptide-coated wells (results not shown). When cytoplasmic extracts from monocytic THP-1 cells were immunoprecipitated and then probed with these antibodies by immunoblotting, 2 bands of 85 kD and 87 kD, apparent molecular weights, were discerned with anti-IKK antibody (Fig 1A, lane 1). In contrast, anti-IKK antibody precipitated and reacted in immunoblotting with the 87-kD band representing IKK (Fig 1B, lane 2). Thus, the anti-IKK antibody was monospecific for IKK and did not cross-react with IKK (Fig 1B, lane 1). These bands were not visualized when antibodies were preincubated with homologous peptides before the immunoblotting (results not shown). View larger version (75K): [in this window] [in a new window]   Fig 1. Detection of IKK and IKK in human monocytic THP-1 cells by immunoprecipitation and immunoblotting. Cytoplasmic lysates from unstimulated cells were immunoprecipitated with anti-IKK NH2-terminal peptide antibody (lane 1) and anti-IKK COOH-terminal peptide antibody (lane 2). Immunoprecipitated proteins were resolved by (A) SDS-PAGE and immunoblotted with anti-IKK NH2-terminal peptide antibody, with (B) anti-IKK COOH-terminal peptide antibody (NS represents nonspecific band). Stimulus-dependent activation of IKK and IKK in human monocytic THP-1 cells.   Having established the presence of IKK and IKK in the cytoplasmic fraction of human monocytic THP-1 cells, we examined the kinase activity in immunoprecipitates obtained from LPS-stimulated cells as compared with those from TNF--stimulated cells (Fig 2A). Stimulated and nonstimulated THP-1 cells were lysed, and the cytoplasmic fraction was prepared by differential centrifugation. Immunoprecipitated IKK and IKK were assayed for kinase activity with 32P--ATP and a fusion protein made of IB residues 1-54 attached to GST.17,22 Nonstimulated THP-1 cells had no detectable kinase activity in immunoprecipitates obtained with anti-IKK antibodies (Fig 2A, lane 1). Low-level basal kinase activity was detectable in anti-IKK antibody immunoprecipitates (Fig 2A, lane 4). When cells were stimulated with LPS from E coli 0127:B8 (Difco) (1 µg/mL for 30 minutes), kinase activity was readily detectable by phosphorylation of GST-IB (residues 1-54) fusion protein in immunoprecipitates produced by 2 antibodies (Fig 2A, lanes 3 and 6). Phosphoimager analysis of these bands indicated that the LPS-responsive kinase activity immunoprecipitated by monospecific anti-IKK antibody exceeds the activity precipitated by anti-IKK antibody. Similar, albeit weaker, kinase activity of immunoprecipitated IKK and IKK was evoked by TNF- (100 U/mL for 5 minutes) (Fig 2A, lanes 2 and 5). Kinase activity of immunoprecipitated IKK and IKK was specific for serines 32 and 36 of IB because a fusion protein containing mutant IB segment with serines 32 and 36 replaced by alanine was not phosphorylated (Fig 2B). Activation of IB kinase complex containing IKK and IKK by LPS in THP-1 cells was concentration dependent. At least 10 ng/mL of LPS from E coli 0127:B8 (DIFCO, Detroit, MI) was sufficient to induce detectable IKK and IKK activity (Fig 3). By comparison, the lowest concentration of TNF- inducing similar activation was 30 U/mL or approximately 1 ng/mL. All preparations of LPS tested induced activation of IKK and IKK. These included LPS derived from E coli 0127:B8, P aeruginosa, and S minnesota (Fig 4). The activation of IKK and IKK by structurally diverse LPS from different gram-negative bacterial species indicates that IKK complex responds to all endotoxic LPS tested. However, the method of extraction of LPS derived from the same E coli 0127:B8 serotype results in different IKK complex activating potency of LPS (Fig 4, lanes 2 and 3). View larger version (30K): [in this window] [in a new window]   Fig 2. Stimulus-dependent activation of IKK and IKK in human monocytic THP-1 cells. (A) Nonstimulated cells (lanes 1, 4), TNF--stimulated (100 U/mL)(5 minutes) cells (lanes 2, 5), and LPS E coli 0127:B8 (1 µg/mL)-stimulated (30 minutes) cells (lanes 3, 6). Lanes 1-3 represent immunoprecipitates obtained with anti-IKK antibody; and lanes 4-6 represent immunoprecipitates obtained with anti IKK antibody. The IKK kinase assay was done as described in Materials and Methods by using IB-GST fusion protein as the substrate. (B) IKK and IKK activity toward wild type IB substrate and IB mutant (S32A and S36A) substrate prepared as GST fusion proteins. The cells were stimulated with TNF- (100 U/mL) for 5 minutes and with LPS (1 µg/mL) for 30 minutes. View larger version (28K): [in this window] [in a new window]   Fig 3. Concentration-dependent activation of IKK and IKK by LPS and TNF-. Human monocytic THP-1 cells were stimulated with increasing concentrations of LPS for 30 minutes or TNF- for 5 minutes and cytoplasmic lysates were prepared with anti-IKK antibody. The IKK kinase assay was done as described in Materials and Methods; the substrate was a fusion protein made of wild-type IB (amino acids 1-54) and GST. View larger version (28K): [in this window] [in a new window]   Fig 4. Activation of IKK and IKK in human monocytic THP-1 cells by LPS derived from different gram-negative bacteria. Lane 1, Unstimulated cells; Lane 2, LPS from E coli 0127:B8 extracted by Boivin method (DIFCO); Lane 3, LPS from E coli 0127:B8 prepared by phenol extraction and gel filtration chromatography (Sigma); Lane 4, LPS from P aeruginosa serotype 10. LPS from S minnesota. Human monocytic THP-1 cells were stimulated with different preparations of LPS at 1 µg/mL for 30 minutes and the IKK kinase assay was done as specified in Materials and Methods.The kinase complex was immunoprecipitated with anti-IKK antibody. IKK and IKK are differentially activated by LPS and TNF-.   Stimulation of human monocytic THP-1 cells by LPS or TNF- was followed by determination of enzymatic activity of IKK and IKK in cytoplasmic extracts of stimulated cells prepared at different time intervals. Three NF-B inhibitors, IB, IB, and IB, presented as GST fusion proteins were tested as phosphorylation substrates in standard in vitro kinase assays with immune complex containing IKK. LPS stimulation of THP-1 cells for 10 minutes was sufficient to induce measurable kinase activity, which peaked at 30 minutes (Fig 5). The significant increase in IB kinase activity at 10 minutes is consistent with phosphorylation and degradation of IB in LPS-treated THP-1 cells after 10 minutes stimulation as documented by immunoblotting with specific IB antipeptide antibody. Concurrently, the nuclear import of NF-B monitored by the electrophoretic mobility shift assay shows progressive increase reaching maximum at 30 minutes (Fig 6A and B). Significantly, all three inhibitory proteins, IB, IB, and IB, used as GST fusion proteins at equivalent concentrations were phosphorylated with similar kinetics in response to LPS. In contrast, TNF- stimulation of THP-1 cells resulted in much faster kinetics of phosphorylation of IB-GST fusion protein reaching peak at 3 to 5 minutes and declining after 20 minutes (Fig 5). This pattern of rapid activation by TNF- is consistent with much faster degradation of endogenous IB and nuclear import of NF-B reaching maximum at 10 minutes in TNF--stimulated THP1 cells (Fig 6A and B). The 5 times lower concentration of TNF- (20 U/mL) did not change the rapid course of IKK/IKK activation (results not shown). Thus, the differences in activation kinetics of IB kinase complex induced by LPS and TNF- suggest that these proinflammatory agonists evoke different signaling pathways resulting in activation of IKK-containing and IKK-containing complex in human monocytic cells. View larger version (51K): [in this window] [in a new window]   Fig 5. Time course of LPS-induced activation of IKK and IKK as compared with that induced by TNF-. (A) LPS-induced phosphorylation of 1µg IB-GST, IB-GST, and IB-GST fusion proteins; (B) TNF--induced phosphorylation of IB-GST fusion protein. The IKK kinase assay was done as specified in Materials and Methods and the kinase complex was immunoprecipitated with anti-IKK antibody. View larger version (41K): [in this window] [in a new window]   Fig 6. Time course of LPS-induced degradation of IB and nuclear import of NF-B. (A) Immunoblotting of IB in nonstimulated (time 0) and TNF--stimulated or LPS-stimulated THP-1 cells. (B) Electrophoretic mobility shift assay of NF-B in nuclear fractions isolated from nonstimulated (time 0) and TNF--stimulated or LPS-stimulated THP-1 cells. LPS from E coli 0127:B8 was used in concentration of 1 µg/mL and TNF- at 100 U/mL.     DISCUSSION TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES The results presented here have several important implications for the cellular and molecular pathogenesis of septic shock induced by LPS. First, our studies provide evidence that IB kinase complex containing IKK and IKK is an intracellular target for LPS produced by different gram-negative bacteria (enterobacteriaceae and nonenterobacteriaceae). Second, IKK appears to be a more LPS-responsive signal transducer than IKK based on observed kinase activity (Fig 2). This is consistent with recent demonstration that IKK is not required for signaling induced by proinflammatory cytokines, IL-1, or TNF-.23-25 The lowest concentration of LPS that induces detectable activation of IKK and IKK in human monocytic THP-1 cells was 10 ng/mL. Treatment of human peripheral blood mononuclear cells ("adherent monocytes") with 100 ng/mL of E coli 0111:B4 lipopolysaccharide also induces IKK activity, thus validating THP-1 cells as a model for studying LPS-induced IKK activation in human cells of the myelomonocytic lineage.26 Moreover, LPS-stimulated and TNF--stimulated IKK complex can phosphorylate not only inhibitory proteins IB and IB, but also IB. The IB kinases provide a pivotal checkpoint in NF-B-mediated signaling to the nucleus. Thus, activation of IB kinases by LPS in human monocytic cells constitutes an important link in understanding the NF-B recruitment in molecular pathogenesis of septic shock. Whether continuing activation of IB kinases in vivo by LPS derived from different gram-negative bacteria results in persistent nuclear import of NF-B demonstrated in nonsurvivors of septic shock11 remains to be established with in vivo models. Importantly, kinetics of phosphorylation of all three inhibitory proteins, IB , IB, and IB, as demonstrated herein by IKK complex activated by LPS can contribute to progressive activation of the nuclear import of NF-B. Third, the distinct activation kinetics of IKK-containing and IKK-containing complex by LPS and TNF- in human monocytic cells is consistent with different rate of the nuclear import of NF-B in these cells (Fig 6).4,9,27 These agonists signal through distinct receptors (toll-like receptor 2 and 4 v TNF- receptor 1 and 2).28-31 Receptor-proximal steps in intracellular signaling induced by TNF- that interacts with its cognate receptor, TNFR1, and by LPS interacting with its cognate receptor TLR2 differ.29 Whereas TNF--induced signaling requires TRAF2 in mammalian cell transfectants, LPS-induced signaling via TLR2 seems to require TRAF6.29,32 The latter is required for IL-1 induced signaling, which also uses an adaptor protein MyD88.33-35 Dominant-negative mutants of MyD88 and TRAF6 expressed in TLR2- and IL-1R-transfected cells selectively blocked signaling evoked by LPS and IL-1 but remained ineffective toward TNF--induced signaling.29,32 These differences in receptor-proximal signaling may explain the different kinetics of IKK complex activation by LPS and TNF- in human monocytic THP-1 cells used in our experiments. Further dissection of LPS-stimulated and TNF--stimulated specific pathways of IKK complex activation will help in the development of new inhibitors designed to interrupt potentially lethal lipopolysaccharide-induced intracellular mechanism of septic shock.     ACKNOWLEDGMENT The authors thank Traci Tidwell, Susan Rowlinson, and Erica Holleran for assistance in preparation of the manuscript, Zhi-Liang Chu and Min Dai for preparation of GST fusion proteins, and Hiroyasu Nakano, Juntendo University School of Medicine, Tokyo, Japan, for plasmid pGEX-4T-IB (1-61).     FOOTNOTES Submitted December 31, 1998; accepted May 5, 1999. Supported in part by the National Institutes of Health, Grant Nos. R37HL30647, HL45994, and AI33839. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 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