Functional characterization of rat chemokine ... - Springer Link

Inflammation, Vol. 19, No. 1, 1995

FUNCTIONAL CHARACTERIZATION OF RAT CHEMOKINE MACROPHAGE INFLAMMATORY PROTEIN-2 C H A R L E S W. F R E V E R T , I

ANTHONY

FARONE, J

H A D I D A N A E E , l J O S E P H D. P A U L A U S K I S , I and L E S T E R K O B Z I K 1'2 IPhysiology Program, Harvard School of Public Health 2Department of Pathology, Brigham and Women "s Hospital Boston, Massachusetts

Abstract--Expression of mRNA for the C-X-C chemokine, macrophage inflammatory protein-2 (MIP-2), is induced during acute inflammation in rat models of disease. We have characterized the phlogistic potential of rat recombinant MIP-2 (rMIP-2) protein in vitro and in vivo. Recombinant MIP-2 caused marked PMN chemotaxis in vitro, with peak chemotactic activity at 10 nM. Incubation of whole blood with rMIP-2 caused a significant loss of L-selectin and a significant increase in Mac-1 expression on the PMN surface. Under similar conditions rMIP-2 also caused a modest respiratory burst in PMNs. The intratracheal instillation of 10 and 50 t~g of rMIP-2 caused a significant influx of PMNs into the airspace of the lungs. Rat MIP-2 is a potent neutrophil chemotactic factor capable of causing neutrophil activation and is likely to function in PMN recruitment during acute inflammation in rat disease models.

~TRODUCTION

M a c r o p h a g e inflammatory protein (MIP)-2 is a C - X - C family c h e m o k i n e , w h i c h shares identity with the h u m a n gene, g r o / m e l a n o m a growth-stimulating activity ( M G S A ) (1-3). This c h e m o k i n e was first identified in murine m a c r o p h a g e s after exposure to lipopolysaccharide (LPS) (1). W o l p e et al. found that murine M I P - 2 was chemotactic for h u m a n neutrophils and p r o d u c e d localized inflammation after its subcutaneous injection into foot pads o f m i c e (1). H u m a n g r o / M G S A is a m i t o g e n , a neutrophil ( P M N ) c h e m o t a c t i c factor, and causes a slight respiratory burst in h u m a n P M N s (4, 5). T h e expression o f M I P - 2 m R N A has been characterized in several rat 133 0360 3997/95/0200-0133507,50/0 ~) 1995 Plenum Publishing Corporation

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m o d e l s o f i n f l a m m a t i o n . H u a n g et al. (6) h a v e s h o w n t h a t t h e e x p r e s s i o n o f M I P - 2 m R N A i n c r e a s e s in a l v e o l a r m a c r o p h a g e s ( A M s ) f o l l o w i n g b o t h in v i t r o a n d in v i v o e x p o s u r e to L P S . M a c r o p h a g e i n f l a m m a t o r y p r o t e i n - 2 m R N A l e v e l s are also u p - r e g u l a t e d in rat m o d e l s o f c h r o n i c b r o n c h i t i s (7) a n d e n d o t o x i n i n d u c e d l u n g injury (8). I n o r d e r to f u r t h e r i n v e s t i g a t e t h e role o f M I P - 2 in rat m o d e l s o f i n f l a m m a t i o n , w e h a v e p e r f o r m e d in v i t r o a n d i n v i v o c h a r a c t e r i z a t i o n o f r M I P - 2 p r o t e i n effects o n P M N f u n c t i o n .

MATERIALS

AND METHODS

Cloning and Expression of rMIP-2. A set of composite oligonucleotides was synthesized containing specific restriction endonuclease sites and corresponding to the 5' and 3' sequences of the protein-coding region of rat MIP-2 cDNA (K. Driscoll, EMBL). The 5' primer was 5'-GCGGGATCCGATGGCCCCTCCCACTCGC-3' and the 3' primer was 5'-CATGGAATTCCTTCCCAGGTCAGTTAGC-3'. Total RNA, isolated from LPS-stimulated rat AMs (6), was reverse-transcribed into cDNA using M-MuLV reverse transcriptase (Gibco BRL, Gaithersburg, Maryland) at 37~ for 90 min. The cDNA was subjected to 30 cycles of amplification with 2.5 units AmpliTaq polymerase (Perkin-Elmer Corp., Norwalk, Connecticut) in 25 mM Tris HC1 (pH 8.3), 50 mM KCI, 2.5 mM MgCI2, 5 mM DTT, and 1 mM each of both 5' and 3' primers. Each cycle consisted of denaturation, 94~ annealing, 55~ min, and extension, 72~ min. The PCR product was electropboresed in 1% low-melting-point agarose (Gibco BRL), and the resulting cDNA of expected size was excised and purified with glass beads (GeneClean II, Bio 101, La Jolla, California). For expression of recombinant protein, the purified product was restriction cut with BamHI and EcoRI (underlined sequences above), gel-purified with GeneClean II was above, and ligated into pVL1393 plasmid DNA (PharMinGen, San Diego, California). The resulting plasmid was cotransfected by calcium precipitation with linearized baculovirus DNA containing a lethal deletion (BaculoGold, PharMinGen) into Sf-9 cells derived from Spodopterafrugiperda. The reading frame of the constmct was confirmed by sequencing with the dideoxy nucleotide chain termination method (9). The second virus-amplification supematant was used to infect Sf-9 cells in protein-free medium (Sf-900II, Gibco BRL, Grand Island, New York). At 96 h postinfection, the medium was centrifuged at 40,000g for 1 h at 4~ and the resulting supernatant was dialyzed against 50 mM NaC1/10 mM Tris (pH 8.6) for 48 h. The supematant from the baculovirus system was concentrated 10-fold by lyophilization, resuspended in sterile, distilled water, and analyzed by SDS-polyacrylamide gel electrophoresis using a 16% tricine-sodium polyacrylamide mini-gel electrophoresis system (Novex San Diego, California) (10). Macrophage inflammatory protein-2 has also been expressed in a bacterial system (Peprotech, Inc., Rocky Hill, New Jersey) using the cDNA described above. In preliminary studies, similar functional results were obtained with either type of rMIP-2. Except as otherwise noted, we report on results with the commercially available rMIP-2. Neutrophil ChemotaxisAssay. The ability of rMIP-2 to promote PMN chemotaxis was measured with a Neuroprobe 96-well microchemotaxis chamber as previously described (11). Briefly, PMNs were obtained from a glycogen-induced peritoneal exudate and purified (>94% PMNs) utilizing a discontinuous, two-step Nycodenz (Nycomed AS., Diagnostics Division, Oslo, Norway) gradient (12). Following purification, PMNs were stained with 2',7'-2-carboxyethyl-5-and-6-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) (Molecular Probes Inc., Eugene, Oregon), and 44 ~1 of the BCECF-AM-labeled PMNs (5 • 106 cells/ml) were added to the top wells of the chemotaxis chamber. A 10-/zm-thick, 3-#m-porosity black (reduced fluorescence) polycarbonate

Characterization of Rat MIP-2

135

filter (Neuroprobe, Cabin John, Maryland) separated the top and bottom wells, which contained 15 #1 of rMIP-2 (1, 10, 20, and 50 nM), zymosan-activated serum (ZAS) (positive control), or RPMI 1640 media (negative control). The chemotaxis chamber was incubated for 30 rain at 37~ in humidified air with 5 % CO 2. Following incubation, nonmigrating cells were removed from the side of the filter facing the top wells by gently washing it three times with phosphate-buffered saline (PBS) (Pyrogen-free, Sigma Chemical, St. Louis, Missouri). The filter was then air-dried in the dark. Migration of cells into the filter was quantitated in a Cytoflur 2300 96-well plate reader (Millipore, Bedford, Massachusetts), excitation filter - 4 8 5 / 2 0 nm, emission filter - 5 3 0 / 3 0 nm, and sensitivity of 3. Samples were normalized to maximal chemotaxis towards the positive control (i.e., ZAS, 1 • 10 -~ dilution) after the negative control (i.e., RPMI) had been subtracted. The normalized values have been reported as a percentage of maximal chemotaxis. Neutrophil Adhesion Molecule Assay. The characterization of adhesion molecule expression on PMNs was done in vitro by incubating whole blood with treatment (i.e., negative control), rMIP-2 (10, 20, and 50 nM), and phorbol myristate acetate (PMA) at 10 - 7 M (i.e., positive control). Normal whole blood, anticoagulated with sodium citrate was collected by intracardiac puncture from untreated rats as a source of PMNs. Five hundred microliters of whole blood was used in each group and incubated for 30 min at 37~ Following incubation, leukocytes were rapidly prepared for flow cytometry. Briefly, red blood cell lysis was performed using hypotonic saline. Following lysis, each sample was centrifuged at 200g at 4~ for 8 min in order to remove platelets. The supernatant was discarded and the leukocyterich pellet was washed twice in basic salt solution with Ca 2+ and Mg 2§ (BSS+), containing 0.1% bovine serum albumin (BSA) chilled to 4~ The quantitation of adhesion molecules was done on unfixed cells since the epitope for the L-selectin monoclonal antibody (MAb) appears to be destroyed by fixation (unpublished results). Primary MAb (100 ~1) (Table 1) were added to 5 • 105 white blood cells (WBCs) and incubated for 30 min at 4~ Following this incubation, the samples were centrifuged and washed twice in the cold wash buffer (BSS § with 0.1% BSA). Two hundred microliters of the appropriate 2 ~ antibody was added to the WBCs and incubated for 30 min on ice. A 1/50 dilution of fluorescent anti-mouse IgG F(ab')2, (Boehringer Mannheim Corp., Indianapolis, Indiana) was added to the WBC pellets labeled with the murine MAb for C D I l a and C D l l b , while a 1/40 dilution of a fluorescent anti-hamster IgG (H + L) (Caltag Laboratories Inc,. San Francisco, California) was added to WBC pellets labeled with the hamster MAb for L-selectin. Following incubation these mixtures were centrifuged and washed twice and resuspended in 500/zl of BSS § containing 0.1% BSA. All samples were stored at 4~ in the dark prior to analysis on the flow cytometer. Fluorescence and light scattering of labeled cells was measured with an Ortbo 2151 Cyto-

Table 1. Antibodies Used to Assess Changes in Adhesion Molecules on PMNs following Intravenous Administration of LPS a

Clone

Rat PMN ligand

Source of Antibody

Concentration of Antibody

HRL3 WT. 1 OX-42

L-selectin CD1 la CD1 lb

Seikagaku (14) Serotec (14) Serotec (15)

1 : 40 10 #g/ml 1 : 100

aThe concentration of antibodies used for this study were determined in experiments to characterize the optimal titer for this system. Seikagaku (Tokyo, Japan), Serotec (Oxford, England), Sigma Chemical (St.Louis, Missouri).

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fluoragraf system equipped with an air-cooled laser (488 nm emission, 15 mW output). Green fluorescence was recorded using a 530-nm long-pass filter. PMNs were identified by their forward and 90 ~ light scattering ability (11). Data from 3000 cells per sample were recorded and stored. The data were expressed as the mean channel fluorescence minus the fluorescence of the nonbinding isotype control (i.e., background) as well as the percentage of the control group (i.e., no treatment in vitro). Neutrophil Respiratory Burst Assay. Whole blood, anticoagulated with sodium citrate, was collected from untreated rats by cardiac puncture while under halothane anesthesia (Halocarbon Labs, North Augusta, South Carolina). Dextran sedimentation was performed and the leukocyteenriched upper layer was added to BSS § containing 50/~m of dichlorofluorescin (DCFH) -diacetate (Molecular Probes Inc., Eugene, Oregon) (13). The leukocyte-rich cell suspensions were then incubated at 37 ~ for 20 rain. After DCFH loading, cells were treated in vitro in one of the following groups: (1) negative control--no treatment, (2) positive control--PMA at 10 -7 M, and (3) rMIP-2 at concentrations ranging from 0.001 to 100 nM. Samples were incubated at 37~ for 15 min and then placed on ice and stored in a dark place prior to analysis with flow cytometry as described above. Protocol for In Vivo Characterization of rM1P-2. Vires-antigen-free (VAF) male CD rats (170-200 g) (Charles River, Kingston New York) were used. Intratracheal treatments were performed using a 3.5-in. needle while rats were under halothane anesthesia. Neutrophil recmitment into the lungs was determined in the following treatment groups: (1) no treatment, (2) PBS, (3) 1 /zg rMIP-2, (4) 10/~g rM1P-2, and (5) 50/~g rMIP-2. Bronchoalveolar lavage (BAL) was performed 6 h after IT treatments. Bronchoalveolar Lavage and Analysis. Adult male rats were humanely killed with intraperitoneal pentobarbital sodium (50 mg/kg), and lung lavage was performed with 12 4-ml lavages using PBS-EDTA. Total cell counts and viability were determined by hemacytometer counts of samples diluted in trypan blue solution. Differential cell counts were performed on cytospin-prepared slides stained with Diff-Quik (American Scientific Products, McGaw Park, Illinois). A total of 200 cells were counted per sample, and the number of PMNs and AMs calculated as the total cell count times the percentage of the respective cell type in the BAL sample. Statistical Analysis. Unless otherwise stated, statistical analysis was done by comparing differences between groups with one-factor ANOVA using the Scheff6 F test to separate means. Values were considered significant when P < 0.05. All values have been reported as mean + standard error (SE). Statistical analysis was done on a Macintosh computer using StatView SE+Graphics (Abacus Concepts, Inc., Berkeley, California).

RESULTS

SDS-Polyacrylamide Gel Electrophoresis.

Recombinant protein from both

t h e b a c u l o v i m s - i n s e c t cell s y s t e m a n d t h e b a c t e r i a l s y s t e m m i g r a t e d as a s i n g l e b a n d at - 6 . 4 k D a o n S D S - P A G E . W e h a v e a l s o n o t e d s o m e c r o s s - r e a c t i v i t y between an anti-KC polyclonal antibody and rMIP-2 on western blot analysis w h e r e M I P - 2 a g a i n w a s s h o w n to b e m i g r a t i n g at

-6.4

kDa (unpublished

results). The estimated molecular weight of rMIP-2 on SDS-PAGE to t h e v a l u e s r e p o r t e d f o r m u r i n e M I P - 2 (1).

is s i m i l a r

MIP-2 is a Potent Chemotactic Factor f o r Rat Neutrophils In Vitro. N e u t r o p h i l c h e m o t a x i s w a s m e a s u r e d in a m o d i f i e d B o y d e n c h a m b e r


137

assay at 1, 10, 20, and 50 nM concentrations of rMIP-2 and has been normalized to chemotaxis towards ZAS ( 1 : 1 0 dilutions) and plotted as a percentage of maximal chemotaxis (Figure 1). Peak chemotactic activity for rMIP-2 was found to occur at 10 nM (i.e., 60 ng/ml), similar to values reported for other C-X-C cytokines (1, 5).

MIP-2 Causes Loss of L-Selectin and Up-Regulation of Mac-1 on Rat Neutrophils. To determine if rMIP-2 could cause PMN activation as manifested by changes in expression of adhesion molecules, the levels of L-selectin, Mac1, and LFA-1 were quantitated on the PMN surface (Figure 2). There was a significant decrease (P < 0.05) in the expression of L-selectin when PMNs were incubated with 20 and 50 nM concentrations of rMIP-2 (Figure 2a). In contrast, Mac-1 levels on PMNs in the negative control group were low and were significantly increased (P < 0.05) by 50 nM rMIP-2 (Figure 2b). The positive control, PMA at 10 -7 M, also caused a significant loss of L-selectin (mean channel fluorescence = 12 + 7) and an increase in Mac-1 (mean channel fluorescence = 155 + 18) levels on the PMN surface compared to control (data not illustrated). The expression of LFA-1 on the PMN surface was unchanged at all three doses of rMIP-2 (Figure 2c). Macrophage inflammatory protein-2 is, therefore, capable of influencing the expression L-selectin and Mac-1 on the PMN surface. 120-

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MIP-2 Concentration (nM) Fig. 1. Macrophage inflammatoryprotein-2 is a potent chemotactic factor for rat neutrophilsin vitro. Neutrophil chemotaxis was measured at four concentrationsof rMIP-2 (1, 10, 20, and 50 nM) to determinethe concentrationthat caused maximalchemotaxis. All samples were normalized to and reported as a percentage of chemotaxis towards ZAS at a 1 : 10 dilution (1 nM rMIP-2 = 47.2 _+ 10% maximal chemotaxis, 10 nM rMIP-2 = 93.5 +_ 12% maximal chemotaxis, 20 nM rMIP-2 = 64.4 + 7.4% maximal chemotaxis, and 50 nM rMIP-2 = 68.0 + 9.7% maximal chemotaxis, N = 3 experiments). Values are the mean +_SE.

138

Frevert et al.

2a

L-Selectin o t-

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Concentration

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10 nM

20 nM

50 nM

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2c

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7550250121

10 nM MIP-2

20 nM

50 nM

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Fig. 2. Macrophage inflammatory protein-2 causes loss of L-selectin and up-regulation of Mac-1 on rat neutrophils. Normal PMNs were incubated in vitro for 30 min at 37~ in one of the following four treatment (Tx) groups: (1) ~ no treatment (i.e., control group); (2) 10 nM, 10 nM MIP-2; (3) 20 nM, 20 nM rMIP-2; and (4) 50 nM, 50 nM rMIP-2. The receptor levels for three adhesion molecules, L-selectin (a), Mac-1 (b), and LFA-1 (c), were determined by flow cytometry and are expressed as mean channel fluorescence. There was a significant decrease in the expression of L-Selectin (mean fluorescence channel = 41 + 16 vs. 91 + 13, 20 nM rMIP-2 vs. control, N = 3) and a significant increase in Mac-1 levels (mean fluorescence channel = 43 5 : 6 vs. 10 + 4, 50 nM rMIP-2 vs. control, N = 3) on PMNs following their incubation in rMIP-2. The asterisk denotes groups significantly different from the control group using one-factor ANOVA and P < 0.05. Values are the mean ++_SE.


139

MIP-2 Causes Modest Respiratory Burst in Neutrophils. Neutrophils loaded with D C F H - D A w e r e treated in one o f the f o l l o w i n g groups: (1) negative c o n t r o l - - n o treatment, (2) positive c o n t r o l - - P M A at 10 -7 M , and (3) r M I P - 2 at concentrations ranging f r o m 0.001 to 100 n M . F o l l o w i n g the treatment o f P M N s in vitro, intracellular fluorescence was m e a s u r e d with flow cytometry (Figure 3). W h e n c o m p a r e d to the negative control (mean channel fluorescence = 141 • 18), there was a significant increase (P < 0.05) in the respiratory burst in P M N s incubated at 1 n M (244 + 20), 10 n M (243 + 16), and 100 n M (244 ___ 18) concentrations o f rMIP-2. T h e treatment o f P M N s with P M A at 10 -7 M caused a m o r e substantial increase in their oxidative m e t a b o l i s m (mean channel fluorescence = 930 • 8.5). M I P - 2 is, therefore, capable o f causing a m o d e s t respiratory burst in P M N s . MIP-2 Results in In Situ Recruitment of Neutrophils. T h e ability o f rMIP-2 protein to p r o m o t e P M N migration in v i v o was determined by quantitating the n u m b e r o f P M N s that migrated into the airways o f the lungs in response to an

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MIP-2 Concentration (nM) Fig. 3. Macrophage inflammatory protein-2 causes a modest respiratory burst in neutrophils. PMNs loaded with DCFH-DA were treated in one of the following groups: (1) ~ , no treatment; and (2) MIP-2 at concentrations ranging from 0.001 to 100 riM. Following the treatment of PMNs in vitro, intracellular fluorescence, a measure of oxidative metabolism was measured with flow cytometry. In order to make comparisons between treatment groups, oxidative metabolism (i.e., respiratory burst) has been expressed as the mean channel fluorescence of 3000 cells (N = 3 experiments). When compared to the negative control (mean channel fluorescence = 141 + 18), there was a significant increase (P < 0.05) in the respiratory burst in PMNs incubated at 1 nM (mean channel fluorescence = 244 -l- 20), 10 nM (mean channel fluorescence = 243 5: 16) and 100 nM (mean channel fluorescence = 244 _ 18) concentrations of rMIP-2. The asterisk denotes groups significantly different from the control group (i.e., ~ ) using one-factor ANOVA and P < 0.05. Values are the mean 5: SE.

140

Frevert et al.

IT challenge with rMIP-2. Neutrophil influx was measured 6 h after the IT instillation of rMIP-2 at 1 /zg (N = 3), 10/zg (N = 3), and 50/zG (N = 2) per rat (Figure 4). When compared to rats receiving IT PBS (0.70 + 0.26 x 106 PMNs), there was a significant increase (P < 0.05) in PMNs recovered with BAL following the IT administration of 10/xg (46.33 __+ 7.94 • 106 PMNs) and 50 /zg (34.43 + 6.46 x 106 PMNs) of rMIP-2. Differential cell counts were also determined and PMNs made up 1.7 + 3.2%, 77 + 3.2%, and 74 + 2.5% of the cells recovered with BAL for the PBS, 10 #g rMIP-2, and 50 #g rMIP-2 groups, respectively. MIP-2 is, therefore, capable of promoting PMN migration in situ.

DISCUSSION Macrophage inflammatory protein-2 mRNA is elevated in several rat models of disease, including a model of chronic bronchitis (7) and a model of endotoxininduced lung injury (8). Since MIP-2 has been implicated in the development of several inflammatory diseases, we characterized the ability of this chemokine to modify PMN function in vitro and in vivo. In vitro we characterized the

601 50

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40-

30.

z

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20-

10-

OPBS

Intratracheal

1 ug

10 ug

50 ug

Treatment

Fig. 4. Macrophage inflammatoryprotein-2 results in the pulmonary recruitmentof neutrophils. Neutrophil recruitmentinto the alveolar space was determined followingthe IT administrationof rMIP-2 at a total dose per rat of 1 /zg, 10 #g, and 50/~g. Rats treated with 10 #g rMIP-2 and 50 ~g rMIP-2 per rat had a significantincrease in the recovery of PMNs in the BAL fluid when compared to either no treatment (~) or IT PBS (PBS). The asterisk denotes groups significantly different from the control (i.e., IT PBS) using one-factorANOVA and P < 0.05. Values are the mean + SE.


141

ability of MIP-2 to cause PMN chemotaxis; to alter L-selectin, Mac-l, and LFA-1 expression on PMNs; and to cause a respiratory burst in PMNs. In vivo we evaluated PMN recruitment into the lungs in response to an intratracheal challenge of rMIP-2. MIP-2 was found to be a potent PMN chemotactic factor in vitro, with maximal chemotaxis occurring at 10 nM (60 ng/ml) (93.48 + 12% of maximal chemotaxis towards ZAS). Murine MIP-2 and human gro/MGSA protein share identity with rat MIP-2, and both have been shown to be potent chemotactic agents for PMNs in the 10- to 50-ng/ml range (1, 5). Rat MIP-2, therefore, has the ability to cause in vitro chemotaxis at concentrations similar to that observed with human MGSA and murine MIP-2. Incubation of whole blood with rMIP-2 resulted in a significant loss of L-selectin and up-regulation of Mac-1 expression (Figure 2). This assay was done in whole blood to mimic the dynamics of in vivo events. We also wanted to decrease in vitro manipulation since this can change the expression of the/3 2 integrins on the PMN surface (10). MIP-2 (50 nM) caused a 2.4-fold decrease in L-selectin levels and a 4.3-fold increase in Mac-I levels when compared to control levels. Hence, rat MIP-2 causes phenotypic changes associated with PMN activation. The in vitro incubation of rMIP-2 in whole blood caused a modest respiratory burst by PMNs (Figure 3). We have also evaluated the ability of recombinant rat KC, human MGSA, and human IL-8 to cause a respiratory burst in rat and human PMNs using a similar assay. We found that rKC induced a respiratory burst in rat neutrophils similar to that caused by its human structural homolog, MGSA, on human neutrophils but less than that caused by IL-8 on human PMNs (Frevert, unpublished results). Moreover, rKC and rMIP-2 caused similar increases in the respiratory burst of rat PMNs. In contrast, it has been reported that murine MIP-2 did not cause a respiratory burst in human PMNs possibly due to species specificity or differences in technique (1). In vivo findings show that the IT instillation of 10/zg and 50 t~g of rMIP-2 results in the pulmonary recruitment of PMNs (Figure 4). These findings show that MIP-2 is not only a potent chemotactic factor in vitro but is capable of neutrophil recruitment in situ. Our findings that MIP-2 is capable of causing PMN recruitment and activation implicate this chemokine in the accumulation of PMNs during acute inflammation. These experiments extend observations made at the mRNA level to further implicate MIP-2 as a potentially important cytokine in rat models of acute inflammation in the lung and other sites.

Acknowledgments--Wewould like to thank Amy Imrich for her technical expertise and assistance in the preparation of this manuscript. This research was supported by NIH HL02374, HL19170 NIEHS ES 00002, and a Parker B. Francis Fellowship to J. P.

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