Role of CC chemokines (macrophage inflammatory protein-1β, monocyte chemoattractant protein-1,...

Role of CC Chemokines (Macrophage InflammatoryProtein-1b, Monocyte Chemoattractant Protein-1, RANTES) inAcute Lung Injury in Rats 1

Nicolas M. Bless,* Markus Huber-Lang,*† Ren-Feng Guo,† Roscoe L. Warner,† Hagen Schmal,*Boris J. Czermak,*† Thomas P. Shanley,‡ Larry D. Crouch, § Alex B. Lentsch,¶ Vidya Sarma,†

Michael S. Mulligan,† Hans Peter Friedl,* and Peter A. Ward2†

The role of the CC chemokines, macrophage inflammatory protein-1b (MIP-1b), monocyte chemotactic peptide-1 (MCP-1), andRANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats wasdetermined. Rat MIP-1b, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed.mRNA and protein expression for MIP-1b and MCP-1 were up-regulated during the inflammatory response, while mRNA andprotein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats withanti-MIP-1 b Ab significantly decreased vascular permeability by 37% (p 5 0.012), reduced neutrophil recruitment into lung by65% (p 5 0.047), and suppressed levels of TNF-a in bronchoalveolar lavage fluids by 61% (p 5 0.008). Treatment of rats withanti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally withblocking Abs to MCP-1, RANTES, or MIP-1b, significant reductions in the bronchoalveolar lavage content of these chemokinesoccurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1b, but not RANTES or MCP-1,caused enhancement of the lung vascular leak. These data indicate that MIP-1b, but not MCP-1 or RANTES, plays an importantrole in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggestthat in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundantfunction. The Journal of Immunology,2000, 164: 2650–2659.

T he in vivo requirements for various CC chemokines inacute lung inflammatory responses is not well defined.Macrophage inflammatory protein-1 (MIP-1)3 is a low

m.w., heparin binding protein consisting of a doublet that containsMIP-1a and MIP-1b (1). These subunits are highly homologous.Because both MIP-1a and MIP-1b show in vitro chemotactic ac-tivity for monocytes and demonstrate nearly identical bindingcharacteristics for macrophages together with an ability to cross-compete for binding to monocytes, a common receptor on mono-cytes for MIP-1a and MIP-1b has been suggested (2). MIP-1a, butnot MIP-1b, stimulates the secretion of TNF-a, IL-1a, and IL-6from peritoneal macrophages in vitro (3). MIP-1a has been shownto contribute to leukocyte recruitment and lung injury in models of

lung inflammation induced by deposition of IgG immune com-plexes (4) and by airway instillation of bacterial LPS (4, 5). Underthese conditions MIP-1a seems to function as an autocrine stim-ulator of macrophages, causing enhanced secretion of TNF-a. Lit-tle is known about the functions of MIP-1b during in vivo inflam-matory reactions.

Monocyte chemotactic protein-1 (MCP-1) is derived frommononuclear cells and other cell sources, including alveolar mac-rophages, and has chemotactic activity for monocytes (6–9). Lunginflammatory injury in rats induced by IgA immune complexes ismacrophage dependent but neutrophil independent. In this model,blockade of MCP-1 attenuates the development of lung injury (7).It has been suggested that MCP-1 may also enhance the inflam-matory response to other stimuli (10). However, whether MCP-1affects neutrophil-dependent acute lung inflammation is unknown.

RANTES has been characterized as a chemoattractant for mono-cytes and lymphocytes (11). In a model of endotoxemia in mice,RANTES was shown to be up-regulated in lung after i.p. injectionof LPS (12). Furthermore, the expression of RANTES in vivo wasdependent on the production of TNF-a.

In the current study we sought to determine whether the rat CCchemokines, MIP-1b, MCP-1, and RANTES, were involved in theacute inflammatory response following intrapulmonary depositionof IgG immune complexes. All three chemokines were present inBAL fluids during lung inflammation. Studies employing blockingAbs indicated that MIP-1b contributed significantly to lung pro-duction of TNF-a and to recruitment of neutrophils and full de-velopment of lung injury. In contrast to the effects of in vivo block-ade of MIP-1b, Ab-induced blockade of MCP-1 or RANTES hadno effect on lung neutrophil recruitment or the severity of lunginjury, even though it could be shown in vivo that the BAL levels

*Department of Trauma Surgery, University of Freiburg, Freiburg, Germany;†De-partment of Pathology, University of Michigan Medical School, Ann Arbor, MI48109;‡Department of Pediatrics, University of Cincinnati School of Medicine, Cin-cinnati, OH 45229;§Department of Physiology, University of Nebraska School ofDentistry, Lincoln, NE 68198; and¶Department of Surgery, University of LouisvilleSchool of Medicine, Louisville, KY 40292

Received for publication July 30, 1999. Accepted for publication December 13, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grant HL31963.2 Address correspondence and reprint requests to Peter A. Ward, Department of Pa-thology, University of Michigan Medical School, M5240 Medical Science I, Box0602, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail address: [email protected] Abbreviations used in this paper: MIP-1, macrophage inflammatory protein-1;MCP-1, monocyte chemotactic protein-1; MPO, myeloperoxidase; rr, rat recombi-nant; CINC, cytokine-induced neutrophil chemoattractant.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

of MCP-1 and RANTES were substantially reduced in the pres-ence of these Abs. In companion experiments exogenous admin-istration of MIP-1b, but not of MCP-1 or RANTES, caused en-hanced lung injury. Thus, these data demonstrate a role for MIP-1b, but not for MCP-1 or RANTES, in IgG immune complex-induced lung injury, suggesting that the biological functions of CCchemokines in lung inflammatory reactions are not necessarilyinterchangeable.

Materials and MethodsAntibodies

Rabbit polyclonal IgG anti-BSA was purchased from ICN Biomedicals(Costa Mesa, CA). Rabbit polyclonal Ab to rat MCP-1 was produced asdescribed previously (7). Rabbit polyclonal IgG anti-rat RANTES was pro-duced in rabbits and purified as IgG using protein G affinity chromatog-raphy. By Western blot analysis, anti-MCP-1 did not cross-react withMIP-1a or RANTES, and anti-RANTES did not cross-react with MIP-1aor MCP-1. Goat polyclonal IgG anti-murine MIP-1b was purchased fromR&D Systems (Minneapolis, MN). The ability of anti-MCP-1 IgG to blockthe in vitro chemotactic activity for mononuclear cells has been reported(7), while the ability of anti-RANTES to cause delayed rejection of cardiacallografts and xenografts in rats has also been noted (13, 14).

IgG immune complex-induced alveolitis

Specific pathogen-free male Long-Evans rats (275–300 g; Charles RiverBreeding Laboratories, Portage, MI) were anesthetized with ketamine HCl(150 mg/kg i.p.). In positive control animals 2.5 mg of rabbit polyclonalIgG anti-BSA in a volume of 300ml of PBS, pH 7.4, was instilled via anintratracheal catheter during inspiration. Immediately thereafter, 10 mg ofBSA in 0.5 ml of PBS was injected i.v. Negative control rats received 2.5mg of anti-BSA IgG intratracheally in the absence of an i.v. infusion of 10mg of BSA. For analysis of pulmonary vascular permeability, traceamounts of125I-labeled BSA were injected i.v. Four hours after initiationof the inflammatory reactions, rats were exsanguinated, the pulmonary cir-culation was flushed via the pulmonary artery with 10 ml of phosphate(PBS), and the lungs were surgically removed. The extent of lung injurywas quantified by calculating the lung permeability index (dividing theamount of radioactivity ([125I]BSA) in the PBS-perfused lungs by theamount of radioactivity in 1.0 ml of blood obtained from the inferior venacava at the time of sacrifice). Effects of in vivo blockade of MIP-1b orRANTES in the lung injury model were assessed by the intratracheal in-stillation of 400mg of anti-MIP-1b, anti-RANTES, or nonspecific goat orrabbit IgG together with anti-BSA. In vivo blockade of MCP-1 wasachieved by i.v. injection of 0.5 ml of anti-MCP-1 serum or 0.5 ml ofnormal rabbit serum just before infusion of BSA, because this is the pro-tocol that caused suppression of other types of inflammatory injury in lungmodels of granulomatous vasculitis or in IgA immune complex-inducedalveolitis (7, 15). In other experiments, where indicated, 400mg of rabbitanti-rat MCP-1 IgG or preimmune IgG were given intratracheally togetherwith anti-BSA. For the animal studies, then values noted in the figurelegends represent the number of rats used in each group, indicated byvertical bars. In other studies experimental results were indicative of sim-ilar findings from at least two separate and independent experiments.

RNA extraction for cloning

Whole lungs were dissected and immediately frozen in liquid nitrogen 2–4h after initiation of immune complex deposition. Total RNA was extractedfrom lung homogenates using a guanidinium isothiocyanate/chloroform-based technique (RNA STAT-60, Tel-Test, Friendswood, TX) followed byisopropanol precipitation. Poly(A) mRNA was purified on oligo(dT)-cel-lulose. First-strand cDNAs were constructed from 1.0mg of poly(A)mRNA in a RT reaction primed with a poly(dT) primer (cDNA Cycle Kit,Invitrogen, San Diego, CA). Cloning of rat MIP-1b cDNA was obtainedusing these first strands as templates by PCR with the following oligonu-cleotide primers: 59 primer (59-ATG AAG CTC TGC GTG TCT-39) and 39primer (59-TCA GTT CAA CTC CAA GTC A-39). The PCR product of thepredicted size (279 bp) was ligated into a pCRII vector (TA Cloning Kit,Invitrogen) and sequenced followed by submission of the results to Gen-Bank (accession no. UO6434).

MIP-1b protein expression

NdeI (59) andBamHI (39) restriction enzyme sites were added to the 59 and39 ends of the cDNA encoding the mature protein for MIP-1b by PCR forligation into the pET15b expression vector (Novagen, Madison, WI). After

ligation, the pET-rMIP-1b plasmids were transformed into competentNova BlueEscherichia coli. After plasmid purification proper orientationof the insert was confirmed by restriction digest analysis and sequencing.Purified pET-rMIP-1b plasmid was then used for transformation of theE.coli strain BL21(DE3)pLysS. Following induction of the protein with 0.4mM isopropyl b-D-thiogalactopyranoside (Life Technologies, Grand Is-land, NY), the cells were lysed into 50 mM NaH2PO4 (pH 8.0), 300 mMNaCl, and 10 mM imidazole. The supernatant fluid was applied to a Ni-NTA column (Qiagen, Santa Clarita, CA), and the protein was eluted ac-cording to the manufacturer’s instructions, dialyzed in PBS, and concen-trated using Centricon concentrators (Amicon, Beverly, MA). Rat PBMCand alveolar macrophages were evaluated for chemotactic responses to CCchemokines as previously described (4). FMLP was used as a reference asa positive control.

RT-PCR cloning of rat RANTES

First-strand cDNAs were constructed using 1 mg of mRNA with a RTreaction primed with poly(dT) primer (cDNA Cycle Kit, Invitrogen). Am-plification was performed using these first strands as templates in a PCRemploying the following primers: 59 primer (59-ACC AATG AAG ATCTCT GCA GCT-39) and 39 primer (59-ATC CTA GCT CAT CTC CAAATA-39). PCR products were then ligated into a pCRII vector according tothe manufacturer’s instructions (TA Cloning Kit, Invitrogen). TA One ShotcompetentE. coli cells (TA Cloning Kit, Invitrogen) were transformedwith ligation reaction mixtures and grown up for plasmid purification. Plas-mids that provided a template for a successful PCR were sequenced by theUniversity of Michigan Core Facility using the T7 and SP6 promoter re-gions of the pCRII vector and compared with the known human and murinesequences. The procedure was duplicated on a product cloned from mRNAobtained from lungs of a second rat undergoing intrapulmonary depositionof IgG immune complexes.

Recombinant expression of rat RANTES

HindIII (59) and BamHI (39) restriction enzyme sites were added to theends of the cDNA encoding the mature protein for rat RANTES by PCR.The amplified product was then used for ligation into the pET23a vector(Novagen). Competent Nova BlueE. coli was transformed with the ligatedpET-rRANTES plasmids. After confirmation of proper orientation by re-striction digest analysis, purified plasmids were used for transformation ofBL21(DE3)pLysSE. coli strains. These bacterial cultures were used tooptimize isopropylb-D-thiogalactopyranoside-stimulated expression of re-combinant rat RANTES.

Rabbit polyclonal anti-rat RANTES

Polyclonal rabbit anti-rat RANTES was raised against the expression prod-uct in 3-kg New Zealand White rabbits repeatedly immunized with 50–500mg of rat RANTES emulsified in Freund’s adjuvant. For determination ofserum titers of anti-RANTES Ab, an indirect ELISA was used. Briefly,96-well Immulon 4 ELISA plates (Dynex Technologies, Chantilly, VA)were coated with soluble recombinant rat RANTES (5mg/ml) overnight at4°C. The plates were blocked with 2% BSA in PBS for 1 h before additionof serial dilutions of rabbit anti-RANTES serum. The plates were washed,and 100ml/well HRP-conjugated goat anti-rabbit Ab (1/3000 dilution; Bio-Rad, Richmond, CA) was added to the wells. Reactions were developedwith o-phenylenediamine dihydrochloride substrate and stopped by addi-tion of 3 M H2SO4. Titers were determined by measuring absorbance at490 nm. The ability of this Ab to neutralize recombinant rat and humanRANTES was demonstrated by the chemotaxis assay (as described below).

Pulmonary expression of MIP-1b, MCP-1, and RANTES mRNA

Pulmonary cytoplasmic RNA was isolated as previously described (4),fractionated electrophoretically in a 1% formaldehyde gel, and transferredto a nylon membrane (Micron Separations, Westboro, MA). For analysis ofMIP-1b mRNA, a MIP-1b-specific oligomer (59-GGC CAC AAG CAGGAG GAG AGA GAA GGC AGA CAC-39) was end labeled with[g-32P]ATP. For analysis of MCP-1 or RANTES mRNA, plasmids con-taining the confirmed rat cDNAs for MCP-1 or RANTES were used togenerate radiolabeled [32P]dCTP probes by PCR using specific oligoprimerpairs. The primer sequences for MCP-1 were 59-ATC AGC TAG CCTCCA CCA CTA TGC-39 and 59-CTA AAC CTT ACA CTA CGA TCGGGT GG-39. The primer sequences for RANTES were 59-ACC ATG AAGATC TCT GCA GCT-39 and 59-ATA AAC CTC TAC TCG ATC CTA-39.Northern blots were hybridized at 65°C for 16 h. Autoradiography of theblots was performed at270°C on Kodak BioMax MR film (EastmanKodak, Rochester, NY).

2651The Journal of Immunology

Pulmonary expression of MIP-1b, MCP-1, and RANTESproteins

Lung expression of MIP-1b, MCP-1, and RANTES was assessed by West-ern blot analysis. Lungs were obtained from rats 4 h after intrapulmonarydeposition of IgG immune complexes (positive controls) or from rats 4 hafter receiving anti-BSA intratracheally but with the omission of i.v. in-fused BSA (negative controls). The lungs were homogenized, and the ma-terial was centrifuged for 30 min at 14,0003 g. Supernatant fluids weresubjected to electrophoretic separation in a nonreducing 10% polyacryl-amide gel and transferred to a nitrocellulose membrane. Nonspecific bind-ing sites were blocked with TBS (40 mM Tris, pH 7.6, and 300 mM NaCl)containing 5% nonfat dry milk for 12 h at 4°C. Membranes were thenincubated with 2mg/ml IgG Ab to the relevant CC chemokine in TBS with0.1% Tween 20 (TBST). After three washes in TBS with 0.1% Tween 20,membranes were incubated in a 1/25,000 dilution of HRP-conjugated don-key anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove,PA) for detection of MIP-1b. For RANTES and MCP-1, HRP-conjugatedgoat anti-rabbit IgG was employed. Immunoreactive proteins were detectedby enhanced chemiluminescence according to manufacturer’s instructions.

Chemotaxis assay

This assay was described previously (4, 7). Rat PBMC and rat alveolarmacrophages were labeled with 29,79-bis-[2-carboxyethyl]-5-[and 6]-car-boxy-fluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR).Labeled cells (44ml; 2.25 3 105 cells/well) were then loaded into uppercompartments of 96-well minichambers (NeuroProbe, Cabin John, MD).Test samples (in 33ml) were added to the bottom compartments. The topand bottom compartments were separated by a polycarbonate membranewith a pore size of 3mm, and the chambers were incubated at 37°C for 2 h.The numbers of cells that migrated through the polycarbonate filter (on thebottom surface of the membrane) were measured with a cytofluorometer(Cytofluor II, PerSeptive Biosystems, Framingham, MA), using an excita-tion wavelength of 485 nm and an emission wavelength of 530 nm andexpressed as fluorescence units.

Lung myeloperoxidase (MPO) content

Whole lungs obtained at the time of sacrifice were immediately frozen inliquid nitrogen. After mechanical homogenization in a Polytron homoge-nizer (Tebmar, Cincinnati, OH) at an instrument setting of 4 for 40 s at 5°C,MPO content was quantitated by measuring the rate of decomposition ofH2O2 (per minute) in the presence ofO-dianisidine at an absorbance of 460nm (16).

TNF-a levels in BAL fluids

The BAL fluid content for TNF-a was measured using the highly sensitiveWEHI cell cytotoxicity assay as previously reported (17).

Analysis of CC chemokines in BAL fluids in the presence ofblocking Abs

To determine whether the blocking Abs to CC chemokines reached theirtargets, 400mg of Ab IgG to MCP-1, RANTES, or MCP-1 or 400mg of

preimmune IgG were instilled intratracheally with the anti-BSA IgG fol-lowed by i.v. infusion of 10 mg of BSA, according to procedures describedabove. Negative controls received 400mg of preimmune IgG intratrache-ally together with the anti-BSA, but the i.v. infusion of BSA (10 mg) wasomitted. At the time of sacrifice, BAL fluids (3.0 ml) were collected (seeabove). To remove IgG from the BAL fluids, because IgG would interferewith the ELISA measurement, BAL fluids were subjected to centrifugationin Ultrafree centrifugal filter and tube devices (Millipore, Bedford, MA)with a cut-off of 50 kDa. The pass-through fluid was then analyzed for theappropriate CC chemokine by indirect ELISA as described above. All re-agents were purchased from R&D Systems. For MIP-1b detection, coatingAb (1 mg/ml) followed by biotinylated Ab (200 ng/ml) was used. ForMCP-1 detection, coating Ab and biotinylated Ab (2mg/ml and 200 ng/ml,respectively) were used. An ELISA detection kit was used for RANTESdetection, using instructions of the manufacturer.

Exogenous administration of CC chemokines

To extend studies featuring the in vivo use of Abs to MIP-1b. MCP-1 andRANTES, recombinant CC chemokines (5.0mg each), were added to in-tratracheally administered anti-BSA, and the permeability index was mea-sured at 4 h according to technical details provided above and inResults.Recombinant mouse MIP-1b was obtained from R&D, while recombinantrat MCP-1 and RANTES were obtained from PeproTech (Rocky Hill, NJ).Densitometric analysis

Northern blots were exposed for 3 days on a phosphorimaging screen andquantitated by laser densitometry using ImageQuant software and Phos-phorImager 445 SI (both obtained from Molecular Dynamics,Sunnyvale, CA).

Statistical analysis

All values are expressed as the mean6 SEM. Data were analyzed with aone-way ANOVA, and individual group means were compared using Stu-dent-Newman-Keuls test. Differences were considered significant whenp , 0.05. For calculation of the percent change, negative control valueswere subtracted from positive control group.

ResultsCloned rat MIP-1b

Cloned cDNA for rat MIP-1b (GenBank accession no. UO6434)had a 279-nucleotide open reading frame that shared 89% homol-ogy with the published murine oligonucleotide sequence (1). Theopen reading frame encoded a 93-aa protein with a putative 26-aasignal peptide; the mature protein consisted of 67 aa. The deducedamino acid sequence shared 84% homology with the publishedsequence of the murine protein.

Expression of rat recombinant (rr) MIP-1b

rrMIP-1b was expressed in BL21(DE3)pLysSE. coli, analyzed by15% SDS-PAGE, and stained with Coomassie blue. The expressed

FIGURE 1. A, SDS-PAGE analysis of rrMIP-1b. The predominant MIP-1b band was present in a position aligned with the 14.3-kDa marker.B, Westernblot analysis of rmMIP-1b (lane 1) and rrMIP-1b (lane 2) revealed slightly different electrophoretic positions.

2652 CC CHEMOKINES AND LUNG INJURY

product showed a band that aligned closely with the 14.3-kDamarker together with slower migrating species, perhaps represent-ing multimers (Fig. 1A), formation of which would be promoted bythe four cysteine residues present in MIP-1b. Western blot analysisshowed a band for rrMIP-1b (Fig. 1B, lane 2) that closely alignedwith the 14.3-kDa marker compared with the position of rmMIP-1b, which appeared between the 6.2- and 14.3-kDa markers (Fig.1B, lane 1).

Chemotactic activity of rrMIP-1b

Rat rMIP-1b was purified from BL21(DE3)pLysSE. coli super-natants at a final stock concentration of 300mg/ml. The chemo-tactic activity of rrMIP-1b for rat PBMC was assessed and com-pared with the PBML responses to FMLP. Rat rMIP-1b hadsignificant chemotactic activity for rat PBMB over a range from3.8 3 10213 to 3.83 1029 M, with peak activity at 3.83 10210

M (Fig. 2). The characteristic fall in chemotactic activity at highconcentrations of chemoattractant was found. PBMC demonstratedpeak chemotactic responses to 10 nM FMLP.

Expression of MIP-1b mRNA during IgG-immune complex lunginjury

Pulmonary expression of MIP-1b mRNA during IgG immunecomplex-induced injury was determined as a function of time(0–48 h) by Northern blot analysis. Equal loading of RNA wassuggested by the 28S marker (Fig. 3A). MIP-1b mRNA was un-detectable in lungs of animals sacrificed at time zero (Fig. 3,B andC). However, up-regulation of MIP-1b mRNA was observed asearly as 30 min after the intrapulmonary deposition of immunecomplexes. Peak expression of MIP-1b mRNA occurred at 6 h,similar to the time courses for expression of mRNA for MIP-2,MIP-1a, and cytokine-induced neutrophil chemoattractant (CINC)in inflamed rat lung (18–20). Enhanced expression of mRNA forMIP-1b was observed for as long as 24 h. By 48 h, MIP-1b mRNAreturned to nearly undetectable levels. A similar pattern of expres-sion was noted in two additional, independent experiments.

MIP-1b protein expression in lung

Expression of MIP-1b in lung was assessed by Western blot anal-ysis of whole lung homogenates obtained 4 h after initiation oflung injury. Homogenates (50, 100, 300, and 500mg total protein)were separated in SDS-PAGE under nonreducing conditions. Inthese experiments, 25 ng of rmMIP-1b was used as a positivecontrol. Anti-MIP-1b recognized a protein in rat lung homoge-nates (300 and 500mg) in a position that aligned with the slowestmigrating of three bands for rmMIP-1b (Fig. 4). It is assumed thatthe rmMIP-1b preparation contained isoforms of MIP-1b. Westernblot analysis of MIP-1b in lung homogenates (Fig. 4) also revealedthe presence of slower migrating species of material reactive withanti-MIP-1b. Homogenates (500mg of protein) obtained fromlungs of rats undergoing IgG immune complex deposition but sac-rificed at time zero failed to demonstrate reactivity for MIP-1b inWestern blots.

FIGURE 2. Monocyte chemotactic activity of rrMIP-1b. Fluorescein-labeled rat PBMC (labeled with 29,79-bis-[2-carboxyethyl]-5-[and 6]-car-boxy-fluorescein acetoxymethyl ester (BCECF) as described inMaterialsand Methodsand Ref. 6) were exposed to increasing concentrations ofFMLP (1–1000 nM) or rrMIP-1b (0.05–500 nM). The amount of cells thatmigrated through a filter containing pores of 3mm was assessed by mea-suring fluorescence in a cytofluorometer, using excitation at 485 nm andemission at 530 nm. Values represent the mean6 SEM (n 5 4/time point)and are characteristic of data from three separate experiments.

FIGURE 3. A, Northern blot analysis of mRNA for MIP-1b from ho-mogenates of IgG immune complex-injured lungs. Samples were collectedover 48 h after initiation of the lung inflammatory responses, with loadingas determined by ethidium bromide staining of 28S RNA.B, The presenceof mRNA for MIP-1b in lung homogenates was established by Northernblot analysis using an MIP-1b-specific oligomer end labeled with[g-32P]ATP. C, The OD of the Northern blot was determined in a Phos-phorImager. For this and subsequent Northern blots, animals received anti-BSA intratracheally (2.5 mg) and BSA (10 mg) i.v. and were sacrificedfrom 0 h to thevarious time points indicated.

FIGURE 4. Pulmonary expression of MIP-1b during IgG immune com-plex-induced lung injury. Homogenates of rat lungs harvested 4 h afterinitiation of lung injury were analyzed by SDS-PAGE (under nonreducingconditions), using goat anti-MIP-1b polyclonal Ab. Fifty, 100, 300, and500mg of protein from lung homogenate were loaded. Mouse rMIP-1b (25ng) was added as a reference control.


Effects of anti-MIP-1b on IgG immune complex-induced lunginjury

To determine whether MIP-1b contributed to IgG immune com-plex-induced lung injury, rats were treated with MIP-1b blockingAb. Administration of 400mg of anti-MIP-1b (given intratrache-ally with the anti-BSA) significantly reduced the permeability in-dex by 37% (p 5 0.012) compared with otherwise unmanipulatedpositive controls or compared with positive controls also receiving400mg of preimmune IgG intratracheally with the anti-BSA (Fig.5A). Anti-MIP-1b reduced IgG immune complex-induced in-creases in lung MPO by 65% (p 5 0.047) compared with other-wise unmanipulated positive controls (Fig. 5B). Compared withthe positive control group receiving preimmune IgG, which causedelevated lung levels of MPO, treatment with anti-MIP-1b de-pressed the MPO content by nearly 80% (Fig. 5B). BAL fluidsfrom negative control lungs (in which the i.v. infusion of BSA wasomitted) contained virtually no detectable TNF-a (Fig. 5C). Incontrast, levels of TNF-a in BAL fluids from IgG immune com-plex-injured lungs (at 4 h) were decreased by 61% (p 5 0.008) inthe presence of anti-MIP-1b (Fig. 5C), whereas BAL content of

TNF-a was not significantly reduced by treatment with 400mg ofpreimmune goat IgG compared with that in the otherwise nonma-nipulated positive controls. The data suggest that MIP-1b, likeMIP-1a (4), is required for the full generation of TNF-a and thesubsequent recruitment of neutrophils and full development oflung injury.

MCP-1 mRNA and protein expression during IgG-immunecomplex-induced lung injury

Pulmonary expression of MCP-1 mRNA during IgG immune com-plex-induced lung injury was determined as a function of time(0–8 h) by Northern blot analysis. Equal loading of RNA wassuggested by the 28S marker (Fig. 6A). MCP-1 mRNA was un-detectable in lungs at time zero (Fig. 6,B andC). Up-regulation ofMCP-1 mRNA was detected by 1 h and progressively increased,reaching maximal levels 4 h after induction of lung injury. Peakexpression of MCP-1 mRNA was maintained for as long as 8 h.Pulmonary expression of MCP-1 protein was assessed by ELISAanalysis of BAL fluids. Levels of MCP-1 protein were virtuallyundetectable in negative controls (,2 ng/ml; Fig. 6D) in which i.v.infusion of BSA was omitted. Four hours after initiation of IgGimmune complex-induced lung injury, MCP-1 levels in BAL fluidsincreased to 12.36 0.3 ng/ml (p , 0.05).

Failure of anti-rat MCP-1 to protect against IgG immunecomplex-induced lung injury

To determine whether MCP-1 contributed to lung injury inducedby IgG immune complexes, rats were injected i.v. with 0.5 ml ofnormal rabbit serum or 0.5 ml of rabbit anti-rat MCP-1 serum justbefore infusion of BSA. This protocol has been shown to protectagainst MCP-1-dependent models of acute lung injury in rats (7,

FIGURE 5. In vivo blockade of MIP-1b with intratracheally adminis-tered 400mg of polyclonal goat IgG anti-MIP-1b or 400mg of preimmunegoat IgG. Parameters of injury were vascular permeability (A), lung contentof MPO (B), and TNF-a content in BAL fluids (C). Values represent themean6 SEM (n 5 5/group). In this and all other figures employing animalgroups, negative controls received anti-BSA intratracheally, but the i.v.infusion of BSA was omitted. Positive controls received anti-BSA intra-tracheally and 10 mg of BSA i.v. in addition to any of the other indicatedmanipulations. The MPO determination was made by measuring the rateper minute of decomposition of H2O2 in the presence ofO-dianisidine atthe absorbance of 460 nm.

FIGURE 6. A, MCP-1 expression during IgG immune complex-inducedlung injury. Loading of RNA for Northern blot analysis was confirmed bymethylene blue staining of 28S RNA.B, Appearance of mRNA for MCP-1in lung after IgG immune complex-deposition.C, Densitometric analysisof Northern blot.D, Presence of MCP-1 protein in BAL fluids in negativecontrols and positive controls following IgG immune complex deposition.Values represent the mean6 SEM (n 5 6/group). In the negative controlgroup, rats received anti-BSA intratracheally, but the i.v. infusion of BSAwas omitted. Animals in the two groups shown inD were killed at 4 h.


15). Treatment with anti-MCP-1 did not reduce IgG immune com-plex-induced increases in the lung permeability index (Fig. 7A).Similarly, anti-MCP-1 had no effect on lung recruitment of neu-trophils as determined by lung MPO content (Fig. 7B). Additionalstudies were performed with intratracheal administration of 400mg of anti-MCP-1 IgG (rabbit) or preimmune rabbit IgG. Similarto the protocols for in vivo use of anti-MCP-1 and anti-RANTES,400mg of anti-MCP-1 or 400 mg of preimmune IgG was added tothe anti-BSA preparation, and the mixture was instilled intratra-cheally. Using the procedures described above, the permeabilityindex was measured at 4 h in twopositive control groups receivingeither preimmune IgG or anti-MCP-1 IgG. Six animals were ineach group. The permeability index values for the positive controlgroups receiving either preimmune IgG or anti-MCP-1 IgG were0.656 0.05 and 0.696 0.05, respectively (nonsignificant). Thus,although MCP-1 mRNA is up-regulated during IgG immune com-plex-induced lung inflammation (Fig. 7), it apparently does notcontribute to the development of lung injury in the lung injurymodel.

Expression of RANTES mRNA during IgG-immune complex lunginjury

Pulmonary expression of RANTES mRNA during IgG immunecomplex-induced lung injury was determined by Northern blotanalysis as a function of time (0–8 h). Equal loading of RNA wassuggested by the 28S marker (Fig. 8A). RANTES mRNA wasconstitutively expressed in lungs of animals undergoing immunecomplex deposition but sacrificed at time zero (Fig. 8,B andC).Changes in RANTES mRNA were not consistently detected in

lung extract over an 8-h period following induction of the lunginflammatory reactions.

Failure of anti-rat RANTES to protect against IgG immunecomplex-induced lung injury

Intratracheal administration of 400mg of anti-rat RANTES (coin-stilled with anti-BSA) did not reduce the increase in the lung per-meability index in the lung inflammatory model compared with theeffect of intratracheal coinstillation (with anti-BSA) of 400mg ofpreimmune IgG (Fig. 8D). No protection from injury was founddespite the fact that anti-rat RANTES blocked the chemotacticproperties of rrRANTES (Table I), and, as shown (below) in TableII, the presence of anti-RANTES IgG prevented detection of RAN-TES in BAL fluids from inflamed lungs. As shown in Table I, bothrat PBMC and rat alveolar macrophages responded chemotacti-cally to FMLP and rrRANTES, although the chemotactic re-sponses of alveolar macrophages to rrRANTES were more impres-sive. In part this was due to the relatively high background values(in the presence of the buffer, HBSS) found with rat PBMC. In thepresence of anti-RANTES, the chemotactic responses to RANTESwere profoundly suppressed. This same Ab significantly prolongedthe rejection time of cardiac allografts and xenografts in rats (13,14). The data suggest that although RANTES is constitutively ex-pressed in lung at the mRNA level, its levels are not altered duringIgG immune complex-induced lung injury. The failure of anti-RANTES to reduce neutrophil influx and to protect against lunginjury suggests that RANTES does not contribute directly or in-directly to the development of lung injury in the model employed.

FIGURE 7. Effects of anti-MCP-1 on induction of IgG immune com-plex-induced lung injury. Anti-MCP-1 serum was administered i.v. (0.5ml) just before IgG immune complex deposition. The effects of anti-MCP-1 on lung permeability index (A) and lung MPO (B) are shown.Values represent the mean6 SEM (n 5 6/group). Other experiments involv-ing intratracheal instillation of anti-MCP-1 IgG are described in the text.

FIGURE 8. A, RANTES expression during IgG immune complex-in-duced lung injury.B, Loading of RNA for Northern blot analysis wasdemonstrated by methylene blue staining of 28S RNA.C, Appearance ofmRNA for RANTES in lung after IgG immune complex-deposition.D,Densitometric analysis of Northern blot. The effects of 400mg anti-RANTES, administered intratracheally, on lung permeability index areshown. The negative and positive controls are similar to those described inFig. 7D.


Effects on lung injury of exogenously administered CCchemokines

To extend the results with blocking Abs to MIP-1b, MCP-1, orRANTES (see below), another series of experiments was com-pleted in which 5.0mg of MIP-1b, MCP-1, or RANTES was addedto the anti-BSA IgG, which was administered intratracheally. Thepermeability index was determined 4 h later. As shown by the datain Table III (Expt A), the copresence of RANTES did not alter thepermeability index in a statistically significant manner, whileMIP-1b significantly increased (by 44%;p 5 0.02) the lung per-meability index. The copresence of MCP-1 (Table III, Expt B),like that of RANTES, failed to cause any increase in the vascularpermeability index in this lung injury model. These observations,which are the reverse of the effects of blocking Abs employed invivo, strengthen the conclusion in this model of lung injury thatMIP-1b, but not MCP-1 or RANTES, participates in events lead-ing to lung injury.

Blocking Ab-induced changes in BAL chemokines

Animals (n 5 4/group) undergoing IgG immune complex deposi-tion received 400mg of preimmune IgG or 400mg of anti-MCP-1,anti-RANTES, or anti-MIP-1b IgG intratracheally together withthe anti-BSA, as described above. At the time of sacrifice, BALfluids were obtained, and ELISA was performed for quantitation ofthe three CC chemokines (MCP-1, MIP-1b, and RANTES). Ex-perimental protocols are described above. The results are shown inTable II. As shown by the data, the BAL levels of MCP-1 werereduced from 46.16 1.91 to 17.46 1.77 when the latter groupreceived intratracheal instillation of anti-MCP-1; this differencewas statistically significant and represented nearly an 80% reduc-tion when the negative control values are subtracted from the pos-itive control groups. In the case of RANTES the increase from

74.0 6 4.02 to 3126 10.4 occurring in negative and positivecontrols, respectively, was totally eliminated in the presence ofanti-RANTES. In fact, BAL levels of RANTES fell to less thandetectable levels (,3 pg/ml). In the case of MIP-1b, BAL levelsin the negative and positive controls were 2.756 0.35 and 7.5061.41 pg/ml, respectively. In the presence of anti-MIP-1b, levelsfell to 0.73 6 0.04 pg/ml. Thus, it appears that intratracheal de-livery of anti-CC chemokines found their targets in lung, causingsubstantial reductions in BAL levels of these CC chemokines.

To determine whether the administration of anti-MIP-1b af-fected BAL levels of MIP-1a (which is known to be required forthe full expression of injury in this model (18)), BAL fluids (asdescribed in Table II, in which IgG anti-MIP-1b IgG was em-ployed in vivo) were evaluated by ELISA for MIP-1a content. Thenegative control values for MIP-1a were 4.006 0.35 pg/ml, whilethe positive control values in animals treated with preimmune IgGor anti-MCP-1 IgG (400mg intratracheally) were 1106 10.5 and1036 6.15 pg/ml, respectively. In positive controls receiving 400mg of anti-MIP-1b intratracheally, the MIP-1a values fell to34.7 6 5.52 pg/ml, suggesting that full in vivo expression ofMIP-1a depends on MIP-1b.

DiscussionProperties of the CC chemokine family generally center around theability to chemotactically attract monocytes and lymphocytes intoinflammatory sites (21). However, it has recently been shown thatlung expression of the CC chemokine, MIP-1a, is up-regulatedduring IgG immune complex-induced lung injury (4). In thismodel the development of lung injury is dependent upon the in-trapulmonary recruitment of neutrophils (22). Treatment with

Table I. Inhibition of RANTES-directed chemotaxis by anti-RANTES IgGa

Cell Population Treatment Fluorescence

% Inhibition ofRANTES-Directed

Chemotaxis

Rat PBMC FMLP (1mM) 12266 107HBSS (buffer) 3476 13Rat RANTES (100 ng/ml) 4166 26Rat RANTES (100 ng/ml)1 anti-RANTES (100mg/ml) 3436 9 100 (p 5 0.035)Preimmune IgG (100mg/ml) 3856 14

Rat alveolar macrophages FMLP (1mM) 5306 61HBSS (buffer) 1546 1Rat RANTES (100 ng/ml) 4806 25Rat RANTES (100 ng/ml)1 anti-RANTES (100mg/ml) 1806 11 94 (p 5 0.001)Preimmune IgG (100mg/ml) 6056 59

a Chemotactic experiments employed rat PBMC or rat alveolar macrophages using procedures described inMaterials and Methods.Values represent mean6 SEM. For eachassay, five replicate samples were assessed. The data in this table are representative of data from two separate experiments.

Table II. Effects of blocking Abs on BAL content of chemokinesa

Groupb

BAL Content (pg/ml)

MIP-1b MCP-1 RANTES

Negative control1 400 mg preimmune IgG 2.756 0.35 8.656 2.12 74.0 6 4.02Positive control1 400 mg preimmune IgG 7.506 1.41 w

v46.16 1.91 w

v312 6 10.4 w

vp , 0.05 p , 0.05 p , 0.05

Positive control1 400 mg Ab IgG 0.736 0.04 wv 17.46 1.77 wv ,3 wv

a See text for details. Abs used were directed against MIP-1b, MCP-1, or RANTES.b Negative controls received 400mg preimmune IgG together with the anti-BSA, which was given intratracheally; the i.v. infusion of BSA (10 mg) was omitted. Other details

are given in the text.


blocking Ab to MIP-1a significantly reduced both lung recruit-ment of neutrophils and the increases in vascular permeability in-duced by formation of IgG immune complexes (4). These effectswere associated with marked reductions in BAL levels of TNF-a,suggesting that MIP-1a functions as an autocrine activator of al-veolar macrophages, facilitating their production of TNF-a. Thereduction of lung neutrophil recruitment observed under condi-tions of MIP-1a blockade could in part be attributed to neutral-ization of MIP-1a, which also possesses chemotactic activity forneutrophils (23). As well, reduced lung neutrophil accumulationmediated by MIP-1a blockade may be a result of reduced intrapul-monary production of TNF-a and subsequent up-regulation of vas-cular adhesion molecules. Generation of TNF-a (and IL-1) duringIgG immune complex-induced lung injury is the primary stimulusfor the up-regulation of the adhesion molecules, ICAM-1 and E-selectin, on pulmonary vascular endothelial cells (24, 25). In thismodel both ICAM-1 and E-selectin are required for lung neutro-phil recruitment (26). The current study demonstrates a similar rolefor MIP-1b in IgG immune complex-induced lung injury. UnlikeMIP-1a, MIP-1b demonstrates no chemotactic activity for neutro-phils (27). Thus, it is likely that, under conditions of MIP-1bblockade, reduced lung neutrophil recruitment is due to reducedpulmonary production of TNF-a and subsequent reduction in theup-regulation of adhesion molecules in the pulmonary vasculature.Our in vivo data contrast with previous in vitro studies that failedto demonstrate an effect of MIP-1b on macrophage production ofTNF-a (3). Together with the current findings the fact that theearlier studies used peritoneal macrophages could suggest organ-specific macrophage responses to MIP-1b.

The role of MCP-1 during acute inflammation is unclear. In IgAimmune complex-induced lung injury, blockade of MCP-1 by i.v.administration of anti-MCP-1 greatly reduced the extent of tissueinjury (7). In this model lung injury is monocyte/macrophage de-pendent, suggesting that MCP-1 may function as an activator ofpulmonary macrophages. Also, in pulmonary granulomatous vas-culitis occurring after the i.v. infusion of glucan, this inflammatoryreaction was greatly reduced by Ab to rat MCP-1 (15). Duringacute endotoxemia in mice, i.v. administration of MCP-1 reducedplasma levels of TNF-a and improved survival rates that wereassociated with increased IL-10 production (28). In the same stud-ies neutralization of MCP-1 augmented TNF-a production, re-duced IL-10 production, and decreased survival, suggesting that

MCP-1 may under special circumstances have anti-inflammatoryeffects. In the current study we show that MCP-1 is up-regulated inlung shortly after the deposition of IgG immune complexes. How-ever, blockade of MCP-1 had no effect on pulmonary neutrophilrecruitment or the extent of lung injury. Whether the biologicaleffects of MCP-1 are species specific or are specific for the type ofinflammatory condition under scrutiny remains to be determined.

Expression of RANTES mRNA is constitutive in lung (Fig. 8).Induction of lung inflammation by IgG immune complexes did notlead to measurable up-regulation of RANTES mRNA beyond theconstitutive level. RANTES demonstrates chemotactic activity formonocytes, T lymphocytes of the memory/helper phenotype, andeosinophils (11, 29). No significant chemotactic activity for neu-trophils has been detected, but RANTES has the ability to activatebasophils and cause histamine release (30). Ab to rat RANTES hascaused significant prolongation in the survival of hearts allograftedfrom Brown Norway rats into Lewis rats (13) and of hamsterhearts xenografted into rats (14). However, in the IgG immunecomplex model of lung injury described in the current studies,treatment with anti-RANTES failed to reduce lung neutrophil ac-cumulation and did not affect the increases in lung vascular per-meability. The lack of an effect of anti-RANTES on acute lunginflammation occurred despite the fact that this preparation wasalso shown to potently block the chemotactic activity of rrRAN-TES for PBMC and alveolar macrophages and to reduce BALlevels of RANTES below measurable levels. Intravenous protocolsfor anti-MCP-1 and anti-RANTES blocked the production of IgAimmune complex-induced lung injury (7) and rejection of al-lografted hearts in rats, respectively (see above). Although it ispossible that in the IgG immune complex model of lung injuryinsufficient Ab gained access to lung RANTES and MCP-1, thisseems unlikely in view of the effects of these Abs on BAL levelsof these two chemokines. Therefore, it appears that RANTES aswell as MCP-1 have no demonstrable role in the development ofacute lung inflammation induced by IgG immune complexes.

The experiments featuring the exogenous administration ofMIP-1b, RANTES, or MCP-1 (together with the anti-BSA) indi-cate that only MIP-1b enhances lung injury as measured by thechange in lung vascular extravasation of [125I]albumin (Table III).These observations correlate inversely with the blocking effects ofanti-MIP-1b on reducing parameters of inflammatory lung injury(Fig. 6), whereas anti-MCP-1 (Fig. 7) and anti-RANTES (Fig. 8)

Table III. Effects of exogenous CC chemokines on Lung Vascular Permeability

Expt. Conditionsa Mean Permeability Index Percent Changeb

A Negative controls 0.156 0.01Positive controls 0.426 0.01 w

vwvvvv

p, NS

Positive controls1 RANTES 0.476 0.07 wv 118p 5 0.02

Positive controls1 MIP-1b 0.546 0.03 wv 144

B Negative controls 0.146 0.03Positive controls 0.646 0.06 w

vp, NS

Positive controls1 MCP-1 0.636 0.04 wv 22

a Negative controls received anti-BSA IgG intratracheally in the absence of an i.v. infusion of 10 mg BSA. Positive controlsreceived anti-BSA intratracheally (1.5 mg in Expt. A and 2.5 mg in Expt. B) with or without 5.0mg recombinant CC chemokine,and 10 mg BSA i.v. The permeability index was calculated at the 4 h interval of time. For each group,n 5 4.

b Percent change was calculated by subtracting negative control values from positive control values, then determining thepercent difference between the positive control groups receiving or not receiving exogenous CC chemokine.


failed to influence the intensity of inflammatory lung injury trig-gered by intrapulmonary deposition of IgG immune complexes.Collectively, these data suggest that in this model of lung injuryonly one of the three CC chemokines participates in the pathogen-esis of lung injury.

In acute lung inflammatory injury in rats, there appears to be asomewhat late peak in the expression of mRNA for MIP-1a (18),MIP-1b and MCP-1 (this report), MIP-2 (19, 20), and CINC (20).Despite this, in the case of MIP-2 and CINC proteins, which wereprecisely quantitated by ELISA in BAL fluids, the expression ofCINC protein peaked at 2 h, while the peak for MIP-2 was between2 and 4 h. The apparent discrepancy between lung mRNA levelsand BAL chemokine content could well be due to the fact that theBAL content of chemokines probably derives from alveolar mac-rophages, while much of the mRNA measured in whole lung ex-tracts derives from the interstitial compartment, which includesmacrophages and other cell types capable of expressing chemo-kines. It should also be noted that beyond 4 h after initiation of IgGimmune complex-induced lung injury in rats, no further increasesoccur in injury (measured as [125I]albumin extravasation), in lungbuildup of neutrophils (myeloperoxidase content), and in BAL lev-els of TNF-a (20). Accordingly, the focus on changes occurringbetween 0 and 4 h seems most relevant to the pathogenesis ofevents linked to inflammatory injury in the lung.

This report as well as previously published data indicate thatsome CC chemokines, namely MIP-1a and MIP-1b, play signif-icant roles in the induction of acute inflammatory reactions in lung.The data suggest that the functions of MIP-1a and MIP-1b duringacute lung inflammation induced by IgG immune complexes maybe separate from their leukocyte chemoattractant properties. It ap-pears that both MIP-1a and MIP-1b operate as autocrine activatorsof alveolar macrophages, facilitating the acute inflammatory pro-cess. In contrast, other CC chemokines, including MCP-1 andRANTES, do not appear to have any relevant role in the develop-ment of acute lung injury, despite being constitutively expressed(RANTES) or up-regulated (MCP-1) during lung inflammation.

The pathogenesis of the lung inflammatory reactions triggeredby intrapulmonary deposition of IgG immune complexes in rats isprobably dependent on engagement of FcRs in lung macrophages.Knockout mice lacking FcgR showed profoundly depressed acuteinflammatory responses in reversed passive Arthus reactions com-pared with those of wild-type mice (31). Engagement of FcRs inthese inflammatory models would be expected to be required foractivation of tissue macrophages and their generation of a wave ofcytokines and chemokines. In the lung inflammatory reactions de-scribed in the current report, BAL macrophages showed immuno-staining for MIP-1b (data not shown), similar to our earlier studiesof MIP-2 and CINC in the same inflammatory model (20). Re-garding the role of complement in these inflammatory models,complement depletion involving reduction of serum C3 to,3% ofnormal levels or blockade of C5a with Ab profoundly suppressedthe lung-damaging inflammatory response, reduced BAL levels ofTNF-a, and greatly diminished up-regulation of vascular ICAM-1(32, 33). Why C3-deficient (knockout) mice appear to respond toimmune complex-induced alveolitis (34) is unclear, but it is pos-sible that in the absence of C3, noncomplement-derived C5-cleav-ing enzymes that are known to be abundant in neutrophils (35, 36)may replace the complement-generated C5 convertases.

AcknowledgmentsWe thank Audrey Fleming of R&D Systems for her help with the Westernblot, Robin G. Kunkel for her assistance with preparation of the illustra-tions, and Beverly Schumann and Peggy Otto for their secretarialassistance.

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