Rat KC cDNA cloning and mRNA expression in lung macrophages and fibroblasts

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RAT KC cDNA CLONING AND mRNA EXPRESSION IN LUNG MACROPHAGES FIBROBLASTS Songlih Huangl, Joseph D. Paulauskislpf,

AND

and Lester Kobzikl~*~(

‘Department of Environmental Health, Harvard School of Public Health Boston, Massachusetts 02115 *Department of Pathology, Brigham and Women’s Hospital Boston, Massachusetts 02115 Received

March

13,

1992

We have isolated and sequenced overlapping cDNA clones for rat KC* The 0.93 kb cDNA has a single open reading frame of 288 nucleotides, and substantial sequence identity with the platelet-factor 4 family members mouse KC, hamster gro, and human gro. Using cloned cDNA as a probe, expression of KC mRNA in lavaged rat alveolar macrophages (AMs) increased after lipopolysaccharide (LPS) treatment. We also studied expression in vitro by a rat fetal lung fibroblast cell line, RFL-6. Expression of KC mRNA in RFL-6 cells increased after treatment with interleukin 1 or with PXSS,Inc. conditioned medium from rat AMs treated with LPS. 0 199?Acadsmlc

Cytokines produced at the focus of inflammation may attract and/or activate neutrophils. Members of the platelet factor-4 (PF-4) family include cytokines implicated as powerful neutrophil chemoattractants and activators. The prototype and best characterized member of this family is human interleukin (IL)-8, as reviewed by Leonard and Yoshimura (1). Other members of the PF-4 family, including neutrophilactivating peptide- (2), MGSAIgro protein (3) macrophage inflammatory protein-2 (4) and a protein secreted by rat glomerular cells, reported as cytokine-induced neutrophil chemoattractant (CINC) (5) also have neutrophil chemotactic/activating properties.

*Sequence data from this article GenBank Libraries under Accession

have been deposited No. M86536.

with

the

EYBL/

t Parker B. Francis Fellow in Pulmonary Research 1 To whom correspondence should be addressed at Respiratory Biology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Abbreviations; AM, alveolar macrophage; bp, base pairs; IL, interleukin; CINC, cytokine-induced neutrophil chemoattractant; CM, conditioned medium; M-MuLV, Moloney murine leukemia virus; PDGF, platelet-derived growth factor; LPS, lipopolysaccharide; MGSA, melanoma growth stimulatory activity; NAP, neutrophil activating protein; PF-4, platelet factor-4; TNF, tumor necrosis factor.

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The peptide sequence

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of CINC showed substantial

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identity with another PF-4 family

member, the mouse KC gene product, which was first identified as an early gene in BalbfC 3T3 fibroblasts stimulated by platelet-derived growth factor (6). Studies in animal models of inflammation

will be needed to define the role of

PF-4 cytokines in viva. The rat offers convenient and relevant models of acute and chronic inflammation in the lungs. We report here the cDNA cloning and full-length sequence of rat KC. This cDNA was used to investigate KC gene expression pulmonary cells in response to inflammatory

in rat

stimuli.

METHODS Cell isolation and culture: Rat alveolar macrophages (AMs) were collected by sterile bronchoalveolar lavage with pyrogen-free saline (Abbott Laboratories, North Chicago, IL). Yields and viability were determined by hemocytometer counts of aliquots diluted in trypan blue solution. Cells were cultured in RPM1 1640 (Sigma Chemical, St. Louis, MO), with 0.1% bovine serum albumin at 0.6 x 1O6 cells/ml. Bacterial lipopolysaccharide (Escherichia co/i, Sigma Chemical) was added at a final concentration of 10 ug/ml. Cells were cultured at 37°C in 5% CO2 for 4 h. RFL-6 rat fetal lung fibroblast cells were obtained from American Type Culture Collection (Rockville, MD). The rat AM cell line, NR8383, was generously provided by Dr. R. Helmke (7). RFLS cells were grown to confluence in RPM1 1640 with 10% fetal calf serum prior to experiments, and in treatment groups recombinant human IL-l a or IL-l p (Genzyme Corp., Boston, MA) was added to a final concentration of 10 rig/ml. The conditioned medium of NR8383 cells or lavaged rat AMs was collected after culturing NR8383 cells or lavaged rat AMs at 6 x 10s cells/l00 mm polystyrene dish, in RPM1 1640 alone or with 10 ug/ml LPS for 4 or 24 h,. .as indicated. . PNA punflcatron : Total cell RNA was isolated from rat AMs and RFL-6 cells using a modified guanidinium method (8). Cells were washed 2 X with DEPC-treated PBS and lysed in 4 M guanidine thiocyanateI25 mM Na Citrate (pH 7.0)/0.5% N-lauroylsarcosine/O.l M 2-mercaptoethanol. The mixture was layered on 5.7 M CsClIO.1 M Na2EDTA and centrifuged at 47,000 rpm (268,000 X g), 4 h, in a Beckman SW55 Ti rotor. Pelleted RNA was resuspended in DEPC-treated TE (10 mM Tris/l mM EDTA, pH 7.4) buffer. Rat AM cDNA librarv construction: Total RNA from 40 x 10s rat AMs stimulated with 10 ug/ml LPS for 4 h was purified and poly(A)+ RNA isolated by oligo(dT)-cellulose chromatography (Collaborative Research, Inc., Lexington, MA). Five ug of poly (A)+ RNA was used for cDNA library construction by a modification of the Gubler and Hoffman method (9) in the k ZAP II vector (Stratagene, La Jolla, CA). Briefly, mRNA was reverse-transcribed by M-MuLV reverse transcriptase using a poly T linker-primer containing an Xho I restriction sequence in a reaction buffer containing 5-methyl dCTP. The second strand was synthesized with RNase H and DNA polymerase I. Double-stranded cDNA was resolved by agarose gel electrophoresis and cDNA larger than 500 bp electroeluted. Following ligation of an EcoRl adaptor, cDNA was directionally cloned into h ZAP II phagemid and packaged (Gigapak II Gold, Stratagene). Approximately 3 x lo6 independent clones were obtained with an average insert size of 820 bp. Sof A set of oligonucleotides were synthesized corresponding to sequences in the protein coding region of mouse KC cDNA (10). The 5’ primer was 5’-GCCA(G/A)TGA(G/A)CTGCGCTGTCA(G/A)TGC-3’ ( bases 83-l 06) and the 3’ primer was S-CTTGGGGACACCTTTTAGCATCTT-3’ ( bases 266-289). Total RNA, isolated from LPS-stimulated rat AMs, was reverse transcribed by M-MuLV 923

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reverse trancriptase (GIBCO BRL, Gaithersburg, MD) at 37°C for 90 min. The cDNA was subjected to 30 cycles of amplification with 2.5 U AmpliTaq polymerase (PerkinElmer Corp., Norwalk, CT) in 25 mM Tris-HCI (pH 8.3) 50 mM KCI, 2.5 mM MgCl*, 0.01% gelatin, 5 mM DlT, and 1 mM each of both 5’ and 3’ primers. Each cycle consisted of denaturation, 94”C/l min, annealing, 55”C/2 min, and extension, iYC/3 min. The PCR product was electrophoresed in 1.2% low-melting-point agarose (GIBCO BRL), and the resulting cDNA of expected size excised and purified on a PrimeErase column (Stratagene). For sequencing, the purified product was cloned into pCR1000 plasmid (Invitrogen, San Diego, CA), and sequenced by the dideoxy nucleotide chain termination method (1 l), using Sequenase (United States Biochemical Corp., Cleveland, OH). Sequencing primers corresponded to T7 promoter and Ml 3-forward sequences in pCR1000. The PCR product was labeled with random primer labeling (GIBCO BRL) and [32P]-dATP with the addition of 0.1 ug of the 3’ PCR primer in the labeling reaction. Librarv screenina: Approximately 10,000 recombinant phages were plated on each of five 15 mm petri dishes, and then blotted onto nitrocellulose filters (Millipore, Bedford, MA). The filters were prehybridized in 6 X SSC/O.O5% SDS&X Denhardt’s solution, and 100 ug/ml salmon sperm DNA at 65°C for 3 h, then hybridized with the radiolabeled PCR product in 6X SSC/O.5% SDS at 65°C for 16 h. Filters were washed in 3X SSC/ 0.2% SDS 25’C/15 min, 1X SSC/O.2% SDS 37”C/15 min, 1X SSC/O.2% SDS 50°C/15 min, and 0.2X SSC/O.2% SDS 65”C/25 min before autoradiography. After secondary screening, positive phages were resuspended in SM buffer (85 mM NaCI, 8 mM MgS04, 50 mM Tris, pH 7.5, 0.01% gelatin). Five ul of this buffer for 10 independent clones was boiled for 5 min, and subjected to PCR (same conditions as above) with primers corresponding to T3 and T7 sequences on pBluescript. The PCR products having the largest insert sizes (-1 .l kb) from two independent clones, were purified by PrimeErase column, subcloned into the pCR1000 vector and sequenced as described using universal primers or a series of synthesized 17-mer oligonucleotide primers. . . . : Total RNA from rat AMs or RFLS cells (10 ug/lane) Northern blottina was denatured, resolved in 0.8% agarose-formaldehyde gels, transferred to Nytran filters (Schleicher & Schuell, Keene, NH), and UV crosslinked to the membrane. The quantity of RNA loaded in each lane was comparable as evaluated by ethidium bromide staining of ribosomal RNA. We have previously determined that the mRNA transcript size for KC is approximately 1.3 kb (Huang et al., submitted). The -1 .l kb PCR product was purified as above and random primer labeled for use in Northern blot analysis. Prehybridization and hybridization were carried out in 0.5 M NaP04/1 mM EDTA/7% SDS/150 ug/ml tRNA (12) at 65°C. Blots were washed 2 x 15 min each in 0.1 X SSC/l% SDS at 37°C and 52°C before autoradiography.

RESULTS Using the rat KC RT-PCR product to screen the rat AM cDNA library, approximately 300 positive plaques were identified from 50,000 plaques screened. Two independent clones were selected for further study and sequenced in both directions. The first clone (KC-l) is almost full-length, missing the first 6 bases of the 5’ untranslated region, identified in the second clone (KC-2). Cloned KC-2 consisted of bases -51 to 454. The cDNA (Fig. 1) is 0.93 kb in length. Relative to the first ATG, nucleotides at positions -3 to -1 are ACC, and position +4 is G, conforming to the eukaryotic consensus sequence for translation initiation (13). Rat KC has 51 nucleotides in the 5’ untranslated region, 288 in translated region, and 590 in the 3’ untranslated region. The deduced protein has 96 amino acids (MW = 10.3 kD) with 4 924

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A CCTAAACCAGCTCCAGCACTCCAGACTCCAGCCACACTCCAACAGAGCACC

-51

1 I 3 52 18

97 33

ATG GTC met val

TCA GCC ACC CGC TCG CTT CTC TGT GCA GCG CTG CCT GTG CTG GCC ser ala thr arg ser leu leu cys ala ala leu pro val leu ala ACC AGC CGC CAA GCC ACA GGG GCG CCC thr ser arg gln ala thr gly ala pro t AGT CAG TGC CTG CAG ACA GTG GCA GGG cys gln cys leu gln thr val ala gly

GTC GCC AAT val ala asn

GAG CTG CGC glu leu arg

ATT CAC TTC AAG AAC ATC ile his phe lys gln ile

142 48

CAG AGT TTG AAG GTG ATG CCG CCA GGA CCC CAC TGC ACC CAA ACC gln ser leu lys val met pro pro gly pro his cys thr gln thr

187 63

GAA GTC ATA GCC ACA CTC AAG AAT GGT CGC GAG GCT TGC CTT GAC glu val ile ala thr leu lys asn gly arg glu ala cys leu asp

232 78

CCT GAA GCC CCC ATG GTT CAG AAG ATT GTC CAA AAG ATG CTA AAG pro glu ala pro met val gln lys ile val gin lys met leu lys

271

GGT GTC CCC AAG TAA TGGAGAAAGAAGATAGATTGCACCGATGGCGTCTGTCTG gly val pro lys ***

93 331

390 449 508 567 626 685 144 803 862

GTGAACGCTGGCTTCTGACACACTAGTTTTACACATTTTACGATTTCTATTGAGGGTC CT-TTTTATGTTCCACCAAGTGTGTGGTTTTTATTTTACATTAAT ATTTAACGATGTGGATGCGTTTCATCGATGGTCGTTCAATA GATGGTAGGCGTTAAATATCTCGTTAAATTAATATTTATTGGGAGACCATTAGGTGTCA ACCACTGTGCTAGAAGGTGTTGAGCGGGAAGAAGGGGCGGAGAGATGAGAGTCTGGGATC GTGTTTTGTGTTAGGGTGAGGAATGTGTGTGAGAGGCTATGTTGTATGTTTTGA~G~TG TTATTTATTGAAAGTTGTCTTTCATATTTTATGGTCAACATTGATGTGTTGAAGCTTCC CTTGGACATTTTATGTCTAGTTTGTAGGGCACAATGCCTTTG CTCCTTCTCGTCTCAGGACAGAG~GTTCAAAGGACTGTAA =I$GTTTTATTAAAAAATA(,,

B 1 1 1 1 27

27 31 37 63 63

67 73

MVS----ATRSL-.IP----.....--....----.LL.....L..... .AP----.....--.R.pLLLL.LL.....L..... .ARAALS.AP.

LCAA----LPVLATSRQATGAP NPR.LRVALLL.LLV.AG.R.A..S

VANELRCQCLQTVAGIHFKQIQSLKVMPPGPHCTQT 1...........f.,....L.N......L.S.,..... . . . . . . . . . . ..MT.V.L.N.E....T......... ..T......,..LQ...P.N...VN.KS.....A.. EVIATLKNGREACLDPEAPMVQKIVQKMLK-GVPK . . . . . . . . . . . . . . . . . . . . . . . . ..Q....)J...............~.~R. . . . . . . . . ..K...N.AS.I.K..IE...NSDKSN

RAT MOUSE HAMSTER HUMAN RAT MOUSE HAMSTER HUMAN

..L..........-....

Fiq. 1. (A) Nucleotide and deduced amino acid sequence of rat KC cDNA. The determination of the translation initiation site and signal peptide cleavage site (arrow) is described in the text. ATTTA and TTAllTAT sequences characteristic of many cytokine cDNAs are underlined and the two putative polyadenylation signals AATAAA are double underlined. (B) Comparison of peptide sequence of rat KC, mouse KC, hamster gro, and human gro. Amino acids identical to the rat counterpart are represented by dots. Aligned to reveal homology; dash (-) represents assigned spacing of amino acid.

925

RAT MOUSE HAMSTER HUMAN

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cysteines at positions characteristic

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of the PF-4 family. It also contains a hydrophobic

signal sequence and the rule of von Heijne (14) predicts the cleavage site after glycine 24. The deduced mature protein has 72 amino acid residues and is identical to the previously reported partial sequence for CINC protein (5). The mature protein has 90% identity to mouse KC (lo), 83% identity to hamster gro, and 65% identity to the human gro gene product (15). Six repeats of the mRNA instability ATTTA sequence

found in the 3’ untranslated

(16) were

region, and two of them are part of the TTATTTAT motif

(17). When the nucleotide sequence of the translated region was compared to all GenBank sequences by the program BLAST (18) the sequences identified and percent identity were 92% to mouse KC, 88% to hamster gro, 77% to human gro, and 81% to mouse macrophage

inflammatory

protein-e.

A DNA probe prepared from KC-l was used to study the expression mRNA in rat AMs and fibroblasts. LPS (Fig. 2). The size difference

of KC

Expression of KC in rat AMs is markedly induced by between KC cDNA insert and mRNA estimated from

Northern blot analysis is most likely the result of polyadenylation and estimate error. Hybridization of RNA from LPS-stimulated hamster AMs with KC-l also detected a transcript size of 1.3 kb (not shown). These results are comparable

to the reported size

of mouse KC (1.2 kb) (6).

1.3

C

kb

LPS

Northern blot analysis of rat alveolar macrophage (AM) total RNA probed with RNA (10 pg/lane) was electrophoresed on 0.8% agarose under denaturing conditions. Compared to AMs in control medium (C), LPS stimulation (10 pg/ml, 4 h) caused a marked increase in KC mRNA expression. Fia. KC-l.

926

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lh

4h

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16h

-1.3

kb

-1.3

0 4

123

kb

45

Fig. 3. Northern blot analysis of KC mRNA expression in RFL-6 ceils treated with recombinant human IL-1 czor IL-l p for times indicated. Cells in control medium (C) did not express significant levels of KC mRNA. Treating RFL-6 with IL-l a or IL-l p caused increased expression of KC that was detectable as early as 1 h and lasted for at least 16 h, the last time point examined. Fig. Northern blot analysis of KC mRNA expression in RFL-6 cells treated with various conditioned media (CM): Lane 1, control medium RPM1 1640; lane 2, 3, CM of rat alveolar macrophages treated with LPS (10 g/ml) for 4 and 24 h, respectively; lane 45 CM of unstimulated NR8383 and NR l 383 cells stimulated with LPS for 4 h, respectively.

The expression

of KC mRNA in the rat fetal lung fibroblast cell line, RFL-6, was

also studied. KC mRNA was not detectable in unstimulated

RFL-6 cells. Recombinant

human IL-la and IL-lp at 10 rig/ml induced a rapid and marked increase in its expression

(Fig. 3), which was detected at all three time points examined

(1 h, 4h, and

16h). The presence of 10% fetal bovine serum in the culture medium enhanced

the

stimulatory effect of IL-l (not shown). To examine the possible effect of alveolar macrophages

on lung fibroblasts,

medium from LPS-stimulated

RFL-6 cells were cultured in diluted conditioned

rat AMs or the rat pulmonary alveolar macrophage

cell

line NR8383 (7). LPS-stimulated AM and NR8383 cells secreted factors to the medium that enhanced the expression of KC (Fig. 4). Treating RFL-6 cells with LPS (10 ug/ml) alone caused weak but detectable

expression

of KC (not shown).

DISCUSSION These results predict the cDNA sequence of rat KC and also demonstrate substantial

increases in KC mRNA expression

by rat AMs and fibroblasts

upon

exposure to inflammatory mediators in vitro. The KC gene product is likely to have strong chemotactic activity for neutrophils based on the: 1) marked chemoattraction cause by rat CINC protein, identified as rat KC by our cDNA data and available protein 927

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sequence (5, 19); 2) the potent neutrophil chemotactic activity (3) of the human counterpart of rodent KC, the gro gene, independently identified as MGSA (20) and NAP-3 (21). The interaction of structural cells and inflammatory cells has been the focus of recent investigations, which have indicated that epithelial cells, fibroblasts, and endothelial cells all participate actively in the process of inflammation. Although human monocytes and macrophages produce IL-8 in response to LPS, epithelial cells (22) and fibroblasts do not (23). It was postulated that IL-l and/or TNF secreted by mononuclear inflammatory cells are necessary for fibroblasts to produce IL-8 in vivo. In contrast, KC expression in RFL-6 cells and Balb/C 3T3 fibroblasts (24) can both be enhanced by LPS stimulation. However, it is clear that the expression of KC is one of the pathways through which rat AMs can regulate the inflammatory process in the lung. KC and JE were identified as early genes transcriptionally activated by PDGF (6) but unlike other competent genes that encode factors involved in cell division, the JE product is a macrophage chemotactic protein, MCP-1 (25) and the KC product turned out to be a neutrophil chemotactic protein. The coexpression of JE, KC and the early competence genes involved in cell division such as c-fos demonstrated the close relationship between inflammation and cell proliferation. However, since the human gro gene has growth stimulatory activities for melanoma cells and normal melonocytes (26) it is possible that KC may also participate directly in cell proliferation on yet unidentified target cells, or in a more intricate manner involving collaboration with other growth factors.

ACKNOWLEDGMENTS We thank Michele Sullivan, Marshall Katler, and Amy Colby for technical support. This work is supported by NIH HL-01761, HL-19170, and NIEHS-0002.

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