a role for b2 integrins (cd11/cd18) in the regulation of cytokine gene expression of...

A role for b2 integrins (CD11/CD18) in the regulationof cytokine gene expression of polymorphonuclearneutrophils during the inflammatory response

BARBARA WALZOG,1 PAMELA WEINMANN, FRANK JEBLONSKI,KARIN SCHARFFETTER-KOCHANEK,* KURT BOMMERT,† AND PETER GAEHTGENSDepartment of Physiology, Freie Universitat, D-14195 Berlin, Germany; *Department of Dermatology,Universitat zu Koln; and †Max Delbruck Center for Molecular Medicine, D-13122 Berlin-Buch,Germany

ABSTRACT Growing evidence supports the ideathat adhesion via b2 integrins not only allows cellulartargeting, but also induces intracellular signaling,which in turn activates functional responses of adher-ent cells. This study investigates whether b2 integrin-mediated adhesion of human polymorphonuclear neu-trophils (PMN) has a functional impact on cytokineproduction. Aggregation of the b2 integrin Mac-1(CD11b/CD18) by antibody cross-linking was found toinduce substantial de novo synthesis of IL-8 mRNA asmeasured by semiquantitative RT-PCR and Northernblotting technique, respectively. Induction of IL-8mRNA was also observed upon adhesion of PMN toimmobilized fibrinogen, a functional equivalent of itsclotting product fibrin that serves as a native ligand ofMac-1. Results were confirmed using PMN derivedfrom CD18-deficient mice, which were unable to pro-duce MIP-2 mRNA, a homologue of human IL-8, inthe presence of immobilized fibrinogen. In contrast, asubstantial increase of MIP-2 mRNA was observedwhen wild-type PMN were incubated on immobilizedfibrinogen. In human PMN, ELISA technique showedthat the gene activation that required tyrosine kinaseactivity resulted in a substantial production and secre-tion of biologically active IL-8 and IL-1b. In contrast,no TNF-a or IL-6 production was found, revealing thatb2 integrins mediate differential expression of proin-flammatory cytokines. The biological relevance of thepresent findings was confirmed in an in vivo model ofacute inflammation. Altogether, the present findingsprovide evidence for a functional link between clot-ting and inflammatory responses that may contributeto the recruitment and/or activation of PMN andother cells at sites of lesion.—Walzog, B., Weinmann,P., Jeblonski, F., Scharffetter-Kochanek, K., Bommert,K., Gaehtgens, P. A role for b2 integrins (CD11/CD18) in the regulation of cytokine gene expressionof polymorphonuclear neutrophils during the inflam-matory response. FASEB J. 13, 1855–1865 (1999)

Key Words: inflammation z adhesion z host defense z interleu-kin 8 z interleukin 1

Several cytokines serve as important mediators offunctional responses of human polymorphonuclearneutrophils (PMN).2 The chemokine interleukin 8(IL-8) is an especially potent activator of this cell typeby inducing various PMN functions, e.g., chemoc-tatic migration, exocytosis, and the respiratory burst(1). PMN have the ability to produce IL-8 as well asother cytokines by de novo mRNA synthesis in re-sponse to different soluble agonists such as thebacterial tripeptide f-Met-Leu-Phe (fMLP), the cyto-kine tumor necrosis factor a (TNF-a), or the plate-let-activating factor (PAF), respectively (2). This isthought to contribute to para- or autocrine cellactivation and may serve as a feed-forward signal forrecruitment of additional PMN to their target re-gions at sites of lesion (3).

The b2 integrin family (CD11/CD18) of leukocyteadhesion molecules plays a dominant role in thisrecruitment process. The b2 integrins CD11a/CD18(LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18(gp150/95), and CD11d/CD18, which are constitu-tively expressed on the surface of leukocytes, areheterodimers consisting of a common b subunit(CD18) and an a subunit (CD11a, CD11b, CD11c, orCD11d) (4). They bind specific ligands, which allowsPMN to localize at their target surface by mediatingfirm adhesion: after the reversible phase of selectin-mediated rolling of PMN along the activated micro-

1 Correspondence: Freie Universitat Berlin, Department ofPhysiology, Arnimallee 22, D-14195 Berlin, Germany. [email protected]

2 Abbreviations: BSA, bovine serum albumin; ELISA, en-zyme-linked immunoassay; FITC, fluorescin isothiocyanate;fMLP, N-formyl-Met-Leu-Phe; mAb, monoclonal antibody;ICAM, intercellular adhesion molecule(s); Ig, immunoglob-ulin; IL, interleukin; i.p., intraperitoneally; MIP-2, macro-phage inflammatory protein-2; PAF, platelet-activating factor;PBS, phosphate-buffered saline; PE, phycoerythrin; PMN,polymorphonuclear neutrophils; TNF, tumor necrosis factor;RT-PCR, reverse transcription-polymerase chain reaction;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis; TNF, tumor necrosis factor; WBC, white bloodcells.

18550892-6638/99/0013-1855/$02.25 © FASEB

vascular endothelium, PMN are activated by endo-thelium-derived soluble mediators such as platelet-activating factor or IL-8, which trigger the functionalup-regulation of the ligand binding activity of the b2

integrins (5). This allows the b2 integrins to effi-ciently bind their counter receptors on the microvas-cular endothelium. LFA-1 is thought to play thepivotal role in this recruitment process by binding tothe intercellular adhesion molecules 1 and 2(ICAM-1, -2), allowing firm adhesion, spreading, andsubsequent emigration of the PMN (6, 7). Mac-1 isalso known as a receptor for ICAM-1 (8), but severalreports suggest a subordinate role in PMN adhesionto endothelial cells as compared to LFA-1 (9, 10).Mac-1 serves as the predominant receptor for C3bi aswell as fibrinogen and its clotting product fibrin (11,12). gp150/95 binds C3bi and fibrinogen as well (13,14), but the physiological impact of these interac-tions seems less important due the low surfaceexpression on PMN when compared to the highlyabundant Mac-1 (15). A large body of evidence existsfor the role of Mac-1 as receptor for C3bi during hostdefense by facilitating phagocytosis of opsonizedparticles (16). In contrast, the exact physiologicalrole for the strong affinity of Mac-1 to fibrinogen orfibrin, respectively, is still not defined precisely.

Clotting and thrombus formation as a conse-quence of endothelial cell injury, for example, isknown to trap PMN and other white blood cells. ThePMN are thought to play an important role in thesubsequent inflammatory responses (17, 18). Thephysiological significance of PMN in thrombogenicprocesses is further strengthened by different mech-anisms allowing a recruitment of circulating PMNinto a forming thrombus: While adherent to fibrin,PMN (19) and activated adherent platelets (20, 21)are able to capture free-flowing PMN. Although thisallows the initial interaction of circulating PMN witha forming thrombus, further activation is required toinduce functional responses of recruited PMN.

Adhesive interactions of b2 integrins are able toinitiate intracellular signaling events and participatein the activation of various PMN functions (22–24).These effects are known to require integrin aggrega-tion, which is induced by binding to immobilized,but not soluble, ligands (25). In this context, it seemslikely that the formation of fibrin during clottingmay provide an appropriate matrix to induce theseb2 integrin-mediated outside-in signaling events,which may regulate and/or activate PMN functions.Therefore, the present study was undertaken toinvestigate whether b2 integrin-mediated interac-tions of human PMN are sufficient to activate rele-vant PMN responses. Since cytokines are good can-didates for in vivo recruitment and activation ofPMN, the effect of b2 integrin-mediated adhesion toimmobilized fibrinogen is studied on gene induction

of human IL-8, IL-6, IL-1b, and TNF-a, respectively,using semi-quantitative reverse transcription-poly-merase chain reaction (RT-PCR), Northern blottingtechnique, and enzyme linked immunoassay(ELISA) technique, respectively. The biological sig-nificance of the observed effects is analyzed, respec-tively in an in vivo model of acute inflammation inmice.

MATERIALS AND METHODS

Isolation of human PMN and culture of HL-60 cells

PMN were isolated from heparinized blood (10 I.E./ml) ofhealthy donors. After erythrocyte sedimentation in 40% (v/v)autologous plasma, the leukocyte-rich plasma was layeredonto a discontinuous Percoll gradient, as described (27), andcentrifuged at 600 3 g for 20 min. The PMN-containing bandwas collected and washed in Dulbecco’s phosphate-bufferedsaline (PBS). Cells were resuspended in PBS supplementedwith 0.25% bovine serum albumin (BSA) and 0.1% glucose.PMN viability was . 97% as assessed by the trypan blueexclusion test; purity was .98% as analyzed by microscopyusing Hemacolor staining (Merck, Darmstadt, Germany).The human leukemia cell line HL-60 (ATCC CCL 240) wasgrown in RPMI 1640 medium supplemented with 10% fetalcalf serum, 1% glutamine, and antibiotics (50 U/ml penicil-lin, 50 mg/ml streptomycin) in 5% CO2 at 37°C.

Animals

Mutant mice deficient in CD18 (28) or wild-type controlanimals of the same genetic background (mixed 129/Sv andC57BL/6J) were used. Animal experiments were subject toinstitutional approval.

Isolation of murine PMN

Bone marrow cells were harvested from tibias and femurs andincubated overnight in DMEM medium supplemented with20% fetal calf serum, 15% cell culture supernatant derivedfrom Wehi-3b cells (ATCC TIB-68), 1% glutamine, andantibiotics (50 U/ml penicillin, 50 mg/ml streptomycin) in5% CO2 at 37°C. PMN were washed and resuspended in PBSsupplemented with 0.25% BSA and 0.1% glucose. Prior toadhesion experiments, PMN were analyzed for expression ofCD18 and Gr-1, a marker of mature PMN, using flow cytom-etry.

Peritonitis model

Mice were injected intraperitoneally (i.p.) with 1 ml of 3%sterile thioglycollate. After 4 h, animals were killed by CO2inhalation and injected i.p. with 5 ml PBS. Peritoneal fluidwas collected, and emigrated white blood cells (WBC) werewashed twice with PBS. Peripheral blood was collected fromthe animals by resection of the tip of the tail prior to killing.After lysis of the erythrocytes by incubation of an aliquot (250ml) of heparinized blood for 5 min at RT with 1 ml of 0.15 MNH4CL, 1 mM KHCO3, and 0.1 mM EDTA, peripheral WBCwere washed in PBS.

1856 Vol. 13 October 1999 WALZOG ET AL.The FASEB Journal

Antibodies

The monoclonal antibody IB4 (mAb IB4) [mouse anti-hu-man CD18, immunoglobulin G2a (IgG2a) (11)] was isolatedfrom hybridoma supernatants (ATCC 10164-HB) by proteinA-Sepharose. Purity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); saturatingconcentration was determined by flow cytometry. F(ab)2fragments of IB4 were prepared by pepsin digestion, followedby protein A-Sepharose purification. The F(ab)2 preparationsof IB4 showed a uniform molecular size of ;110 kDa onSDS-PAGE under nonreducing conditions. The mAbsMHM24 (anti-human CD11a, IgG1), 2LPM19c (anti-humanCD11b, IgG1), KB90 (anti-human CD11c, IgG1), andMHM23 (anti-human CD18, IgG1) were obtained from Da-kopatts, Glostrup, Denmark. The F(ab9)2 fragments of thesecondary polyclonal goat anti-mouse IgG were purchasedfrom Sigma, Deisenhofen, Germany. The anti-human IL-8mAb (clone 4.1.3) was a generous gift from Dr. J. Baker,Genentech (San Francisco, Calif.). The phycoerythrin- (PE)labeled rat anti-mouse CD18 antibody (clone C71/16) andthe fluorescin isothiocyanate- (FITC) labeled rat anti-Gr-1antibody (clone RB6–8C5) were obtained form PharMingen(San Diego, Calif.).

Flow cytometry

Murine PMN (5 3 105/20 ml) were stained using the PE-labeled anti-mouse CD18 antibody and the FITC-labeledanti-Gr-1 antibody in a final concentration of 25 mg/ml. Afterantibody incubation for 1 h at 4°C in the dark, cells werewashed and treated with FACS lysing solution according tosupplier’s instructions (Becton Dickinson). In each sample,104 cells were counted (FACScan, Becton Dickinson) andanalyzed using CellQuest software.

Integrin aggregation and PMN adhesion

PMN (5 3 106/ml) were incubated with 10 mg/ml of theintact monoclonal anti-CD11/CD18 antibodies or theirF(ab9)2 fragments in PBS supplemented with 0.25% BSA, and0.1% glucose for 20 min at room temperature under gentlerotation. After two washes, PMN were suspended in PBS (5 3106/ml) supplemented with 0.25% BSA and 0.1% glucose.Integrin aggregation was induced by cross-linking of theprimary mAb using F(ab9)2 fragments of the secondary anti-body in a final dilution of 1:20. For positive control, PMNwere stimulated with 100 nM fMLP. For adhesion experi-ments, 500 ml aliquots of PMN (5 3 106/ml) in HEPES buffer(20 mM HEPES and 0.9% NaCl) supplemented with 0.1%(w/v) glucose were seeded onto petri dishes (2 cm diameter)coated with either human or murine fibrinogen at a finalconcentration of 250 mg/ml at 4°C overnight, followed by twoextensive washes with PBS. Adhesion was induced by theaddition of divalent cations (1.2 mM Ca21, 1 mM Mg21, 0.2mM Mn21). In the absence of divalent cations only minimaladhesion was observed (data not shown).

RT-PCR

Total RNA was isolated using the guanidine isothiocyanatemethod (29) using Trizol (Life Technologies, Eggenstein,Germany). RNA (500 ng) was transcribed into cDNA using0.5 mg oligo(dT) primers (Life Technologies) and 50 Ureverse transcriptase MMLV (Promega, Madison, Wis.). PCRamplification of human IL-8 cDNA was carried out usingspecific primers that yield a 335 bp product. (upstreamprimer: 59-GGA CAA GAG CCA GGA AGA AAC C, down-

stream primer: 59-CTT CAA AAA CTT CTC CAC AAC (TIPMOLBIOL, Berlin, Germany). For control, specific primersets for human b2-microglobulin (upstream primer: 59-CCAGCA GAG AAT GGA AAG TC, downstream primer: 59-GATGCT GCT TAC ATG TCT CG) or human GADPH (upstreamprimer: 59-GGT CGG AGT CAA CGG ATT TGG T, down-stream primer: TGT GGG CCA TGA GGT CCA CCA C) wereused that yield 300 bp and 977 bp products, respectively. PCRamplification of cDNA of murine macrophage inflammatoryprotein 2 (MIP-2), a homologue of human IL-8 (30), wascarried out using specific primers that yield a 302 bp product(upstream primer: 59-ATG GCC CCT CCC ACC TGC CG,downstream primer: 59-TCA GTT AGC CTT GCC TTT GT).For control, specific primer sets for murine GADPH (up-stream primer: 59-ATG GTG AAG GTC GGT GTG AA,downstream primer: TTA CTC CTT GGA GGC CAT GT) orb-actin (upstream primer: 59-ATG GGT CAG AAG GAC TCCTA; downstream primer: 59-CTA GAA GCA CTT GCG GTGCA) were used that yield 1001 bp and 989 bp products,respectively. PCR (25 cycles: 1 min 94°, 1 min 60°C, 1 min72°C) was performed using 1.25 U AmpliTaq DNA polymerase(Perkin Elmer, Weiterstadt, Germany). PCR products wereanalyzed by gel electrophoresis and visualized with ethidiumbromide under UV light.

Northern blot

RNA (3 mg) was separated on 1.2% agarose/1.1% formalde-hyde gels and transferred onto positively charged polyamidemembranes (Schleicher & Schuell, Dassel, Germany) over-night. IL-8 probes were generated from cloned PCR products(pBluesript SKII, Stratagene, Heidelberg, Germany) and se-quenced for control. Detection was performed using fluor-12-dUTP labeling combined with a chemiluminescence detec-tion system (Stratagene) and subsequent autoluminographyby exposure to X-ray films (XOMAT-AR, Kodak, Germany).

ELISA

Cell culture supernatants of adherent human PMN wereharvested from petri dishes, centrifuged, and immediatelyused or stored at 220°C. Cytokine concentration was ana-lyzed in duplicate using ELISA kits (KHC0082 for hIL-8,KHC0012 for hIL-1b, KHC0062 for hIL-6, and KHC3012 forhTNFa) from Biosource (Ratingen, Germany), performedaccording to supplier’s instructions.

Chemotaxis

Human PMN (2 3 106/sample) were allowed to transmigratefor 1 h at 37°C through transwell filters (6.5 mm diameter, 3mm pore size, Corning Costar, Cambridge, Mass.) in responseto cell culture supernatants derived from human PMN, whichadhered to fibrinogen for 24 h or 10 nM fMLP for positivecontrol, respectively. Transmigrated PMN were harvestedfrom the lower chamber in the presence of 5 mM EDTA andcounted under a microscope. The assay was done in dupli-cate.

Statistical analysis

Data shown represent mean 6 sd where applicable. Statisticalsignificance was determined using Student’s t test; P,0.05 wasconsidered statistically significant.

1857b2 INTEGRINS REGULATE CYTOKINE GENE EXPRESSION

Reagents

BSA, cycloheximide, human and murine fibrinogen, fMLP,pepsin A from porcine stomach mucosa, Percoll, and cyano-gen bromide-activated protein A insolubilized on SepharoseCL-4B were obtained from Sigma. Actinomycin D, herbimy-cin A, and genistein were obtained from Calbiochem (LaJolla, Calif.). Buffers were obtained from Biochrom (Berlin,Germany).

RESULTS

A role of b2 integrins (CD11/CD18) in IL-8 geneinduction

To determine whether b2 integrin (CD11/CD18)-me-diated adhesive interactions are sufficient to triggercytokine gene expression in human PMN, adhesionwas mimicked by antibody cross-linking of CD18 on thecell surface: PMN were treated with the anti-CD18 mAbIB4 or its F(ab9)2 fragments, which were subsequentlycross-linked using F(ab9)2 fragments of a secondarygoat anti-mouse IgG. The expression of IL-8 was de-tected by semiquantitative RT-PCR using specific prim-ers for IL-8 and b2 microglobulin for control, respec-tively. Prior to RT-PCR, the quality of isolated RNA wastested on a RNA check-gel. In each experiment, RNApreparations were confirmed to be negative forgenomic DNA by PCR (data not shown). Using thisapproach, evidence was obtained that aggregation ofb2 integrins induced de novo synthesis of IL-8 mRNA(Fig. 1). RT-PCR for IL-8 revealed that unstimulatedPMN express IL-8 mRNA at a low basal level. This wastrue for most experiments in which a PCR product wasdetectable after 25 cycles. Although up to 30–35 PCR

cycles were required to clearly detect IL-8 mRNA insome experiments (data not shown), IL-8 mRNA wasfound in all unstimulated samples, revealing that thisgene has some basal activity in isolated human PMN.Treatment of PMN with the anti-CD18 mAb caused aslight increase of IL-8 mRNA within 2 h after stimula-tion when compared with unstimulated cells. Uponintegrin aggregation by cross-linking, the anti-CD18mAb by F(ab9)2 fragments of a secondary antibody,IL-8 mRNA, were markedly increased within the ob-served time period. This effect was quantitatively com-parable to the effect of 100 nM fMLP, a bacterial-derived tripeptide used for positive control. ThemRNA level of the constitutively expressed housekeep-ing gene b2 microglobulin was unaffected on PMNstimulation by CD18 cross-linking, as demonstrated bysimilar amounts of the PCR products obtained. Thus,integrin aggregation was sufficient to activate the IL-8gene in human PMN.

Kinetics and specificity of b2 integrin-mediated IL-8 gene induction

The b2 integrin-mediated IL-8 gene expression wasmarkedly increased within 2 h after integrin aggre-gation and declined within 4 h after stimulation (Fig.2A). For control, expression of b2 microglobulinmRNA was detected and found to be almost unaf-fected by CD18 cross-linking during the observedperiod, demonstrating that integrin aggregation in-duced transient up-regulation of IL-8 mRNA. Tostudy the engagement of Fc receptors in the ob-served effect, integrin aggregation was induced byF(ab9)2 fragments of both the primary anti-CD18mAb IB4 and the secondary antibody (Fig. 2B). Thesubsequent increase of IL-8 mRNA expression seenby the use of F(ab9)2 fragments was similar in extentto that observed when the intact primary antibodywas used. In addition, an irrelevant isotype-matchedcontrol of the primary antibody showed no effect onIL-8 mRNA expression (data not shown). Thus, theinduction of IL-8 mRNA was transient, specific forintegrin aggregation, and independent of Fc recep-tor engagement.

Immobilized fibrinogen: a matrix that activatesPMN

Adhesion of human PMN to immobilized fibrino-gen, a native ligand of the b2 integrins CD11b/CD18and CD11c/CD18, respectively, induced an increaseof IL-8 mRNA similar to the effect observed byantibody cross-linking of CD18. This was measuredby RT-PCR (Fig. 3A) and Northern blotting tech-nique, respectively (Fig. 3B): Both methods revealeda strong increase of IL-8 mRNA within 2 h inadherent PMN as compared to unstimulated sus-

Figure 1. Aggregation of b2 integrins induces de novo synthesisof IL-8 mRNA. DNA gel electrophoresis of RT-PCR productsof IL-8 or b2 microglobulin (MG). Human PMN were stimu-lated for 2 h at 37°C with the anti-CD18 mAb IB4 alone(anti-CD18), by cross-linking of the anti-CD18 mAb usingF(ab9)2 fragments of a secondary antibody (CD18 X link), leftuntreated (control), or stimulated by 100 nM fMLP, respec-tively. M: DNA size marker (Gibco BRL). Results are repre-sentative of three independent experiments. Integrin aggre-gation by cross-linking CD18 using the anti-CD18 mAbMHM23 gave similar results (data not shown).


pended control cells within the same time period.The housekeeping gene GAPDH was used as internalstandard to prove that equal amounts of mRNA werepresent in all RT-PCR samples. Use of the Northernblotting technique confirmed the results: adhesionfor 2 h markedly increased the IL-8 mRNA level ascompared to suspended control cells. In the absenceof divalent cations, which were used to induce adhe-sion, very poor adhesion/spreading of PMN to fi-brinogen was observed, and IL-8 production wasfound to be almost absent (data not shown). How-ever, a slight up-regulation of IL-8 mRNA was detect-able in unstimulated suspended PMN after 2 h inculture when compared to freshly isolated PMN (0h), but this effect was quite small when comparedwith the adhesion-mediated induction of the IL-8gene.

Lack of gene activation in the absence of CD18

To confirm that gene activation is due to engage-ment of the b2 integrins, PMN derived from mutantmice lacking CD18, the b subunit of the b2 integrins,or wild-type control animals of the same geneticbackground were incubated in the presence of diva-lent cations on fibrinogen-coated culture dishes.Cytokine gene induction was analyzed by semiquan-titative RT-PCR of MIP-2 mRNA, a homologue ofhuman IL-8 (Fig. 4A). Within 2 h, adhesion caused amarked induction of MIP-2 mRNA in wild-type PMNwhen compared to suspended control cells. In con-trast, CD18-deficient cells failed to produce substan-tial amounts of MIP-2 mRNA when incubated onimmobilized fibrinogen. This was consistent with theobservation that CD18-deficient PMN failed to ad-

Figure 3. Induction of the IL-8 gene is mediated by PMNadhesion to immobilized fibrinogen. Human PMN wereallowed to adhere to immobilized fibrinogen for 2 h at 37°Cin the presence of 1.2 mM Ca21, 1 mM Mg21, and 0.2 mMMn21. For negative control, PMN were kept in suspension forthe times indicated. A) DNA gel electrophoresis of RT-PCRproducts of IL-8 and GAPDH, respectively. M: DNA sizemarker (Promega). B) Northern blot analysis of IL-8 mRNAexpression. As shown in the agarose gel of the 18 s rRNA,equal amounts of RNA were present in all samples. Results arerepresentative of three independent experiments.

Figure 2. De novo synthesis of IL-8 mRNA is transient andindependent of Fc receptors. DNA gel electrophoresis ofRT-PCR products of IL-8 or b2 microglobulin (MG). A)Human PMN were treated with the anti-CD18 mAb IB4 andstimulated for the times indicated at 37°C by addition ofF(ab9)2 fragments of the secondary antibody. Results arerepresentative of three independent experiments. B) HumanPMN were stimulated for 2 h at 37°C by cross-linking of CD18using the intact primary anti-CD18 mAb IB4 and F(ab9)2fragments of the secondary antibody, left untreated (control),or stimulated by cross-linking of CD18 using F(ab9)2 frag-ments of both the anti-CD18 mAb IB4 and the secondaryantibody, respectively. Results are representative of threeindependent experiments.


here to the fibrinogen matrix (data not shown).Expression of the housekeeping gene GAPDH,which was used as internal standard, demonstratedthat equal amounts of mRNA were present in allsamples. For control, PMN were analyzed by flowcytometry for expression of CD18 and Gr-1, a markerof mature neutrophils (Fig. 4B). Both wild-type andCD18-deficient PMN stained positively for Gr-1, con-firming that mature PMN were present in all exper-iments. As expected, only wild-type PMN stainedpositively for CD18, whereas it was absent on PMNderived from CD18-deficient animals. Together,these data show that adhesion and subsequent gene

activation were critically dependent on expression ofb2 integrins on the cell surface of PMN.

Mac-1 (CD11b/CD18): the predominant activatorof the IL-8 gene

To study the question of which of the three b2

integrins expressed on human PMN are able toactivate the IL-8 gene, the a subunits of LFA-1(CD11a), Mac-1 (CD11b), and gp150/95 (CD11c)were cross-linked by specific mAbs. Using the North-ern blot technique, the b2 integrin Mac-1 (CD11b/CD18) was found to mediate a strong induction ofIL-8 mRNA within 2 h after antibody cross-linking ascompared to the unstimulated control (Fig. 5). Geneinduction by CD11b cross-linking was similar inextent to the effect of CD18 engagement, suggestingthat Mac-1 was the predominant activator of the IL-8gene. In contrast, cross-linking of CD11c led only toa slight induction of the IL-8 gene, and engagementof CD11a had no detectable effect. For negativecontrol, IL-8 mRNA expression was also measured insuspended HL-60 cells without further stimulationand found to be absent. Thus, Mac-1 seems torepresent the dominant activator of cytokine geneexpression in human PMN.

Adhesion-mediated production and secretion ofIL-8

To investigate whether the observed up-regulation ofIL-8 mRNA may have physiological significance, in-duction of IL-8 was studied at the protein level. Inthese experiments, ELISA technique was used tomeasure the IL-8 peptide in the supernatant ofhuman PMN that adhered to immobilized fibrino-gen (Fig. 6). Some basal secretion of IL-8 was ob-served in unstimulated suspended PMN, which in-

Figure 5. Mac-1 (CD11b/CD18) cross-linking mimicks theeffect of PMN adhesion. Northern blot analysis of IL-8expression in PMN and HL-60 cells. The a subunits of LFA-1(CD11a), Mac-1 (CD11b), and gp150/95 (CD11c) were cross-linked on PMN using specific mAbs for 2 h at 37°C. Fornegative control, PMN (control) or HL-60 were left un-treated. For positive control, PMN were stimulated by 100 nMfMLP. As shown in the agarose gel of the 18 s rRNA, equalamounts of RNA were present in all samples. Results arerepresentative of three independent experiments.

Figure 4. A) Lack of gene activation in the absence of CD18.Murine PMN obtained from CD18-deficient or wild-typecontrol animals were incubated on immobilized fibrinogenfor 2 h at 37°C in the presence of 1.2 mM Ca21, 1 mM Mg21,and 0.2 mM Mn21. For negative control, PMN were kept insuspension for 2 h. DNA gel electrophoresis of RT-PCRproducts of MIP-2 and GAPDH is shown. M: 100 bp DNA sizemarker (Promega). B) Flow cytometric analysis of murinePMN obtained from CD18-deficient or wild-type controlanimals, respectively. PMN were stained for expression ofGr-1 and CD18. For negative control, PMN were stained withPE- or FITC-labeled, isotype-matched control antibodies (dot-ted line). Results are representative of three independentexperiments.


creased from ;12 pg/ml to 195 pg/ml within 24 hafter stimulation. However, a strong enhancement ofIL-8 secretion was detected in adherent PMN: within24 h after the onset of adhesion, IL-8 secretionincreased 42-fold to ;8000 pg/ml PMN when com-pared to unstimulated control cells. Adhesion-medi-ated IL-8 production was even greater than the effectinduced by 100 nM fMLP, which resulted in IL-8secretion of ;3600 pg/ml after 24 h of stimulation.Thus, adhesion was more efficient than the solublemediator fMLP and showed a faster kinetic: adher-ent PMN had already produced ;4600 pg/ml of IL-8in the supernatant within 6 h after the onset ofadhesion, whereas fMLP-induced IL-8 productionwas only ;1000 pg/ml within this period. For com-parison, the supernatant of unstimulated suspendedcontrol cells contained ;100 pg/ml of IL-8 within6 h. In further experiments, the effect of costimula-tion by adhesion and simultaneous fMLP applicationwas investigated and found to be simply additive(data not shown).

Requirement for de novo mRNA and proteinsynthesis

To confirm that adhesion-induced secretion of IL-8required both de novo mRNA as well as proteinsynthesis, the effect of actinomycin D and cyclohex-imide was studied on adhesion-induced IL-8 secre-tion (Fig. 7). Both inhibition of RNA synthesis byactinomycin D and inhibition of protein synthesis bycycloheximide resulted in almost complete inhibi-tion of IL-8 secretion. Thus, secreted IL-8 was

formed de novo by activation of the IL-8 gene. Tofurther understand the intracellular mechanismsthat underlie integrin-mediated gene regulation, theeffect of tyrosine kinase inhibitors was investigated.Treatment of PMN with herbimycin A, a potentinhibitor of tyrosine kinases, abolished IL-8 produc-tion almost completely (Fig. 7). Similar results wereobtained when using genistein for inhibition oftyrosine kinases (data not shown). Thus, the inte-grin-mediated cytokine production was dependenton intracellular tyrosine signaling.

Chemotactic migration of PMN by adhesion-mediated IL-8 production

Next we tested whether the secreted entity showedthe characteristic biological activity of IL-8 by elicit-ing chemotactic migration (Fig. 8). Human PMNwere allowed to transmigrate through transwell fil-ters in response to supernatants derived from humanPMN, which exerted adhesive interactions to immo-bilized fibrinogen for 24 h. These supernatants,which contained on average 8 ng/ml IL-8 as deter-mined by ELISA technique (see Fig. 6), inducedchemotactic migration of 22% of total PMN added.This was 63% of the effect induced by 10 nM fMLP,which resulted in ;35% transmigrating cells. Neu-tralization of IL-8 by a monoclonal antibody inhib-ited 60% of the effect induced by the supernatantderived from adherent PMN and resulted in 9%transmigrating cells, showing that the major entitythat exhibited chemotactic activity was indeed IL-8.However, the incomplete inhibition of chemotaxisby the anti-IL-8 mAb suggests that adherent PMN

Figure 7. b2 integrin-mediated IL-8 secretion requires de novomRNA and protein synthesis. PMN were left untreated (con-trol) or treated with 10 mM cycloheximide (CHX), 10 mMactinomycin D (Act. D), or 30 mM herbimycin A (herbi) for30 min at 37°C prior to adhesion to fibrinogen in thepresence of 1.2 mM Ca21, 1 mM Mg21, and 0.2 mM Mn21 for24 h at 37°C. IL-8 was measured in the supernatant of PMN byELISA technique. n 5 4, mean 6 sd, *P,0.05 vs. control.

Figure 6. b2 integrin-mediated adhesion induces secretion ofIL-8. PMN were kept in suspension and left unstimulated(control), stimulated in suspension with 100 nM fMLP, orallowed to adhere to fibrinogen (adhesion), respectively, andincubated for the times indicated at 37°C in the presence of1.2 mM Ca21, 1 mM Mg21, and 0.2 mM Mn21. IL-8 wasmeasured in the supernatant of PMN by ELISA technique. n5 8, mean 6 sd, *P,0.05 vs. unstimulated control.


may generate additional chemotactic factors besidesIL-8. Addition of the antibody also induced a slightbut not statistically significant reduction of the fMLP-induced response.

Differential cytokine expression by b2 integrin-mediated adhesion

To determine whether b2 integrin-mediated generegulation was unique for IL-8 or common to otherproinflammatory cytokine genes in human PMN, theeffect of adhesion was investigated on productionand secretion of IL-1b, IL-6, and TNF-a, respectively.Using ELISA techniques, no significant productionof IL-6 was detectable on adhesion to immobilizedfibrinogen or challenge with 100 nM fMLP within aperiod of 0 h to 24 h (Fig. 9). Similarly, TNF-a wasbelow the detection limit of 10 pg/ml in all experi-ments (data not shown). In contrast, adhesion tofibrinogen induced a substantial production of IL-1bwithin 24 h whereas 100 nM fMLP was unable toaffect IL-1b gene expression during this same pe-riod. IL-1b secretion was abolished when PMN weretreated with cycloheximide or actinomycin D, re-spectively (data not shown). Thus, b2 integrin-medi-ated adhesion caused differential expression of cyto-kine genes in human PMN.

Cytokine gene induction in vivo

To study the biological relevance of the observedeffects, an vivo model of inflammation was used.

Four hours after induction of an acute peritonitis inmice by i.p. injection of 1 ml of 3% thioglycollate,extravasated leukocytes were harvested from theperitoneal cavity and subjected to RT-PCR analysis.As shown in Fig. 10, the MIP-2 mRNA level wasprofoundly up-regulated upon emigration of WBC inresponse to thioglycollate injection when comparedto peripheral WBC or bone marrow cells of the sameanimal. Levels of the constitutively expressed house-keeping gene b-actin analyzed for control were al-most identical, indicating that similar amounts ofmRNA were present in all samples. This demon-strates that leukocyte extravasation in acute inflam-mation is associated with a substantial up-regulationof the MIP-2 gene in mice.

DISCUSSION

In the present study, a novel b2 integrin-mediatedmechanism is reported that triggers the de novo

Figure 9. b2 integrin-mediated adhesion induces productionand secretion of IL-1b but not of IL-6 or TNF-a. PMN werekept in suspension and left unstimulated (control), stimu-lated in suspension with 100 nM fMLP, or allowed to adhereto fibrinogen (adhesion), respectively, and incubated for thetimes indicated at 37°C in the presence of 1.2 mM Ca21, 1mM Mg21, and 0.2 mM Mn21. IL-1b, IL-6, and TNF-a weremeasured in the supernatant of PMN by ELISA technique. n5 5, mean 6 sd, *P,0.05 vs. control. TNF-a was belowdetection limit in all experiments (data not shown).

Figure 8. Adhesion-mediated IL-8 production elicits chemo-tactic migration of PMN. PMN (2 .106/sample) were allowedto transmigrate through transwell filters for 1 h at 37°C inresponse to vehicle (control), 10 nM fMLP, or cell culturesupernatants derived from PMN, which adhered to fibrino-gen for 24 h (supernatant). Transmigration was measured inthe presence or absence of an anti-IL-8 mAb. Transmigratedcells were harvested and counted under the microscope.Chemotaxis was calculated as transmigrated cells in percentof total cell number. n 5 4, mean 6 sd, *P,0.05 vs. control.


synthesis of IL-8 mRNA in human PMN. The effectwas maximal after aggregation of the b2 integrinsinduced by antibody cross-linking of the anti-CD18mAb by F(ab9)2 fragments of a secondary antibody.Binding of the anti-CD18 mAb alone induced only aslight increase in IL-8 mRNA, which agrees withprevious reports demonstrating that efficient inte-grin signaling requires integrin aggregation (25).Although PMN express different types of Fc recep-tors that trigger intracellular signal transductionevents on ligand binding (31), their ligand interac-tion was not required for integrin-mediated cytokinegene induction, as shown by the use of F(ab9)2

fragments of both the primary and secondary anti-bodies for integrin aggregation. The physiologicalsignificance of the observed effect was strengthenedby the fact that PMN adhering to immobilized fibrin-ogen, a matrix that is functionally equivalent tofibrin, elicited the same response. This demonstratesthat b2 integrin-mediated adhesion to a native ma-trix suffices to induce gene activation in humanPMN. The most abundant b2 integrin, Mac-1(CD11b/CD18) (15), which serves as ligand forfibrinogen and fibrin, was confirmed to be responsi-ble for gene induction by antibody cross-linking ofthe a subunits. However, cross-linking of CD11c alsoresulted in a small increase of IL-8 mRNA, suggestingthat gp150/95 is also able to trigger gene activationupon ligand binding. Due to its low surface expres-sion (15), the physiological significance of this effectis probably less important than the induction medi-ated by Mac-1. Since LFA-1 was unable to inducegene activation, Mac-1 seems to represent the dom-inant signaling molecule in human PMN among thethree b2 integrins tested. However, the putative role

CD11d/CD18, the fourth b2 integrin (32), was notinvestigated.

To exert its signaling capacity, Mac-1 requiredaggregation by antibody cross-linking or adhesion toan immobilized ligand, conditions known to causeclustering of the integrins (25, 33). Although thesignal transduction events triggered on b2 integrinclustering remain to be identified, the use of CD18-deficient PMN revealed that engagement of b2 inte-grins was critically required for the adhesion-medi-ated signaling that allowed gene activation. Previousreports presented evidence that b2 integrins partici-pate in the induction of PMN functions, e.g., therespiratory burst, actin polymerization, or degranu-lation (23, 24). The present findings show that b2

integrin engagement not only contributes to thecontrol of short term PMN functions occurringwithin minutes on adhesion, but also affects geneexpression in human PMN. Although PMN are ca-pable to produce all cytokines tested by de novosynthesis (2), only production of IL-8 and IL-1b wasinduced on b2 integrin-mediated adhesion, whereasno IL-6 or TNF-a synthesis was observed. Thus, theb2 integrins induce differential expression of proin-flammatory cytokines. Gene induction was found todepend strongly on intracellular tyrosine signaling.Therefore, proteins that have previously been shownto become tyrosine phosphorylated on b2 integrin-mediated adhesion are good candidates to mediatethis gene activation, e.g., the guanine nucleotideexchange factor vav or the src-kinase family memberp58fgr (26, 34). However, further investigations re-garding adhesion-dependent gene regulation inPMN will need to show whether the signal transduc-tion cascade that is activated on b2 integrin-mediatedadhesion is unique to cytokine genes. Indirect evi-dence that b2 integrins are involved in PMN apopto-sis (35, 36), a process that probably depends on generegulation (37), may support the idea that not onlycytokine genes are regulated on PMN adhesion.

The finding that adhesion to an appropriate ma-trix—namely, fibrin or immobilized fibrinogen—induces production and secretion of the proinflam-matory cytokines IL-8 and IL-1 supports the conceptthat clotting is functionally linked to inflammatoryresponses. During severe inflammation, which isaccompanied with exudation of plasma, clotting canalso occur in the extravascular space, thereby provid-ing an appropriate matrix for b2 integrin-mediatedadhesion of emigrated PMN. Although the lifetimeof mature PMN is short when compared to otherleukocyte population, this cell type has been un-equivocally shown to produce cytokines by de novoRNA and protein synthesis (3). The estimated half-time mature PMN spend in the circulation is ;4 h(38), but the life span of PMN is much longer andlasts ;24 h (37, 39). Moreover, the lifetime of PMN

Figure 10. Leukocyte extravasation is associated with cytokineinduction. White blood cells of wild-type mice were obtainedfrom the peripheral blood (peripheral WBC), from the bonemarrow, or from the peritoneal cavity (emigrated WBC) 4 hafter induction of peritonitis by i.p. injection of 3% thiogly-collate. DNA gel electrophoresis of RT-PCR products ofMIP-2 and b-actin is shown. M: fX174 DNA/HaeIII sizemarker (Promega). Results are representative of three inde-pendent experiments.


is further extended during systemic inflammation byinhibition of apoptosis (40) via cytokines such asgranulocyte-macrophage colony-stimulating factor,etc. (41). Thus, PMN may spend a considerableperiod of time in the tissue. Accordingly, inductionof an acute peritonitis in mice resulted in up-regula-tion of the MIP-2 mRNA in emigrated leukocytes,demonstrating that up-regulation of cytokine genesis not only inducible in vitro, but also occurs in vivowithin several hours after the onset of inflammation.During this period, the majority of emigrating leu-kocytes represents PMN (42).

The observed induction of the proinflammatorycytokines IL-8 and IL-1 in human PMN maystrengthen the inflammatory reaction, since IL-1 iswell known to induce up-regulation of adhesionmolecules and secretion of proinflammatory media-tors by endothelial cells, which both support leuko-cyte emigration during inflammation (43, 44). IL-8 isa potent chemokine for PMN and may thereforecontribute to the chemotactic recruitment of addi-tional PMN to sites of lesion (3). Moreover, IL-1 isknown to affect hemostasis by induction of tissuefactor in monocytes and endothelial cells (45, 46), aswell as wound healing by promoting proliferation offibroblasts (47). Tissue remodeling may be furthersupported by IL-8, a CXC chemokine, which not onlyattracts PMN, but also bears an ELR-motif (Glu-Leu-Arg) and plays a functional role in angiogenesis byinducing neovascularization (48). Thus, the ob-served cytokine induction may not only play a role inacute host defense by strengthening the inflamma-tory response, but may also play a role in activatingthe reorganization cascade elicited on lesion. This isconsistent with the observation that patients suffer-ing from leukocyte adhesion deficiency type I, aninherited defect of the CD18 gene, not only fail toelicit an inflammatory response, but also show im-paired wound healing (49, 50), supporting the ideaof a link between hemostasis, inflammation, tissueremodeling, and subsequent wound repair. Takentogether, the present data show that b2 integrin-mediated adhesion induces the transient activationof the IL-8 and the IL-1b gene, predominantly viaMac-1 (CD11b/CD18), the major ligand of fibrino-gen, and its clotting product, fibrin. This geneactivation, which depends on tyrosine kinase-medi-ated intracellular signaling, results in substantialproduction and secretion of biologically active cyto-kines. Due to the pattern of cytokines secreted, thisresponse may be able to support inflammation andcontribute to the control of hemostasis as well as tothe induction of tissue remodeling and wound heal-ing processes, respectively.

Supported by Deutsche Forschungsgemeinschaft (SFB366/C3 and B3).

REFERENCES

1. Baggiolini, M., and Clark-Lewis, I. (1992) Interleukin-8, a che-motactic and inflammatory cytokine. FEBS Lett. 307, 97–101

2. Cassatella, M. A. (1995) The production of cytokines by poly-morphonuclear neutrophils. Immunol. Today 16, 21–26

3. Cassatella, M. A., Bazzoni, F., Ceska, M., Ferro, I., Baggiolini, M.,and Berton, G. (1992) IL-8 production by human polymorpho-nuclear leukocytes: the chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release ofIL-8 through a pertussis toxin-sensitive pathway. J. Immunol. 148,3216–3220

4. Arnaout, M. A. (1990) Structure and function of the leukocyteadhesion molecules CD11/CD18. Blood 75, 1037–1050

5. Diamond, M. S., and Springer, T. A. (1994) The dynamicregulation of integrin adhesiveness. Curr. Biol. 4, 506–507

6. Dustin, M. L., and Springer, T. A. (1988) Lymphocyte functionassociated antigen-1 (LFA-1) interaction with intercellular ad-hesion molecule-1 (ICAM-1) is one of at least three mechanismsfor lymphocyte adhesion to cultured endothelial cells. J. CellBiol. 107, 321–331

7. de Fougerolles, A. R., Stacker, S. A., Schwarting, R., andSpringer, T. A. (1991) Characterization of ICAM-2 and evidencefor a third counter-receptor for LFA-1. J. Exp. Med. 174, 253–267

8. Diamond, M. S., Staunton, D. E., de Fougerolles, A. R., Stacker,S. A., Garcia-Aguilar, J., Hibbs, M. L., and Springer, T. A. (1990)ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).J. Cell Biol. 111, 3129–3139

9. Lu, H., Smith, C., W., Perrard, J., Bullard, D., Tang, L., Shappell,S. B., and Entman, M. L. (1997) LFA-1 is sufficient in mediatingneutrophil emigration in Mac-1-deficient mice. J. Clin. Invest. 99,1340–1350

10. Lub, M., van Kooyk, Y., and Figdor, C. G. (1996) Competitionbetween lymphocyte function-associated antigen 1 (CD11a/CD18) and Mac-1 (CD11b/CD18) for binding to intercellularadhesion molecule-1 (CD54). J. Leukoc. Biol. 59, 648–655

11. Wright, S. D., Rao, P. E., Van Voorhis, W. C., Craigmyle, L. S.,Iida, K., Talle, M. A., Westberg, E. F., Goldstein, G. W., andSilverstein, S. C. (1983) Identification of the C3bi receptor ofhuman monocytes and macrophages by using monoclonalantibodies. Proc. Natl. Acad. Sci. USA 80, 5699–5703

12. Wright, S. D., Weitz, J. I., Huang, A. J., Levin, S. M., Silverstein,S. C., and Loike, J. D. (1988) Complement receptor type 3(CD11b/CD18) of human polymorphonuclear leukocytes rec-ognizes fibrinogen. Proc. Natl. Acad. Sci. USA 85, 7734–7738

13. Loike, J. D., Sodeik, B., Cao, L., Leukona, S., Weitz, J., Detmers,P. A., Wright, S. D., and Silverstein, S. C. (1991) CD11c/CD18recognizes a domain at the N-terminus of the A-alpha chain offibrinogen. Proc. Natl. Acad. Sci. USA 88, 1044–1048

14. Bilsland, C. A. G., Diamond, M. S., and Springer, T. A. (1994)The leukocyte integrin p150,95 (CD11c/CD18) as a receptorfor iC3b: activation by a heterologous b subunit and localizationof a ligand recognition site to the I domain. J. Immunol. 152,4582–4589

15. Arnaout, M. A., Spits, H., Terhorst, C., Pitt, J., and Todd, R. F.(1984) Deficiency of a leukocyte surface glycoprotein (LFA-1) intwo patients with Mo1 deficiency. Effects of cell activation onMo1/LFA-1 surface expression in normal and deficient leuko-cytes. J. Clin. Invest. 74, 1291–1300

16. Arnaout, M. A., Todd, R. F., Dana, N., Melamed, J., andSchlossman, S. F. (1983) Inhibition of phagocytosis of comple-ment C3- or immunoglobulin G-coated particles and of iC3bibinding by monoclonal antibodies to a monocyte-granulocytemembrane glycoprotein (Mo1). J. Clin. Invest. 72, 171–177

17. Marcus, A. J. (1994) Thrombosis and inflammation as multicel-lular processes: significance of cell-cell interaction. Semin. Hema-tol. 31, 261–269

18. Dosquet, C., Weill, D., and Wautier, J.-L. (1995) Cytokines andthrombosis. J. Cardiovasc. Pharmacol. 25, 13–19

19. Kuijper, P. H. M., Torres, H. I. G., van der Linden, J. A. M.,Lammers, J.-W. J., Sixma, J. J., Zwaginga, J. J., and Koenderman,L. (1997) Neutrophil adhesion to fibrinogen and fibrin underflow conditions is diminished by activation and L-selectin shed-ding. Blood 89, 2131–2138

20. Weber, C., and Springer, T. A. (1997) Neutrophil accumulationon activated, surface-adherent platelets in flow is mediated by


interaction of Mac-1 with fibrinogen bound to alphaIIbbeta3and stimulated by platelet-activating factor. J. Clin. Invest. 100,2085–2093

21. Diacovo, T. G., Roth, S. J., Buccola, J. M., Bainton, D. F., andSpringer, T. A. (1996) Neutrophil rolling, arrest, and transmi-gration across activated, surface-adherent platelets via sequen-tial action of P-selectin and the b2 integrin CD11b/CD18. Blood88, 146–157

22. Walzog, B., Offermanns, S., Zakrzewicz, A., Gaehtgens, P., andLey, K. (1996) b2 integrins mediate protein tyrosine phosphor-ylation in human neutrophils. J. Leukoc. Biol. 59, 747–753

23. Nathan, C., Srimal, S., Farber, C., Sanchez, E., Kabbash, L.,Asch, A., Gailit, J., and Wright, S. D. (1989) Cytokine-inducedrespiratory burst of human neutrophils: Dependence on extra-cellular matrix proteins and CD11/CD18 integrins. J. Cell Biol.109, 1341–1349

24. Walzog, B., Seifert, R., Zakrzewicz, A., Gaehtgens, P., and Ley, K.(1994) Cross-linking of CD18 in human neutrophils induces anincrease of intracellular free Ca21, exocytosis of azurophilicgranules, quantitative up-regulation of CD18, shedding of L-selectin, and actin polymerization. J. Leukoc. Biol. 56, 625–635

25. Miyamoto, S., Akiyama, S. K., and Yamada, K. M. (1995)Synergistic roles for receptor occupancy and aggregation inintegrin transmembrane function. Science 267, 883–885

26. Zheng, L., Sjolander, A., Eckerdal, J., and Andersson, T. (1996)Antibody-induced engagement of b2 integrins on adherenthuman neutrophils triggers activation of p21ras through tyrosinephosphorylation of the protooncogene product Vav. Proc. Natl.Acad. Sci. USA 93, 8431–8436

27. Hjorth, R., Jonsson, A. K., and Vretblad, P. (1981) A rapidmethod for purification of human granulocytes using Percoll. Acomparison with dextran sulfate. J. Immunol. Methods 43, 95–101

28. Scharffetter-Kochanek, K., Lu, H., Norman, K., van Nood, N.,Munoz, F., Grabbe, S., McArthur, M., Lorenzo, I., Kaplan, S.,Ley, K., et al. (1998) Spontaneous skin ulceration and defectiveT cell function in CD18 null mice. J. Exp. Med. 188, 119–131

29. Chomczynski, P., and Sacchini, N. (1987) Single-step method ofRNA isolation by acid guanidinium-phenol-chloroform extrac-tion. Anal. Biochem. 162, 156–159

30. Tecamp-Olson, P., Gallegos, C., Bauer, D., McClain, J., Sherry, B.,Fabre, M., van Deventer, S., and Cerami, A. (1990) Cloning andcharacterization of cDNAs for murine macrophage inflammatoryprotein 2 and its human homologues. J. Exp. Med. 172, 911–919

31. van de Winkel, J. G. J., and Capel, P. J. A. (1993) Human IgG Fcreceptor heterogeneity: molecular aspects and clinical implica-tions. Immunol. Today 14, 215–221

32. Danilenko, D. M., Rossitto, P. V., Van der Vieren, M., Trong,H. L., McDonough, S. P., Affolter, V. K., and Moore, P. F. (1995)A novel canine leukointegrin, alpha d beta 2, is expressed byspecific macrophage subpopulations in tissue and a minorCD81 lymphocyte subpopulation in peripheral blood. J. Immu-nol. 155, 35–44

33. Kornberg, L. J., Earp, H. S., Turner, C. E., Prockop, C., andJuliano, R. L. (1991) Signal transduction by integrins: Increasedprotein tyrosine phosphorylation caused by clustering of beta-1integrins. Proc. Natl. Acad. Sci. USA 88, 8392–8396

34. Berton, G., Fumagalli, L., Laudanna, C., and Sorio, C. (1994) b2integrin-dependent protein tyrosine phosphorylation and acti-vation of the FGR protein tyrosine kinase in human neutrophils.J. Cell Biol. 126, 1111–1121

35. Coxon, A., Rieu, P., Barkalow, F. J., Askari, S., Sharpe, A. H., vonAndrian, U. H., Arnaout, M. A., and Mayadas, T. N. (1996) A

novel role for the b2 integrin CD11b/CD18 in neutrophilapoptosis: a homeostatic mechanism in inflammation. Immunity5, 653–666

36. Walzog, B., Jeblonski, F., Zakrzewicz, A., and Gaehtgens, P.(1997) b2 integrins (CD11/CD18) promote apoptosis of humanneutrophils. FASEB J. 11, 1177–1186

37. Payne, C. M., Glasser, L., Tischler, M. E., Wyckoff, D., Cromey,D., Fiederlein, R., and Bohnert, O. (1994) Programmed celldeath of the normal human neutrophil: an in vitro model ofsenescence. Microsc. Res. Tech. 28, 327–344

38. Nakagava, M., Terashima, T., Dachkova, Y., Bondy, G. P., Hogg,J. C., and Van Eeden, S. F. (1998) Glucocorticoid-inducedgranulocytosis: contribution of marrow release and demargin-ation of intravascular granulocytes. Circulation 98, 2307–2313

39. Walzog, B., Weinmann, P., and Gaehtgens, P. (1999) Bcl-Xl- andBax-a-mediated regulation of apoptosis of human neutrophilsvia caspase-3. Blood 93, 3106–3115

40. Chitinis, D., Dickerson, C., Munster, A. M., and Winchurch,R. A. (1996) Inhibition of apoptosis in polymorphonuclearneutrophils from burn patients. J. Leukoc. Biol. 59, 835–839

41. Cox, G., Gauldie, J., and Jordana, M. (1992) Bronchial epithelialcell-derived cytokines (G-CSF and GM-CSF) promote the sur-vival of peripheral blood neutrophils in vitro. Am. J. Resp. CellMol. Biol. 7, 507–513

42. Walzog, B., Weinmann, P., Jeblonski, F., Scharffetter-Kochanek, K., Bommert, K., and Gaehtgens, P. (1999) Im-pairment of neutrophil emigration in CD18 null mice. Am. J.Physiol. 276, G1125–G1130

43. Collins, T., Read, M. A., Neish, A. S., Whitley, M. Z., Thanos, D.,and Maniatis, T. (1995) Transcriptional regulation of endothe-lial cell adhesion molecules: NF-kappa B and cytokine inducibleenhancers. FASEB J. 9, 899–909

44. Mantovani, A., Bussolino, F., and Dejana, E. (1992) Cytokineregulation of endothelial cell function. FASEB J. 6, 2591–2599

45. Schwager, I., and Jungi, T. W. (1994) Effect of human recom-binant cytokines on the induction of macrophage procoagulantactivity. Blood 83, 152–160

46. Nawroth, P. P., Handley, D. A., Esmon, C. T., and Stern, D. M.(1986) Interleukin-1 induces endothelial cell procoagulantwhile suppressing cell-surface anticoagulant activity. Proc. Natl.Acad. Sci. USA 83, 3460–3464

47. Raines, E. W., Dower, S. K., and Ross, R. (1989) Interleukin-1mitogenic activity for fibroblasts and smooth muscle cells is dueto PDGF-AA. Science 243, 393–396

48. Strieter, R. M., Polverini, P. J., Kunkel, S. L., Arenberg, D. A.,Burdick, M. D., Kasper, J., Dzuiba, J., Van Damme, J., Walz, A.,Mariott, D., et al. (1995) The functional role of the ELR motifin CXC chemokine-mediated angiogenesis. J. Biol. Chem. 270,27248–27357

49. Bowen, T. J., Ochs, H. D., Altman, L. C., Price, T. H., Van Epps,D. E., Brautigan, D. L., Rosin, R. E., Perkins, W. D., Babior,B. M., Klebanoff, S. J., et al. (1982) Severe recurrent bacterialinfections associated with defective adherence and chemotaxisin two patients with neutrophils deficient in a cell-associatedglycoprotein. J. Pediatr. 101, 932–940

50. Hawkins, E. P., Heffelfinger, S. C., and Anderson, D. C. (1992)Leukocyte adhesion deficiency: clinical and postmortem obser-vations. Pediatric Pathol. 12, 119–130

Received for publication November 6, 1998.Revised for publication May 11, 1999.


a role for b2 integrins (cd11/cd18) in the regulation of cytokine gene expression of...

Documents