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(The FASEB Journal. 1999;13:1855-1865.)
© 1999 FASEB

A role for ß2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response

BARBARA WALZOG1, PAMELA WEINMANN, FRANK JEBLONSKI, KARIN SCHARFFETTER-KOCHANEK*, KURT BOMMERT{dagger} and PETER GAEHTGENS

Department of Physiology, Freie Universität, D-14195 Berlin, Germany;
* Department of Dermatology, Universität zu Köln; and
{dagger} Max Delbrück Center for Molecular Medicine, D-13122 Berlin-Buch, Germany

1Correspondence: Freie Universität Berlin, Department of Physiology, Arnimallee 22, D-14195 Berlin, Germany. E-mail walzog{at}zedat.fu-berlin.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growing evidence supports the idea that adhesion via ß2 integrins not only allows cellular targeting, but also induces intracellular signaling, which in turn activates functional responses of adherent cells. This study investigates whether ß2 integrin-mediated adhesion of human polymorphonuclear neutrophils (PMN) has a functional impact on cytokine production. Aggregation of the ß2 integrin Mac-1 (CD11b/CD18) by antibody cross-linking was found to induce substantial de novo synthesis of IL-8 mRNA as measured by semiquantitative RT-PCR and Northern blotting technique, respectively. Induction of IL-8 mRNA was also observed upon adhesion of PMN to immobilized fibrinogen, a functional equivalent of its clotting product fibrin that serves as a native ligand of Mac-1. Results were confirmed using PMN derived from CD18-deficient mice, which were unable to produce MIP-2 mRNA, a homologue of human IL-8, in the presence of immobilized fibrinogen. In contrast, a substantial increase of MIP-2 mRNA was observed when wild-type PMN were incubated on immobilized fibrinogen. In human PMN, ELISA technique showed that the gene activation that required tyrosine kinase activity resulted in a substantial production and secretion of biologically active IL-8 and IL-1ß. In contrast, no TNF-{alpha} or IL-6 production was found, revealing that ß2 integrins mediate differential expression of proinflammatory cytokines. The biological relevance of the present findings was confirmed in an in vivo model of acute inflammation. Altogether, the present findings provide evidence for a functional link between clotting and inflammatory responses that may contribute to the recruitment and/or activation of PMN and other cells at sites of lesion.—Walzog, B., Weinmann, P., Jeblonski, F., Scharffetter-Kochanek, K., Bommert, K., Gaehtgens, P. A role for ß2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response.


Key Words: inflammation • adhesion • host defense • interleukin 8 • interleukin 1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEVERAL CYTOKINES SERVE as important mediators of functional responses of human polymorphonuclear neutrophils (PMN).2 The chemokine interleukin 8 (IL-8) is an especially potent activator of this cell type by inducing various PMN functions, e.g., chemoctatic migration, exocytosis, and the respiratory burst (1) . PMN have the ability to produce IL-8 as well as other cytokines by de novo mRNA synthesis in response to different soluble agonists such as the bacterial tripeptide f-Met-Leu-Phe (fMLP), the cytokine tumor necrosis factor {alpha} (TNF-{alpha}), or the platelet-activating factor (PAF), respectively (2) . This is thought to contribute to para- or autocrine cell activation and may serve as a feed-forward signal for recruitment of additional PMN to their target regions at sites of lesion (3) .

The ß2 integrin family (CD11/CD18) of leukocyte adhesion molecules plays a dominant role in this recruitment process. The ß2 integrins CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (gp150/95), and CD11d/CD18, which are constitutively expressed on the surface of leukocytes, are heterodimers consisting of a common ß subunit (CD18) and an {alpha} subunit (CD11a, CD11b, CD11c, or CD11d) (4) . They bind specific ligands, which allows PMN to localize at their target surface by mediating firm adhesion: after the reversible phase of selectin-mediated rolling of PMN along the activated microvascular endothelium, PMN are activated by endothelium-derived soluble mediators such as platelet-activating factor or IL-8, which trigger the functional up-regulation of the ligand binding activity of the ß2 integrins (5) . This allows the ß2 integrins to efficiently bind their counter receptors on the microvascular endothelium. LFA-1 is thought to play the pivotal role in this recruitment process by binding to the intercellular adhesion molecules 1 and 2 (ICAM-1, -2), allowing firm adhesion, spreading, and subsequent emigration of the PMN (6 , 7) . Mac-1 is also known as a receptor for ICAM-1 (8) , but several reports suggest a subordinate role in PMN adhesion to endothelial cells as compared to LFA-1 (9 , 10) . Mac-1 serves as the predominant receptor for C3bi as well 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 interactions seems less important due the low surface expression on PMN when compared to the highly abundant Mac-1 (15) . A large body of evidence exists for the role of Mac-1 as receptor for C3bi during host defense by facilitating phagocytosis of opsonized particles (16) . In contrast, the exact physiological role for the strong affinity of Mac-1 to fibrinogen or fibrin, respectively, is still not defined precisely.

Clotting and thrombus formation as a consequence of endothelial cell injury, for example, is known to trap PMN and other white blood cells. The PMN are thought to play an important role in the subsequent inflammatory responses (17 , 18) . The physiological significance of PMN in thrombogenic processes is further strengthened by different mechanisms allowing a recruitment of circulating PMN into a forming thrombus: While adherent to fibrin, PMN (19) and activated adherent platelets (20 , 21) are able to capture free-flowing PMN. Although this allows the initial interaction of circulating PMN with a forming thrombus, further activation is required to induce functional responses of recruited PMN.

Adhesive interactions of ß2 integrins are able to initiate intracellular signaling events and participate in the activation of various PMN functions (22 23 24) . These effects are known to require integrin aggregation, which is induced by binding to immobilized, but not soluble, ligands (25) . In this context, it seems likely that the formation of fibrin during clotting may provide an appropriate matrix to induce these ß2 integrin-mediated outside-in signaling events, which may regulate and/or activate PMN functions. Therefore, the present study was undertaken to investigate whether ß2 integrin-mediated interactions of human PMN are sufficient to activate relevant PMN responses. Since cytokines are good candidates for in vivo recruitment and activation of PMN, the effect of ß2 integrin-mediated adhesion to immobilized fibrinogen is studied on gene induction of human IL-8, IL-6, IL-1ß, and TNF-{alpha}, respectively, using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting technique, and enzyme linked immunoassay (ELISA) technique, respectively. The biological significance of the observed effects is analyzed, respectively in an in vivo model of acute inflammation in mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of human PMN and culture of HL-60 cells
PMN were isolated from heparinized blood (10 I.E./ml) of healthy donors. After erythrocyte sedimentation in 40% (v/v) autologous plasma, the leukocyte-rich plasma was layered onto a discontinuous Percoll gradient, as described (27) , and centrifuged at 600 x g for 20 min. The PMN-containing band was collected and washed in Dulbecco's phosphate-buffered saline (PBS). Cells were resuspended in PBS supplemented with 0.25% bovine serum albumin (BSA) and 0.1% glucose. PMN viability was > 97% as assessed by the trypan blue exclusion test; purity was >98% as analyzed by microscopy using Hemacolor staining (Merck, Darmstadt, Germany). The human leukemia cell line HL-60 (ATCC CCL 240) was grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% glutamine, and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin) in 5% CO2 at 37°C.

Animals
Mutant mice deficient in CD18 (28) or wild-type control animals of the same genetic background (mixed 129/Sv and C57BL/6J) were used. Animal experiments were subject to institutional approval.

Isolation of murine PMN
Bone marrow cells were harvested from tibias and femurs and incubated overnight in DMEM medium supplemented with 20% fetal calf serum, 15% cell culture supernatant derived from Wehi-3b cells (ATCC TIB-68), 1% glutamine, and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin) in 5% CO2 at 37°C. PMN were washed and resuspended in PBS supplemented with 0.25% BSA and 0.1% glucose. Prior to adhesion experiments, PMN were analyzed for expression of CD18 and Gr-1, a marker of mature PMN, using flow cytometry.

Peritonitis model
Mice were injected intraperitoneally (i.p.) with 1 ml of 3% sterile thioglycollate. After 4 h, animals were killed by CO2 inhalation and injected i.p. with 5 ml PBS. Peritoneal fluid was collected, and emigrated white blood cells (WBC) were washed twice with PBS. Peripheral blood was collected from the animals by resection of the tip of the tail prior to killing. After lysis of the erythrocytes by incubation of an aliquot (250 µl) of heparinized blood for 5 min at RT with 1 ml of 0.15 M NH4CL, 1 mM KHCO3, and 0.1 mM EDTA, peripheral WBC were washed in PBS.

Antibodies
The monoclonal antibody IB4 (mAb IB4) [mouse anti-human CD18, immunoglobulin G2a (IgG2a) (11) ] was isolated from hybridoma supernatants (ATCC 10164-HB) by protein A-Sepharose. Purity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); saturating concentration was determined by flow cytometry. F(ab)2 fragments of IB4 were prepared by pepsin digestion, followed by protein A-Sepharose purification. The F(ab)2 preparations of IB4 showed a uniform molecular size of ~110 kDa on SDS-PAGE under nonreducing conditions. The mAbs MHM24 (anti-human CD11a, IgG1), 2LPM19c (anti-human CD11b, IgG1), KB90 (anti-human CD11c, IgG1), and MHM23 (anti-human CD18, IgG1) were obtained from Dakopatts, Glostrup, Denmark. The F(ab')2 fragments of the secondary polyclonal goat anti-mouse IgG were purchased from Sigma, Deisenhofen, Germany. The anti-human IL-8 mAb (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) and the fluorescin isothiocyanate- (FITC) labeled rat anti-Gr-1 antibody (clone RB6–8C5) were obtained form PharMingen (San Diego, Calif.).

Flow cytometry
Murine PMN (5 x 105/20 µl) were stained using the PE-labeled anti-mouse CD18 antibody and the FITC-labeled anti-Gr-1 antibody in a final concentration of 25 µg/ml. After antibody incubation for 1 h at 4°C in the dark, cells were washed and treated with FACS lysing solution according to supplier's instructions (Becton Dickinson). In each sample, 104 cells were counted (FACScan, Becton Dickinson) and analyzed using CellQuest software.

Integrin aggregation and PMN adhesion
PMN (5 x 106/ml) were incubated with 10 µg/ml of the intact monoclonal anti-CD11/CD18 antibodies or their F(ab')2 fragments in PBS supplemented with 0.25% BSA, and 0.1% glucose for 20 min at room temperature under gentle rotation. After two washes, PMN were suspended in PBS (5 x 106/ml) supplemented with 0.25% BSA and 0.1% glucose. Integrin aggregation was induced by cross-linking of the primary mAb using F(ab')2 fragments of the secondary antibody in a final dilution of 1:20. For positive control, PMN were stimulated with 100 nM fMLP. For adhesion experiments, 500 µl aliquots of PMN (5 x 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 final concentration of 250 µg/ml at 4°C overnight, followed by two extensive washes with PBS. Adhesion was induced by the addition of divalent cations (1.2 mM Ca2+, 1 mM Mg2+, 0.2 mM Mn2+). In the absence of divalent cations only minimal adhesion was observed (data not shown).

RT-PCR
Total RNA was isolated using the guanidine isothiocyanate method (29) using Trizol (Life Technologies, Eggenstein, Germany). RNA (500 ng) was transcribed into cDNA using 0.5 µg oligo(dT) primers (Life Technologies) and 50 U reverse transcriptase MMLV (Promega, Madison, Wis.). PCR amplification of human IL-8 cDNA was carried out using specific primers that yield a 335 bp product. (upstream primer: 5'-GGA CAA GAG CCA GGA AGA AAC C, downstream primer: 5'-CTT CAA AAA CTT CTC CAC AAC (TIP MOLBIOL, Berlin, Germany). For control, specific primer sets for human ß2-microglobulin (upstream primer: 5'-CCA GCA GAG AAT GGA AAG TC, downstream primer: 5'-GAT GCT GCT TAC ATG TCT CG) or human GADPH (upstream primer: 5'-GGT CGG AGT CAA CGG ATT TGG T, downstream primer: TGT GGG CCA TGA GGT CCA CCA C) were used that yield 300 bp and 977 bp products, respectively. PCR amplification of cDNA of murine macrophage inflammatory protein 2 (MIP-2), a homologue of human IL-8 (30) , was carried out using specific primers that yield a 302 bp product (upstream primer: 5'-ATG GCC CCT CCC ACC TGC CG, downstream primer: 5'-TCA GTT AGC CTT GCC TTT GT). For control, specific primer sets for murine GADPH (upstream primer: 5'-ATG GTG AAG GTC GGT GTG AA, downstream primer: TTA CTC CTT GGA GGC CAT GT) or ß-actin (upstream primer: 5'-ATG GGT CAG AAG GAC TCC TA; downstream primer: 5'-CTA GAA GCA CTT GCG GTG CA) were used that yield 1001 bp and 989 bp products, respectively. PCR (25 cycles: 1 min 94°, 1 min 60°C, 1 min 72°C) was performed using 1.25 U AmpliTaq DNA polymerase (Perkin Elmer, Weiterstadt, Germany). PCR products were analyzed by gel electrophoresis and visualized with ethidium bromide under UV light.

Northern blot
RNA (3 µg) was separated on 1.2% agarose/1.1% formaldehyde gels and transferred onto positively charged polyamide membranes (Schleicher & Schuell, Dassel, Germany) overnight. IL-8 probes were generated from cloned PCR products (pBluesript SKII, Stratagene, Heidelberg, Germany) and sequenced for control. Detection was performed using fluor-12-dUTP labeling combined with a chemiluminescence detection system (Stratagene) and subsequent autoluminography by exposure to X-ray films (XOMAT-AR, Kodak, Germany).

ELISA
Cell culture supernatants of adherent human PMN were harvested from petri dishes, centrifuged, and immediately used or stored at -20°C. Cytokine concentration was analyzed in duplicate using ELISA kits (KHC0082 for hIL-8, KHC0012 for hIL-1ß, KHC0062 for hIL-6, and KHC3012 for hTNF{alpha}) from Biosource (Ratingen, Germany), performed according to supplier's instructions.

Chemotaxis
Human PMN (2 x 106/sample) were allowed to transmigrate for 1 h at 37°C through transwell filters (6.5 mm diameter, 3 µm pore size, Corning Costar, Cambridge, Mass.) in response to cell culture supernatants derived from human PMN, which adhered to fibrinogen for 24 h or 10 nM fMLP for positive control, respectively. Transmigrated PMN were harvested from the lower chamber in the presence of 5 mM EDTA and counted under a microscope. The assay was done in duplicate.

Statistical analysis
Data shown represent mean ± SD where applicable. Statistical significance was determined using Student's t test; P<0.05 was considered statistically significant.

Reagents
BSA, cycloheximide, human and murine fibrinogen, fMLP, pepsin A from porcine stomach mucosa, Percoll, and cyanogen bromide-activated protein A insolubilized on Sepharose CL-4B were obtained from Sigma. Actinomycin D, herbimycin A, and genistein were obtained from Calbiochem (La Jolla, Calif.). Buffers were obtained from Biochrom (Berlin, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A role of ß2 integrins (CD11/CD18) in IL-8 gene induction
To determine whether ß2 integrin (CD11/CD18)-mediated adhesive interactions are sufficient to trigger cytokine gene expression in human PMN, adhesion was mimicked by antibody cross-linking of CD18 on the cell surface: PMN were treated with the anti-CD18 mAb IB4 or its F(ab')2 fragments, which were subsequently cross-linked using F(ab')2 fragments of a secondary goat anti-mouse IgG. The expression of IL-8 was detected by semiquantitative RT-PCR using specific primers for IL-8 and ß2 microglobulin for control, respectively. Prior to RT-PCR, the quality of isolated RNA was tested on a RNA check-gel. In each experiment, RNA preparations were confirmed to be negative for genomic DNA by PCR (data not shown). Using this approach, evidence was obtained that aggregation of ß2 integrins induced de novo synthesis of IL-8 mRNA (Fig. 1 ). RT-PCR for IL-8 revealed that unstimulated PMN express IL-8 mRNA at a low basal level. This was true for most experiments in which a PCR product was detectable after 25 cycles. Although up to 30–35 PCR cycles were required to clearly detect IL-8 mRNA in some experiments (data not shown), IL-8 mRNA was found in all unstimulated samples, revealing that this gene has some basal activity in isolated human PMN. Treatment of PMN with the anti-CD18 mAb caused a slight increase of IL-8 mRNA within 2 h after stimulation when compared with unstimulated cells. Upon integrin aggregation by cross-linking, the anti-CD18 mAb by F(ab')2 fragments of a secondary antibody, IL-8 mRNA, were markedly increased within the observed time period. This effect was quantitatively comparable to the effect of 100 nM fMLP, a bacterial-derived tripeptide used for positive control. The mRNA level of the constitutively expressed housekeeping gene ß2 microglobulin was unaffected on PMN stimulation by CD18 cross-linking, as demonstrated by similar amounts of the PCR products obtained. Thus, integrin aggregation was sufficient to activate the IL-8 gene in human PMN.



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  		Figure 1. Aggregation of ß2 integrins induces de novo synthesis of IL-8 mRNA. DNA gel electrophoresis of RT-PCR products of IL-8 or ß2 microglobulin (MG). Human PMN were stimulated for 2 h at 37°C with the anti-CD18 mAb IB4 alone (anti-CD18), by cross-linking of the anti-CD18 mAb using F(ab')2 fragments of a secondary antibody (CD18 X link), left untreated (control), or stimulated by 100 nM fMLP, respectively. M: DNA size marker (Gibco BRL). Results are representative of three independent experiments. Integrin aggregation by cross-linking CD18 using the anti-CD18 mAb MHM23 gave similar results (data not shown).

Kinetics and specificity of ß2 integrin-mediated IL-8 gene induction
The ß2 integrin-mediated IL-8 gene expression was markedly increased within 2 h after integrin aggregation and declined within 4 h after stimulation (Fig. 2 A). For control, expression of ß2 microglobulin mRNA was detected and found to be almost unaffected by CD18 cross-linking during the observed period, demonstrating that integrin aggregation induced transient up-regulation of IL-8 mRNA. To study the engagement of Fc receptors in the observed effect, integrin aggregation was induced by F(ab')2 fragments of both the primary anti-CD18 mAb IB4 and the secondary antibody (Fig. 2B). The subsequent increase of IL-8 mRNA expression seen by the use of F(ab')2 fragments was similar in extent to that observed when the intact primary antibody was used. In addition, an irrelevant isotype-matched control of the primary antibody showed no effect on IL-8 mRNA expression (data not shown). Thus, the induction of IL-8 mRNA was transient, specific for integrin aggregation, and independent of Fc receptor engagement.



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  		Figure 2. De novo synthesis of IL-8 mRNA is transient and independent of Fc receptors. DNA gel electrophoresis of RT-PCR products of IL-8 or ß2 microglobulin (MG). A) Human PMN were treated with the anti-CD18 mAb IB4 and stimulated for the times indicated at 37°C by addition of F(ab')2 fragments of the secondary antibody. Results are representative of three independent experiments. B) Human PMN were stimulated for 2 h at 37°C by cross-linking of CD18 using the intact primary anti-CD18 mAb IB4 and F(ab')2 fragments of the secondary antibody, left untreated (control), or stimulated by cross-linking of CD18 using F(ab')2 fragments of both the anti-CD18 mAb IB4 and the secondary antibody, respectively. Results are representative of three independent experiments.

Immobilized fibrinogen: a matrix that activates PMN
Adhesion of human PMN to immobilized fibrinogen, a native ligand of the ß2 integrins CD11b/CD18 and CD11c/CD18, respectively, induced an increase of IL-8 mRNA similar to the effect observed by antibody cross-linking of CD18. This was measured by RT-PCR (Fig. 3 A) and Northern blotting technique, respectively (Fig. 3B ): Both methods revealed a strong increase of IL-8 mRNA within 2 h in adherent PMN as compared to unstimulated suspended control cells within the same time period. The housekeeping gene GAPDH was used as internal standard to prove that equal amounts of mRNA were present in all RT-PCR samples. Use of the Northern blotting technique confirmed the results: adhesion for 2 h markedly increased the IL-8 mRNA level as compared to suspended control cells. In the absence of divalent cations, which were used to induce adhesion, very poor adhesion/spreading of PMN to fibrinogen was observed, and IL-8 production was found to be almost absent (data not shown). However, a slight up-regulation of IL-8 mRNA was detectable in unstimulated suspended PMN after 2 h in culture when compared to freshly isolated PMN (0 h), but this effect was quite small when compared with the adhesion-mediated induction of the IL-8 gene.



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  		Figure 3. Induction of the IL-8 gene is mediated by PMN adhesion to immobilized fibrinogen. Human PMN were allowed to adhere to immobilized fibrinogen for 2 h at 37°C in the presence of 1.2 mM Ca2+, 1 mM Mg2+, and 0.2 mM Mn2+. For negative control, PMN were kept in suspension for the times indicated. A) DNA gel electrophoresis of RT-PCR products of IL-8 and GAPDH, respectively. M: DNA size marker (Promega). B) Northern blot analysis of IL-8 mRNA expression. As shown in the agarose gel of the 18 s rRNA, equal amounts of RNA were present in all samples. Results are representative of three independent experiments.

Lack of gene activation in the absence of CD18
To confirm that gene activation is due to engagement of the ß2 integrins, PMN derived from mutant mice lacking CD18, the ß subunit of the ß2 integrins, or wild-type control animals of the same genetic background were incubated in the presence of divalent cations on fibrinogen-coated culture dishes. Cytokine gene induction was analyzed by semiquantitative RT-PCR of MIP-2 mRNA, a homologue of human IL-8 (Fig. 4 A). Within 2 h, adhesion caused a marked induction of MIP-2 mRNA in wild-type PMN when compared to suspended control cells. In contrast, CD18-deficient cells failed to produce substantial amounts of MIP-2 mRNA when incubated on immobilized fibrinogen. This was consistent with the observation that CD18-deficient PMN failed to adhere to the fibrinogen matrix (data not shown). Expression of the housekeeping gene GAPDH, which was used as internal standard, demonstrated that equal amounts of mRNA were present in all samples. For control, PMN were analyzed by flow cytometry for expression of CD18 and Gr-1, a marker of mature neutrophils (Fig. 4B ). Both wild-type and CD18-deficient PMN stained positively for Gr-1, confirming that mature PMN were present in all experiments. As expected, only wild-type PMN stained positively for CD18, whereas it was absent on PMN derived from CD18-deficient animals. Together, these data show that adhesion and subsequent gene activation were critically dependent on expression of ß2 integrins on the cell surface of PMN.



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  		Figure 4. A) Lack of gene activation in the absence of CD18. Murine PMN obtained from CD18-deficient or wild-type control animals were incubated on immobilized fibrinogen for 2 h at 37°C in the presence of 1.2 mM Ca2+, 1 mM Mg2+, and 0.2 mM Mn2+. For negative control, PMN were kept in suspension for 2 h. DNA gel electrophoresis of RT-PCR products of MIP-2 and GAPDH is shown. M: 100 bp DNA size marker (Promega). B) Flow cytometric analysis of murine PMN obtained from CD18-deficient or wild-type control animals, respectively. PMN were stained for expression of Gr-1 and CD18. For negative control, PMN were stained with PE- or FITC-labeled, isotype-matched control antibodies (dotted line). Results are representative of three independent experiments.

Mac-1 (CD11b/CD18): the predominant activator of the IL-8 gene
To study the question of which of the three ß2 integrins expressed on human PMN are able to activate the IL-8 gene, the {alpha} subunits of LFA-1 (CD11a), Mac-1 (CD11b), and gp150/95 (CD11c) were cross-linked by specific mAbs. Using the Northern blot technique, the ß2 integrin Mac-1 (CD11b/CD18) was found to mediate a strong induction of IL-8 mRNA within 2 h after antibody cross-linking as compared to the unstimulated control (Fig. 5 ). Gene induction by CD11b cross-linking was similar in extent to the effect of CD18 engagement, suggesting that Mac-1 was the predominant activator of the IL-8 gene. In contrast, cross-linking of CD11c led only to a slight induction of the IL-8 gene, and engagement of CD11a had no detectable effect. For negative control, IL-8 mRNA expression was also measured in suspended HL-60 cells without further stimulation and found to be absent. Thus, Mac-1 seems to represent the dominant activator of cytokine gene expression in human PMN.



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  		Figure 5. Mac-1 (CD11b/CD18) cross-linking mimicks the effect of PMN adhesion. Northern blot analysis of IL-8 expression in PMN and HL-60 cells. The {alpha} 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. For negative control, PMN (control) or HL-60 were left untreated. For positive control, PMN were stimulated by 100 nM fMLP. As shown in the agarose gel of the 18 s rRNA, equal amounts of RNA were present in all samples. Results are representative of three independent experiments.

Adhesion-mediated production and secretion of IL-8
To investigate whether the observed up-regulation of IL-8 mRNA may have physiological significance, induction of IL-8 was studied at the protein level. In these experiments, ELISA technique was used to measure the IL-8 peptide in the supernatant of human PMN that adhered to immobilized fibrinogen (Fig. 6 ). Some basal secretion of IL-8 was observed in unstimulated suspended PMN, which increased from ~12 pg/ml to 195 pg/ml within 24 h after stimulation. However, a strong enhancement of IL-8 secretion was detected in adherent PMN: within 24 h after the onset of adhesion, IL-8 secretion increased 42-fold to ~8000 pg/ml PMN when compared to unstimulated control cells. Adhesion-mediated IL-8 production was even greater than the effect induced by 100 nM fMLP, which resulted in IL-8 secretion of ~3600 pg/ml after 24 h of stimulation. Thus, adhesion was more efficient than the soluble mediator fMLP and showed a faster kinetic: adherent PMN had already produced ~4600 pg/ml of IL-8 in the supernatant within 6 h after the onset of adhesion, whereas fMLP-induced IL-8 production was only ~1000 pg/ml within this period. For comparison, the supernatant of unstimulated suspended control cells contained ~100 pg/ml of IL-8 within 6 h. In further experiments, the effect of costimulation by adhesion and simultaneous fMLP application was investigated and found to be simply additive (data not shown).



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  		Figure 6. ß2 integrin-mediated adhesion induces secretion of IL-8. PMN were kept in suspension and left unstimulated (control), stimulated in suspension with 100 nM fMLP, or allowed to adhere to fibrinogen (adhesion), respectively, and incubated for the times indicated at 37°C in the presence of 1.2 mM Ca2+, 1 mM Mg2+, and 0.2 mM Mn2+. IL-8 was measured in the supernatant of PMN by ELISA technique. n = 8, mean ± SD, *P<0.05 vs. unstimulated control.

Requirement for de novo mRNA and protein synthesis
To confirm that adhesion-induced secretion of IL-8 required both de novo mRNA as well as protein synthesis, the effect of actinomycin D and cycloheximide was studied on adhesion-induced IL-8 secretion (Fig. 7 ). Both inhibition of RNA synthesis by actinomycin D and inhibition of protein synthesis by cycloheximide resulted in almost complete inhibition of IL-8 secretion. Thus, secreted IL-8 was formed de novo by activation of the IL-8 gene. To further understand the intracellular mechanisms that underlie integrin-mediated gene regulation, the effect of tyrosine kinase inhibitors was investigated. Treatment of PMN with herbimycin A, a potent inhibitor of tyrosine kinases, abolished IL-8 production almost completely (Fig. 7) . Similar results were obtained when using genistein for inhibition of tyrosine kinases (data not shown). Thus, the integrin-mediated cytokine production was dependent on intracellular tyrosine signaling.



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  		Figure 7. ß2 integrin-mediated IL-8 secretion requires de novo mRNA and protein synthesis. PMN were left untreated (control) or treated with 10 µM cycloheximide (CHX), 10 µM actinomycin D (Act. D), or 30 µM herbimycin A (herbi) for 30 min at 37°C prior to adhesion to fibrinogen in the presence of 1.2 mM Ca2+, 1 mM Mg2+, and 0.2 mM Mn2+ for 24 h at 37°C. IL-8 was measured in the supernatant of PMN by ELISA technique. n = 4, mean ± SD, *P<0.05 vs. control.

Chemotactic migration of PMN by adhesion-mediated IL-8 production
Next we tested whether the secreted entity showed the characteristic biological activity of IL-8 by eliciting chemotactic migration (Fig. 8 ). Human PMN were allowed to transmigrate through transwell filters in response to supernatants derived from human PMN, which exerted adhesive interactions to immobilized fibrinogen for 24 h. These supernatants, which contained on average 8 ng/ml IL-8 as determined by ELISA technique (see Fig. 6 ), induced chemotactic migration of 22% of total PMN added. This was 63% of the effect induced by 10 nM fMLP, which resulted in ~35% transmigrating cells. Neutralization of IL-8 by a monoclonal antibody inhibited 60% of the effect induced by the supernatant derived from adherent PMN and resulted in 9% transmigrating cells, showing that the major entity that exhibited chemotactic activity was indeed IL-8. However, the incomplete inhibition of chemotaxis by the anti-IL-8 mAb suggests that adherent PMN may generate additional chemotactic factors besides IL-8. Addition of the antibody also induced a slight but not statistically significant reduction of the fMLP-induced response.



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  		Figure 8. Adhesion-mediated IL-8 production elicits chemotactic migration of PMN. PMN (2 .106/sample) were allowed to transmigrate through transwell filters for 1 h at 37°C in response to vehicle (control), 10 nM fMLP, or cell culture supernatants derived from PMN, which adhered to fibrinogen for 24 h (supernatant). Transmigration was measured in the presence or absence of an anti-IL-8 mAb. Transmigrated cells were harvested and counted under the microscope. Chemotaxis was calculated as transmigrated cells in percent of total cell number. n = 4, mean ± SD, *P<0.05 vs. control.

Differential cytokine expression by ß2 integrin-mediated adhesion
To determine whether ß2 integrin-mediated gene regulation was unique for IL-8 or common to other proinflammatory cytokine genes in human PMN, the effect of adhesion was investigated on production and secretion of IL-1ß, IL-6, and TNF-{alpha}, respectively. Using ELISA techniques, no significant production of IL-6 was detectable on adhesion to immobilized fibrinogen or challenge with 100 nM fMLP within a period of 0 h to 24 h (Fig. 9 ). Similarly, TNF-{alpha} was below the detection limit of 10 pg/ml in all experiments (data not shown). In contrast, adhesion to fibrinogen induced a substantial production of IL-1ß within 24 h whereas 100 nM fMLP was unable to affect IL-1ß gene expression during this same period. IL-1ß secretion was abolished when PMN were treated with cycloheximide or actinomycin D, respectively (data not shown). Thus, ß2 integrin-mediated adhesion caused differential expression of cytokine genes in human PMN.



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  		Figure 9. ß2 integrin-mediated adhesion induces production and secretion of IL-1ß but not of IL-6 or TNF-{alpha}. PMN were kept in suspension and left unstimulated (control), stimulated in suspension with 100 nM fMLP, or allowed to adhere to fibrinogen (adhesion), respectively, and incubated for the times indicated at 37°C in the presence of 1.2 mM Ca2+, 1 mM Mg2+, and 0.2 mM Mn2+. IL-1ß, IL-6, and TNF-{alpha} were measured in the supernatant of PMN by ELISA technique. n = 5, mean ± SD, *P<0.05 vs. control. TNF-{alpha} was below detection limit in all experiments (data not shown).

Cytokine gene induction in vivo
To study the biological relevance of the observed effects, an vivo model of inflammation was used. Four hours after induction of an acute peritonitis in mice by i.p. injection of 1 ml of 3% thioglycollate, extravasated leukocytes were harvested from the peritoneal cavity and subjected to RT-PCR analysis. As shown in Fig. 10 , the MIP-2 mRNA level was profoundly up-regulated upon emigration of WBC in response to thioglycollate injection when compared to peripheral WBC or bone marrow cells of the same animal. Levels of the constitutively expressed housekeeping gene ß-actin analyzed for control were almost identical, indicating that similar amounts of mRNA were present in all samples. This demonstrates that leukocyte extravasation in acute inflammation is associated with a substantial up-regulation of the MIP-2 gene in mice.



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  		Figure 10. Leukocyte extravasation is associated with cytokine induction. White blood cells of wild-type mice were obtained from the peripheral blood (peripheral WBC), from the bone marrow, or from the peritoneal cavity (emigrated WBC) 4 h after induction of peritonitis by i.p. injection of 3% thioglycollate. DNA gel electrophoresis of RT-PCR products of MIP-2 and ß-actin is shown. M: {phi}X174 DNA/HaeIII size marker (Promega). Results are representative of three independent experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, a novel ß2 integrin-mediated mechanism is reported that triggers the de novo synthesis of IL-8 mRNA in human PMN. The effect was maximal after aggregation of the ß2 integrins induced by antibody cross-linking of the anti-CD18 mAb by F(ab')2 fragments of a secondary antibody. Binding of the anti-CD18 mAb alone induced only a slight increase in IL-8 mRNA, which agrees with previous reports demonstrating that efficient integrin signaling requires integrin aggregation (25) . Although PMN express different types of Fc receptors that trigger intracellular signal transduction events on ligand binding (31) , their ligand interaction was not required for integrin-mediated cytokine gene induction, as shown by the use of F(ab')2 fragments of both the primary and secondary antibodies for integrin aggregation. The physiological significance of the observed effect was strengthened by the fact that PMN adhering to immobilized fibrinogen, a matrix that is functionally equivalent to fibrin, elicited the same response. This demonstrates that ß2 integrin-mediated adhesion to a native matrix suffices to induce gene activation in human PMN. The most abundant ß2 integrin, Mac-1 (CD11b/CD18) (15) , which serves as ligand for fibrinogen and fibrin, was confirmed to be responsible for gene induction by antibody cross-linking of the {alpha} subunits. However, cross-linking of CD11c also resulted in a small increase of IL-8 mRNA, suggesting that gp150/95 is also able to trigger gene activation upon ligand binding. Due to its low surface expression (15) , the physiological significance of this effect is probably less important than the induction mediated by Mac-1. Since LFA-1 was unable to induce gene activation, Mac-1 seems to represent the dominant signaling molecule in human PMN among the three ß2 integrins tested. However, the putative role CD11d/CD18, the fourth ß2 integrin (32) , was not investigated.

To exert its signaling capacity, Mac-1 required aggregation by antibody cross-linking or adhesion to an immobilized ligand, conditions known to cause clustering of the integrins (25 , 33) . Although the signal transduction events triggered on ß2 integrin clustering remain to be identified, the use of CD18-deficient PMN revealed that engagement of ß2 integrins was critically required for the adhesion-mediated signaling that allowed gene activation. Previous reports presented evidence that ß2 integrins participate in the induction of PMN functions, e.g., the respiratory burst, actin polymerization, or degranulation (23 , 24) . The present findings show that ß2 integrin engagement not only contributes to the control of short term PMN functions occurring within minutes on adhesion, but also affects gene expression in human PMN. Although PMN are capable to produce all cytokines tested by de novo synthesis (2) , only production of IL-8 and IL-1ß was induced on ß2 integrin-mediated adhesion, whereas no IL-6 or TNF-{alpha} synthesis was observed. Thus, the ß2 integrins induce differential expression of proinflammatory cytokines. Gene induction was found to depend strongly on intracellular tyrosine signaling. Therefore, proteins that have previously been shown to become tyrosine phosphorylated on ß2 integrin-mediated adhesion are good candidates to mediate this gene activation, e.g., the guanine nucleotide exchange factor vav or the src-kinase family member p58fgr (26 , 34) . However, further investigations regarding adhesion-dependent gene regulation in PMN will need to show whether the signal transduction cascade that is activated on ß2 integrin-mediated adhesion is unique to cytokine genes. Indirect evidence that ß2 integrins are involved in PMN apoptosis (35 , 36) , a process that probably depends on gene regulation (37) , may support the idea that not only cytokine genes are regulated on PMN adhesion.

The finding that adhesion to an appropriate matrix—namely, fibrin or immobilized fibrinogen—induces production and secretion of the proinflammatory cytokines IL-8 and IL-1 supports the concept that clotting is functionally linked to inflammatory responses. During severe inflammation, which is accompanied with exudation of plasma, clotting can also occur in the extravascular space, thereby providing an appropriate matrix for ß2 integrin-mediated adhesion of emigrated PMN. Although the lifetime of mature PMN is short when compared to other leukocyte population, this cell type has been unequivocally shown to produce cytokines by de novo RNA 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 and lasts ~24 h (37 , 39) . Moreover, the lifetime of PMN is further extended during systemic inflammation by inhibition of apoptosis (40) via cytokines such as granulocyte-macrophage colony-stimulating factor, etc. (41) . Thus, PMN may spend a considerable period of time in the tissue. Accordingly, induction of an acute peritonitis in mice resulted in up-regulation of the MIP-2 mRNA in emigrated leukocytes, demonstrating that up-regulation of cytokine genes is not only inducible in vitro, but also occurs in vivo within several hours after the onset of inflammation. During this period, the majority of emigrating leukocytes represents PMN (42) .

The observed induction of the proinflammatory cytokines IL-8 and IL-1 in human PMN may strengthen the inflammatory reaction, since IL-1 is well known to induce up-regulation of adhesion molecules and secretion of proinflammatory mediators by endothelial cells, which both support leukocyte emigration during inflammation (43 , 44) . IL-8 is a potent chemokine for PMN and may therefore contribute to the chemotactic recruitment of additional PMN to sites of lesion (3) . Moreover, IL-1 is known to affect hemostasis by induction of tissue factor in monocytes and endothelial cells (45 , 46) , as well as wound healing by promoting proliferation of fibroblasts (47) . Tissue remodeling may be further supported by IL-8, a CXC chemokine, which not only attracts PMN, but also bears an ELR-motif (Glu-Leu-Arg) and plays a functional role in angiogenesis by inducing neovascularization (48) . Thus, the observed cytokine induction may not only play a role in acute host defense by strengthening the inflammatory response, but may also play a role in activating the reorganization cascade elicited on lesion. This is consistent with the observation that patients suffering from leukocyte adhesion deficiency type I, an inherited defect of the CD18 gene, not only fail to elicit an inflammatory response, but also show impaired wound healing (49 , 50) , supporting the idea of a link between hemostasis, inflammation, tissue remodeling, and subsequent wound repair. Taken together, the present data show that ß2 integrin-mediated adhesion induces the transient activation of the IL-8 and the IL-1ß gene, predominantly via Mac-1 (CD11b/CD18), the major ligand of fibrinogen, and its clotting product, fibrin. This gene activation, which depends on tyrosine kinase-mediated intracellular signaling, results in substantial production and secretion of biologically active cytokines. Due to the pattern of cytokines secreted, this response may be able to support inflammation and contribute to the control of hemostasis as well as to the induction of tissue remodeling and wound healing processes, respectively.


   ACKNOWLEDGMENTS
 
Supported by Deutsche Forschungsgemeinschaft (SFB 366/C3 and B3).


   FOOTNOTES
 
2 Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunoassay; FITC, fluorescin isothiocyanate; fMLP, N-formyl-Met-Leu-Phe; mAb, monoclonal antibody; ICAM, intercellular adhesion molecule(s); Ig, immunoglobulin; IL, interleukin; i.p., intraperitoneally; MIP-2, macrophage 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 electrophoresis; TNF, tumor necrosis factor; WBC, white blood cells.

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


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