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(Circulation Research. 2000;87:254.)
© 2000 American Heart Association, Inc.

Integrative Physiology

ß₂-Integrin Blockade Driven by E-Selectin Promoter Prevents Neutrophil Sequestration and Lung Injury in Mice

Ning Xu, Arshad Rahman, Richard D. Minshall, Chinnaswamy Tiruppathi, Asrar B. Malik

From the Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Ill.

Correspondence to Asrar Malik, Department of Pharmacology, College of Medicine, University of Illinois, 835 S Wolcott Ave (M/C 868), Chicago, IL 60612-7343. E-mail abmalik{at}uic.edu

	Abstract

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Abstract—Interaction of CD11/CD18 ß₂ integrins on polymorphonuclear leukocytes (PMNs) with their counterreceptor, intercellular adhesion molecule-1, on the surface of vascular endothelial cells is a critical event mediating stable PMN adhesion and migration across the pulmonary vascular endothelial barrier. Neutrophil inhibitory factor (NIF), a 41-kDa glycoprotein isolated from the canine hookworm (Ancylostoma caninum), binds to the I domain of CD11a and CD11b and inhibits ß₂ integrin–dependent PMN adhesion. We describe a novel strategy using the endothelial cell–specific E-selectin promoter to induce NIF expression in an inflammation-specific manner in pulmonary vascular endothelial cells. A construct containing NIF cDNA driven by the inducible endothelial cell–specific E-selectin promoter (pESNIF) was transfected into human pulmonary artery endothelial cells (HPAECs). Lipopolysaccharide challenge (known to activate E-selectin) resulted in NIF mRNA and protein expression in transfected HPAECs. NIF expression induced by the E-selectin promoter prevented PMN adhesion to the activated HPAECs, whereas PMNs adhered avidly to activated HPAECs in the absence of NIF expression. To address the utility of this approach in conditionally preventing in vivo PMN sequestration, we injected mice intravenously with cationic liposomes containing the pESNIF construct. Analysis of lung tissue showed that intraperitoneal challenge of Escherichia coli resulted in NIF expression. Inflammation-specific NIF expression induced by the E-selectin promoter prevented lung PMN sequestration and vascular injury induced by E coli challenge. These studies suggest the feasibility of conditionally blocking ß₂ integrin function at sites where the endothelium is activated and thereby of locally preventing PMN activation and migration responses that lead to tissue inflammation.

Key Words: neutrophil • CD11 antigen • CD18 antigen • endothelial cell • E-selectin

	Introduction

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Acute lung injury is characterized by polymorphonuclear leukocyte (PMN) accumulation in the pulmonary microcirculation and alveolar spaces, resulting in PMN alveolitis and injury of vascular endothelial and alveolar epithelial barriers.^{1 2 3 4} Interaction of CD11/CD18 ß₂ integrins on PMNs with intercellular adhesion molecule-1 (ICAM-1) expressed on the surface of vascular endothelial cells is a critical event mediating stable PMN adhesion and PMN migration across pulmonary vascular endothelial barrier and tissue infiltration of PMNs.^{5 6 7} Neutrophil inhibitory factor (NIF), a novel glycoprotein isolated from the canine hookworm (Ancylostoma caninum), was identified as a potent inhibitor of PMN adhesion to endothelial cells and adhesion-dependent release of hydrogen peroxide. These effects of NIF resulted from its specific binding to the I-domain CD11/CD18 ß₂ integrins.^{8 9 10 11} We showed that NIF can bind to both CD11b and CD11a integrins and thereby prevent PMN adhesion to endothelial cells to a degree equivalent to anti-CD18 monoclonal antibodies.¹¹ We also showed that introduction of NIF cDNA driven by the constitutively active cytomegalovirus (CMV) immediate-early promoter in mouse pulmonary microvasculature resulted in NIF expression and induced the blockade of PMN ß₂ integrins.¹² NIF expression induced constitutively prevented PMN adhesion to endothelial cells and PMN-mediated lung injury in mice.¹² Because of the potential of preventing inappropriate PMN adhesion and infiltration into tissues, we investigated the possibility of using the inducible endothelial cell–specific E-selectin promoter^{13 14 15} to drive NIF expression in lung tissue and tested the effectiveness of this approach in preventing PMN-mediated lung injury.

E-selectin becomes expressed on activation of the E-selectin promoter in response to cytokines, such as tumor necrosis factor- (TNF-) and interleukin-1ß as well as lipopolysaccharide¹⁶ (LPS) and phorbol esters.^{17 18} Transfection studies in endothelial cells demonstrated that 170-bp 5' regulatory sequences were required for E-selectin expression.¹⁵ Site-directed mutagenesis of this region revealed 2 regulatory elements (-129 to -110 and -99 to -80) essential for maximal promoter activity after cytokine exposure.^{14 19} Protein binding studies with nuclear extracts and recombinant proteins showed that the 2 elements corresponded to 3 nuclear factor-B (NF-B) binding sites (site 1, -126; site 2, -116; and site 3, -94).^{15 19} Because the conditional expression of NIF may be useful in preventing PMN adhesion to activated endothelial cells and resultant lung microvascular injury, we expressed NIF in an inflammation-specific manner using the E-selectin promoter containing the NF-B binding sites in the present study.

	Materials and Methods

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Mice
Pathogen-free male CD-1 mice (25 to 30 g; Harlan Co, Indianapolis, Ind) were anesthetized with an intramuscular injection of ketamine (60 mg/kg) and xylazine (2 mg/kg) in PBS. All animal experiments conformed to the guidelines established by the University of Illinois.

Transgene Constructs
The transgene constructs were as follows: (1) pCMVNIF: NIF cDNA (860 bp) provided by Dr M. Moyle (Corvas, La Jolla, Calif) was subcloned into the EcoRI site of pCR3 (Invitrogen, San Diego, Calif). (2) pESNIF: E-selectin promoter (provided by Dr V. Baichwal, Tularik, San Francisco, Calif) was subcloned into SacI and BamHI sites of PUC 118 vector followed by the subcloning of NIF cDNA downstream of the E-selectin promoter into XbaI and HindIII sites of the same vector. (3) pSPCNIF: NIF cDNA was subcloned downstream of surfactant protein C (SPC) promoter into the EcoRI site of the 3.7SPC/SV40 (provided by Dr J.A. Whitsett, Children’s Hospital Medical Center, Cincinnati, Ohio).

Cell Cultures and Transfections
Human pulmonary artery endothelial cells (HPAECs) and A549 cells were cultured as described.^{20 21} Cells were transfected using SuperFect (Qiagen Inc) and treated with either TNF- (500 U/mL, Promega) or LPS (10 ng/mL, 0111:B4, Sigma Chemical Co) for 6 hours.

DNA/Liposome Preparation and In Vivo Gene Transfer
The transgene (50 µg/mouse) and liposomes were prepared as previously described^{12 22 23} and were intravenously injected in mice. Each mouse received 10⁸ live Escherichia coli bacteria, a dosage that did not result in death within the experimental period. Control mice were injected intraperitoneally with an equal volume of PBS.

Reverse Transcriptase (RT)–Polymerase Chain Reaction (PCR)
Total RNA was isolated from transfected cells or the lungs of transduced mice with the RNeasy mini kit (Qiagen) and pretreated with RNase-free DNase I. RT was performed with Superscript preamplification system (Life Technologies). PCR was carried out using NIF primers (forward primer 5'-ATGGAGGCCTATCTTGTG-3' and reverse primer 5'-TCATAACTCTCGGAATCG-3') with either human GAPDH primers (forward primer 5'-GCGTCTTCACCACCATGG-3' and reverse primer 5'-TGACACGTTGGCAGTGGG-3') or mouse GAPDH primers (forward primer 5'-GGAGATTGTTGCCATCAACG-3' and reverse primer 5'-CATGGACTGTGGTCATGAGC-3') as internal control. The conditions of PCR were as follows: 45 seconds at 94°C, 30 seconds at 60°C, and 1.5 minutes of 72°C for 30 cycles.

Immunofluorescence
HPAECs on coverslips were cotransfected with pESNIF and green fluorescence protein (GFP) and treated with TNF- (500 U/mL) or LPS (10 ng/mL) for 6 hours. Cells without treatment were used as control. Cells were incubated with monoclonal NIF antibody (provided by Dr E. Plow, Cleveland Clinic Foundation) at 4°C overnight followed by Alexa 568 goat anti-mouse secondary antibody (Molecular Probes) at room temperature for 2 hours and then DAPI for 15 minutes. Cells were mounted with ProLong Antifade kit (Molecular Probes).

Immunohistochemistry
Lung sections of mice were prepared as previously described¹² and stained with rabbit anti-NIF antiserum using an ImmunoCruz staining system (Santa Cruz Biotechnology).

PMN Adhesion Assay
Challenged with 500 U/mL TNF- for 6 hours, pCMVNIF- and pESNIF-transfected HPAECs were used for adhesion assay²⁴ with nontransfected or vector-transfected HPAECs as controls. The percentage of PMNs adhering to endothelial cells was determined from the ratio of final reading to initial reading.

Lung PMN Sequestration Assay
Lungs were homogenized for quantification of PMN uptake by myeloperoxidase (MPO) activity as described.^{12 25}

Pulmonary Vascular Permeability and Extravascular Lung Water
At 48 hours after intravenous injection of NIF construct/liposome complex, mice were challenged intraperitoneally with 10⁸ E coli for 6 hours. Mice were then intravenously injected with 1 µCi of ¹²⁵I-labeled albumin and killed after 1 hour. Controls were mice not challenged with E coli. Radioactivities of both blood and whole lung were counted to determine the pulmonary vascular permeability index.²⁶ The extravascular lung water content in the groups was also determined.²⁷

Statistical Analysis
Data are presented as mean±SEM. Comparisons between groups were made by ANOVA with significance of P<0.05.

	Results

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E-Selectin Promoter–Driven NIF Expression Induced in Endothelial Cells
HPAECs (Figure 1A) or A549 cells (Figure 1B) were transfected with pESNIF, pSPCNIF, or pCMVNIF construct or the corresponding pES, pSPC, or pCMV vector. After LPS (10 ng/mL) challenge for 6 hours, total RNA was isolated and pretreated with RNase-free DNase I to eliminate DNA contamination. We observed that the pESNIF construct was active only in the LPS-stimulated HPAECs (lane 5 versus lane 2, Figure 1A). In A549 cells, pSPCNIF construct was constitutively active at low levels (lane 7, Figure 1B), and its expression was upregulated after LPS stimulation (lane 10, Figure 1B). The constitutively active pCMVNIF construct was active in both cell types irrespective of whether the cells were stimulated with LPS (lanes 1 and 4, Figure 1A; lanes 5 and 8, Figure 1B). We observed that the NIF mRNA expression induced by 6 hours of TNF- or LPS challenge resulted in NIF protein expression (Figure 1C). NIF protein expression in pESNIF-transfected HPAECs was induced by either TNF- (500 U/mL) or LPS (10 ng/mL) challenge whereas pESNIF-transfected HPAECs without challenge did not show NIF expression (Figure 1C).

View larger version (34K): [in this window] [in a new window]   Figure 1. Endothelial cell expression of NIF induced by E-selectin promoter. HPAECs (A) or A549 cells (B) were transfected with NIF constructs or with the corresponding vectors. After LPS (10 ng/mL) challenge of cells for 6 hours, total RNA was isolated and analyzed for NIF mRNA expression by RT-PCR. The pESNIF construct was active only in LPS-stimulated HPAECs (lane 5 vs lane 2, Figure 1A). In A549 cells, the pSPCNIF construct was constitutively active at low levels (lane 7, Figure 1B), and its expression was increased after LPS stimulation (lane 10, Figure 1B). The constitutively active pCMVNIF construct was active in both cell types irrespective of whether cells were stimulated with LPS (lanes 1 and 4, Figure 1A; lanes 5 and 8, Figure 1B). hGAPDH was used as the internal control. Results are representative of 2 separate experiments. C, Expression of NIF as determined by immunofluorescence in HPAECs cotransfected with pESNIF and GFP. NIF expression at 6 hours after TNF- or LPS challenge was evident in the pESNIF-transfected HPAECs.

Expression of NIF Induced by E-Selectin Promoter Prevents PMN Adhesion to Endothelial Cells
HPAECs were transfected with pESNIF or pCMVNIF or corresponding pES and pCMV vectors. After TNF- (500 U/mL) challenge of cells for 6 hours, fresh PMNs were added, and adhesion of PMNs to endothelial cells was determined as previously described.²⁴ Some cells were not challenged with TNF- but with phorbol 12-myristate 13-acetate (PMA) (15 nmol/L) added to endothelial cells followed by addition of PMNs. The period of challenge for PMA was 15 minutes, an insufficient period to activate the E-selectin promoter. TNF- challenge resulted in a 5-fold increase in PMN adhesion except in the presence of either constitutive NIF expression (by pCMVNIF) or conditional NIF expression (by pESNIF). These results also indicated that the 15-minute PMA challenge induced an 8-fold increase in PMN adhesion in control cells and a 7-fold increase in pESNIF-transfected cells. Thus, PMN adhesion was only inhibited in the presence of the conditional expression of NIF (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2. E-selectin promoter–induced expression of NIF prevents PMN adhesion to endothelial cells. HPAECs were transfected with NIF constructs or the corresponding vectors. After TNF- (500 U/mL) challenge for 6 hours, PMNs were added, and adhesion of PMNs to HPAECs was determined. Some cells were not challenged with TNF-, but with PMA (15 nmol/L) followed by addition of PMNs for 15 minutes (this short-term PMA exposure does not result in activation of E-selectin promoter). TNF- challenge resulted in a 5-fold increase in PMN adhesion except in the presence of either constitutive NIF expression (by pCMVNIF transfection) or conditional NIF expression (by pESNIF transfection). PMA challenge resulted in an 8-fold increase in PMN adhesion to nontransfected HPAECs and a 7-fold increase in PMN adhesion to the pESNIF-transfected HPAECs not challenged with TNF-. Results are representative of 3 separate experiments. Bars indicate mean±SEM. *P<0.01 from PMN adhesion values in control groups challenged with TNF-.

NIF Expression Induced by E-Selectin Promoter in Lungs After Challenge With E coli
At 48 hours after the intravenous injection of liposomes alone, liposome/pES complex, liposome/pCMV complex, liposome/pESNIF complex, or liposome/pCMVNIF complex, mice were challenged with 10⁸ E coli via IP injection. Total RNA was isolated from lung 6 hours later and pretreated with RNase-free DNase I to eliminate DNA contamination. RT-PCR analysis showed that E coli challenge resulted in expression of NIF when it was driven by the E-selectin promoter. However, NIF expression was constitutive and unaffected by E coli challenge when its expression was driven by the constitutively active CMV promoter (Figure 3A). The lungs of pESNIF-transduced mice were challenged with E coli or PBS alone, inflated with 4% paraformaldehyde in PBS (pH 7.4), and embedded in paraffin. Lung sections (4 µm) were stained with rabbit anti-NIF antiserum using an ImmunoCruz staining system. Expression of NIF was evident in lungs of pESNIF-transduced mouse challenged with E coli (Figure 3B) and not observed in lungs of pESNIF-transduced mouse without E coli challenge (Figure 3B).

View larger version (60K): [in this window] [in a new window]   Figure 3. NIF expression induced by E-selectin promoter in lungs after E coli challenge in mice. A, NIF mRNA expression. At 48 hours after the intravenous injection of liposomes alone, vector/liposome complexes, or NIF construct/liposome complexes, mice were challenged with 108 E coli via intraperitoneal injection. Total RNA was isolated from mouse lung 6 hours later. RT-PCR showed that challenge with E coli resulted in NIF expression in mice receiving the pESNIF construct (lanes 8 and 9), whereas there was no NIF expression without E coli challenge (lanes 6 and 7). pCMVNIF construct resulted in constitutive NIF expression (lanes 12 and 13), which was unaffected by E coli challenge. There was no expression of NIF in either normal control, liposome control, or vector control (lanes 10 and 11) mice. mGAPDH was used as internal control. Results are representative of 4 separate experiments. B, NIF protein expression. pESNIF-transduced mice were challenged with E coli or PBS; lungs were removed, inflated with 4% paraformaldehyde in PBS (pH 7.4), and embedded in paraffin. Lung sections (4 µm) were stained with rabbit anti-NIF antiserum using an ImmunoCruz staining system. Expression of NIF (stained in dark brown) was observed in lung of pESNIF-transduced mouse challenged with E coli but not in lung of pESNIF-transduced mouse without E coli challenge (control).

E coli–Induced NIF Expression Prevents Lung PMN Sequestration and Lung Microvascular Injury in Mice
We developed a mouse model of E coli–induced lung PMN sequestration and vascular injury (Figure 4A). Mice were intraperitoneally injected with either 0.5 mL PBS (pH 7.4) or 10⁸ E coli in PBS and were sacrificed at different time points, 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours (n=5 mice per group). Lungs were isolated, and MPO activity was measured. MPO activity increased in a time-dependent manner with the maximum response observed at 6 hours after E coli challenge. The dosage of E coli was chosen so that no animals died within the experimental period.

View larger version (25K): [in this window] [in a new window]   Figure 4. NIF expression induced by E-selectin promoter prevents PMN sequestration in mouse lungs. A, Mouse model of E coli–induced lung injury. Mice were injected intraperitoneally with either PBS (pH 7.4) or 108 E coli, then killed at 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours (n=5 mice per time point). Lungs were isolated, and MPO activity was measured. Highest MPO activity was observed at 6 hours after E coli challenge. B, Mice were injected intravenously with liposomes alone, liposome/pCMV complex, liposome/pCMVNIF complex, liposome/pES complex, and liposome/pESNIF complex. At 48 hours after injection, mice were challenged intraperitoneally with either PBS (pH 7.4) or 108 E coli and killed 6 hours later. Lungs were isolated, and MPO activity was measured. Expression of NIF in the pESNIF-transduced mice prevented the E coli–induced increase in MPO activity. Similar inhibition of MPO activity was observed in pCMVNIF-transduced mice. In the control groups of mice, E coli resulted in marked increases in MPO activity. Bars indicate mean±SEM; n=5 mice in each group. *P<0.05 from control groups of mice challenged with E coli.

In other experiments, mice were intravenously injected with liposomes alone, liposome/pCMV complex, liposome/pCMVNIF complex, liposome/pES complex, and liposome/pESNIF complex. At 48 hours later, mice were challenged intraperitoneally with either PBS (pH 7.4) or 10⁸ E coli for 6 hours (as described above) and then sacrificed. The results showed that the conditional expression of NIF in pESNIF-transduced mice prevented the increase in MPO activity induced by E coli (Figure 4B). This was also the case in the positive control group of pCMVNIF-transduced mice (Figure 4B).

Following the procedures described above, mice were intravenously injected with 1 µCi of ¹²⁵I-labeled albumin 6 hours after E coli challenge and sacrificed 1 hour after injection of albumin tracer. These results indicated that the conditional expression of NIF prevented the increase in pulmonary microvessel permeability (Figure 5A) and pulmonary edema (Figure 5B) induced by E coli challenge.

View larger version (22K): [in this window] [in a new window]   Figure 5. NIF expression induced by E-selectin promoter prevents lung vascular injury and edema after E coli challenge. At 48 hours after the intravenous injection of liposomes alone, vectors/liposome complex, or NIF constructs/liposome complex, mice were challenged with 108 E coli. After 6 hours, the mice were injected intravenously with 1 µCi 125I-labeled albumin and killed 1 hour later. Mice that did not receive E coli were used as controls. Conditional expression of NIF in the pESNIF-transduced mice prevented the E coli–induced increases in lung microvascular permeability (A) and lung water content (B). Similar inhibition was observed in the pCMVNIF construct–transduced mice. However, in the control groups of mice, E coli resulted in significant increases in pulmonary microvascular permeability and lung water content. Bars indicate mean±SEM; n=4 in each group. *P<0.05 from control groups of mice challenged with E coli.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We describe a novel strategy to express a selective PMN ß₂-integrin antagonist, NIF, in a cell- and inflammation-specific manner using the endothelial cell–selective E-selectin promoter. We first carried out studies in HPAEC and A549 line of human alveolar type II epithelial cells. The cells were transfected with pESNIF, pSPCNIF, and pCMVNIF constructs; ie, cells in which the NIF cDNA was driven by the E-selectin promoter, type II alveolar epithelial cell–specific SPC promoter, or constitutively active CMV promoter, respectively. Analysis showed that expressions of NIF mRNA and protein were induced only in HPAECs, and it was evident only with the inflammatory challenge. The expression of NIF mRNA induced by the CMV promoter was observed predictably in both cell types under basal conditions as well as after inflammatory challenge. However, the expression of NIF driven by the SPC promoter was evident only in A549 cells. These results indicate the specificity of the conditional expression of NIF in endothelial cells using the E-selectin promoter and suggest the feasibility of targeting ß₂-integrin blockade in an inflammation-specific manner.

The results showed that the E-selectin promoter, activated by the transcription factor NF-B, regulated the expression of NIF when the endothelial cells were transduced with pESNIF construct. We also demonstrated that activation of NIF expression induced by the E-selectin promoter was specific to endothelial cells because we did not observe NIF expression in type II alveolar epithelial cells transduced with the E-selectin promoter-NIF construct. This finding is consistent with the endothelial cell–specific nature of E-selectin expression.^{28 29} Interestingly, endothelial cell expression of NIF driven by the E-selectin promoter on activation by TNF- prevented PMN adhesion to these cells. In contrast, there was no diminution of PMN adhesion to endothelial cells in the absence of E-selectin promoter activation, as with a 15-minute exposure to PMA. Thus, expression of NIF in endothelial cells induced by the proinflammatory cytokine TNF- or with LPS is capable of preventing PMN adhesion to activated endothelial cells.

In another series of studies, we determined the in vivo utility of inducing conditional ß₂-integrin blockade in preventing lung PMN sequestration and the resultant microvascular injury and tissue edema. We studied the effects of Gram-negative sepsis that is characterized by sequestration of PMNs in the pulmonary microcirculation and migration of PMNs into the alveolar space as well as the development of pulmonary microvascular injury.^{30 31 32} Binding of CD11/CD18 ß₂ integrins on PMNs to ICAM-1 expressed on the endothelial cell surface stabilizes PMN adhesion and results in lung PMN sequestration and promotes PMN migration across the vascular endothelial barrier.⁵ Because NIF is a selective ß₂-integrin antagonist that inhibits PMN adhesion,^{10 11} we addressed the question whether the conditional expression of NIF and the resultant activation of ß₂-integrin blockade in vivo would interfere with lung PMN sequestration and microvascular injury.

We first developed a reproducible acute lung injury model in mice using live E coli bacteria. We found that the optimal dosage of E coli was 10⁸ per mouse injected intraperitoneally. This challenge did not produce death within the experimental period but did result in a time-dependent and consistent lung PMN sequestration with the maximum increase in lung MPO activity observed at 6 hours after challenge. Using this model, we showed that intraperitoneal challenge of mice with E coli induced NIF expression in lungs of mice transduced with the pESNIF construct. In contrast, NIF expression was not evident in the absence of E coli challenge. In a positive control group of mice receiving the pCMVNIF construct, we observed the constitutive expression of NIF, which was unaffected by E coli challenge. These results indicate the feasibility of using the E-selectin promoter to induce NIF expression and thereby of eliciting conditional ß₂-integrin blockade in vivo.

The NIF expression induced conditionally was functionally active in that the E-selectin promoter-driven NIF expression prevented lung PMN sequestration induced by the E coli challenge. The finding that the expression of NIF prevented the increase in lung microvascular permeability and edema formation in response to E coli challenge indicated that the lung injury was PMN-dependent and required the engagement of ß₂ integrins.

Expression of NIF, and the resultant ß₂-integrin blockade, as regulated by the E-selectin promoter has distinct advantages compared with the constitutive induction of NIF using the CMV promoter. First, as we have shown, NIF is expressed after a proinflammatory stimulus and activation of the E-selectin promoter; thus, NIF expression is activated by the same inflammatory stimulus mediating the PMN adhesion and migration responses. Because NIF expression was coupled to the inflammatory stimulus, this may more effectively abrogate the inflammatory response. Second, because NIF expression is driven by the E-selectin promoter, ß₂-integrin blockade would be localized at sites of E-selectin expression in the pulmonary microcirculation; therefore, this strategy may be useful in preventing PMN adhesion and migration in the inflamed or activated regions of the pulmonary microcirculation. Third, because NIF is released locally at sites of NIF expression,¹² it may not result in generalized loss of host-defense function of phagocytic cells that is characteristic of injection of anti-CD18 monoclonal antibodies.³³ Finally, use of E-selectin promoter to induce NIF expression may be advantageous because it results in a reversible impairment of ß₂ integrin function. The expression of E-selectin peaks between 4 and 6 hours and then decreases within 12 hours.^{34 35 36} We showed a similar pattern of NIF expression in endothelial cells transduced with pESNIF construct; ie, increased NIF expression at 4 hours after TNF- challenge of the transduced endothelial cells and decreased expression at 12 hours (data not shown). Thus, NIF expression induced by the E-selectin promoter is unlikely to produce a sustained loss of PMN function.

In summary, we used the E-selectin promoter to express NIF, a selective ß₂ CD11/CD18 integrin antagonist, in a cell- and inflammation-specific manner. The conditional expression of NIF prevented PMN adhesion to activated vascular endothelial cells and lung PMN sequestration and microvascular injury in mice induced by intraperitoneal E coli challenge. We demonstrated the potential usefulness of blocking PMN ß₂ integrin function at sites of activated endothelium in the pulmonary microcirculation, and thereby of locally inhibiting PMN activation and migration responses that lead to tissue inflammation.

	Acknowledgments

 
This work was supported by National Institutes of Health Grants HL27016, HL45638, HL60678, and T32 HL07829.

Received June 5, 2000; accepted June 26, 2000.

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