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(Journal of Leukocyte Biology. 2000;68:73-80.)
© 2000 by Society for Leukocyte Biology

LFA-1 (CD11a/CD18) triggers hydrogen peroxide production by canine neutrophils

Huifang Lu^*,, Christie Ballantyne and C. Wayne Smith^*,

^* Department of Microbiology and Immunology;
Speros P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics; and
Section of Cardiovascular Science, The Methodist Hospital, Department of Medicine, Baylor College of Medicine, Houston, Texas

Correspondence: C. Wayne Smith, M.D., Section of Leukocyte Biology, Children’s Nutrition Research Center, Room 6014, 1100 Bates, Houston, TX 77030. E-mail: cwsmith{at}bcm.tmc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The respiratory burst of neutrophils stimulated by chemotactic factors is markedly augmented by Mac-1-dependent adhesion such as the interaction of Mac-1 (CD11b/CD18) with intercellular adhesion molecule-1 (ICAM-1; CD54) expressed on the surface of parenchymal cells (e.g., cardiac myocytes). In the current study, we evaluate the hypothesis that lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) can also trigger the respiratory burst in neutrophils. To isolate LFA-1/ICAM-1 interactions from Mac-1/ICAM-1 interactions, full-length chimeric ICAM-1 was developed and expressed in L cells with domains 1 and 2 from canine ICAM-1 and domains 3–5 from human ICAM-1 (C1,2;H3–5). We have shown that canine neutrophils do not bind to human ICAM-1. We demonstrated that chimeric ICAM-1 C1,2;H3–5 supported only LFA-1-dependent adhesion of canine neutrophils and that such adhesion triggered rapid onset of H₂O₂ production from canine neutrophils. The following seven experimental conditions distinguished LFA-1-dependent H₂O₂ production from Mac-1-dependent production: It did not require exogenous chemotactic stimulation; H₂O₂ release was more rapid, but the amount released was <40% of that mediated by Mac-1 adhesion; it was inhibited by anti-CD11a and anti-ICAM-1 antibodies; in contrast to that mediated by Mac-1, it was not inhibited by anti-CD11b antibody, neutrophil inhibitory factor (NIF), or cytochalasin B or H7. Thus, canine neutrophils seem to be able to utilize two members of the ß₂ integrin family to interact with ICAM-1 and signal H₂O₂ production, with LFA-1 at an early stage without prior chemotactic stimulation and Mac-1 at a later stage requiring chemotactic stimulation.

Key Words: adhesion molecules • canine • ICAM-1 • endothelial cells • CD11b/CD18 • Mac-1

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Neutrophils release H₂O₂ when stimulated with chemotactic factors [¹ , ² ]. This response is greatly augmented by Mac-1-dependent adhesion to endothelial cells and cardiac myocytes [³ , ⁴ ]. Although neutrophils express cell-surface lymphocyte function-associated antigen-1 (LFA-1) and utilize this integrin to adhere to intercellular adhesion molecule-1 (ICAM-1) on endothelial cells [⁵ , ⁶ ], anti-CD11a monoclonal antibodies (mAbs), unlike anti- CD11b and anti-CD18 mAbs, do not reduce adhesion-dependent enhancement of H₂O₂ production by chemotactically stimulated neutrophils contacting endothelial cells or cardiac myocytes [³ ]. The ability of Mac-1 to act as a cosignal for enhanced H₂O₂ production is further evidenced by experiments using adhesive surfaces that are solely Mac-1-dependent [¹ , ³ ]. The effects of chemotactic stimulation and Mac-1 adhesion appear to be synergetic in that Mac-1 adhesion requires stimulation of the neutrophil [³ , ⁵ ] and, in turn, greatly augments the respiratory burst [³ , ⁴ ].

Berton et al. [⁷ ] have raised the possibility that LFA-1 may also function as a signaling molecule for the production of H₂O₂ by neutrophils. They found that when neutrophils were allowed to settle onto plastic surfaces coated with anti-CD11a mAb, substantial H₂O₂ release could be detected. This observation has been confirmed by Menegazzi et al. [⁸ ]. The anti-CD11a mAb was apparently sufficient to induce the respiratory burst, because the experiments did not include added stimulants such as chemotactic factors. Signaling through LFA-1 may be biologically relevant, because it is clearly established that neutrophils utilize LFA-1 to adhere to [⁵ ] and migrate through endothelial monolayers [⁵ , ⁶ ]. However, studies of Berton et al. did not provide evidence that LFA-1 signals H₂O₂ production by neutrophils following binding to a natural ligand.

In the current study, we have evaluated H₂O₂ production by neutrophils following LFA-1 binding to ICAM-1³. To reduce possible signaling through Mac-1 adhering to ICAM-1, we developed a chimeric ICAM-1 construct of canine domains 1 and 2 and of human domains 3–5. We found in preliminary experiments that canine neutrophils failed to recognize human ICAM-1 (hICAM-1) but bound efficiently to canine ICAM-1 (cICAM-1). Previous studies have shown that the human LFA-1 binding site on hICAM-1 involves domains 1 and 2 [⁹ ], and the human Mac-1 binding site involves hICAM-1 domain 3 [⁹ ]. We observed that canine neutrophils release H₂O₂ after adhesion to the chimeric ICAM-1 that was blocked by anti-CD11a mAb but not by anti-CD11b mAb. H₂O₂ production occurred without added chemotactic stimulation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents
Zymosan, keyhole limpet hemocyanin (KLH), pepsin, geneticin (G418), human serum albumin (HSA), lipopolysaccharide (LPS), cytochalasin B, scopoletin, and horseradish peroxidase (type II) were from Sigma Chemical Company (St. Louis, MO). Gelatin was from Bio-Rad Laboratories (Richmond, CA). H7 [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine, dihydrochloride] and HA1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride] were from Calbiochem or Seikagaku America, Inc. (Rockville, MD). RPMI 1640 medium, penicillin-streptomycin, Dulbecco’s phosphate-buffered saline (DPBS), and 1x trypsin-ethylenediaminetetraacetate (EDTA) were obtained from GIBCO BRL (Grand Land, NY). Protein A ImmunoaffinityPAK column was from Pierce (Rockford, IL). Recombinant neutrophil inhibitory factor (NIF) was obtained from Dr. Matthew Moyle (Corvas International, San Diego, CA).

mAbs
R15.7 [immunoglobulin G1 (IgG1)], anti-CD18 [¹⁰ ], R7.1 (IgG1), anti-CD11a [¹¹ , ¹² ], R6.1, and CA7 (IgG1) specific for human ICAM-1 domains 2 and 5 [¹³ ] were kindly provided by Dr. R. Rothlein (Boehringer Ingelheim Pharmaceutical, Ridgefield, CT). MY904 [IgG1 and F(ab')₂], anti-CD11b, was obtained from Lilly (Indianapolis, IN). CL18/6 and CL18/1 [IgG1 and F(ab')₂], anti-canine ICAM-1 [¹⁴ ], and F(ab')₂ of R7.1 and R15.7 were made using the ImmunoaffinityPAK kit (Pierce).

Culture of canine endothelial cells
Canine jugular vein endothelial cells (CJVEC) were isolated and cultured as described [¹⁰ ], and confluent monolayers on 0.1% gelatin-coated, 25 mm glass coverslips were prepared from the first through the fourth passages.

Isolation of canine polymorphonuclear neutrophil (PMN)
Neutrophils were isolated from healthy mongrel dogs as described previously [¹⁴ ]. Blood samples were anticoagulated with citrate phosphate dextrose (Abbot, Chicago, IL) (0.14 ml/ml blood) and sedimented in 1% (wt/vol in 0.87% NaCl) Dextran (Spectrum Chemical, New Brunswick, NJ) for 45 min at room temperature. Leukocyte-rich plasma was layered on Ficoll-Hypaque gradients, and neutrophils were recovered, washed, and resuspended in DPBS, pH 7.4, containing 0.2% dextrose. Final leukocyte suspensions contained >95% neutrophils and were used immediately for H₂O₂ assays or maintained at 4°C before use in adhesion assays.

Adhesion assay
Transfected L cells or CJVEC were plated onto 0.1% gelatin-pretreated, 25 mm coverslips and allowed to become visually confluent. A visual static adhesion assay was described in detail previously [¹⁵ ]. In studies designed to evaluate the involvement of ß₂-integrins or ICAM-1 in neutrophil adhesion, cells were preincubated as follows: Coverslip with transfected L cells or CJVEC monolayer was treated with anti-ICAM-1 mAbs at 20 µg/ml concentration in 1 ml of PBS for 30 min at room temperature and was mounted in the adhesion chamber directly. Neutrophils were incubated with antibodies specific for integrin subunits at 2–4 times the saturating concentration at room temperature for 30 min. Chemotactic stimuli of 1% zymosan-activated-serum (ZAS; as previously described) [¹⁰ ] for canine neutrophils was added immediately before injecting the cell mixture into the adhesion chamber.

Measurement of hydrogen peroxide production
Hydrogen peroxide production was quantitated by a modification of the method described by Nathan [¹ ]. Briefly, L-cell plates (Linbro, Flow, McLean, VA) were treated with 200 µl of 0.1% gelatin for 1 h at room temperature before adding trypsinized L-cell transfectants. Cells reached confluency in 2–3 days, as judged by phase contrast microscopy. Plates were washed three times with prewarmed DPBS or Kreb-Ringer Phosphate (KRP) to 37°C by gentle blotting onto a paper towel. For Mac-1-dependent adhesion, plates were coated with 150 µl (slightly greater than final volume of experimental incubation) of a 0.5 mg/ml KLH (Sigma) solution in DPBS for at least 60 min at 37°C [³ ]. Plates were washed three times with DPBS or KRP by flicking out contents. The assay reaction mixture (100 µl/well), prepared from 10x stock solutions, was 24 µM scopoletin, 5 µg/ml horseradish peroxidase, 1 mM sodium azide, and 5 mM glucose in low-phosphate KRP. F(ab')₂ fragments of mAbs were added to wells (20 µl/well) from 7x solutions to achieve final saturating concentrations. Unless otherwise indicated, 10–20 µg/ml was used. For canine experiments, 1.4 µl ZAS was added to 1% of the final volume. Neutrophils were added in a volume of 20 µl to achieve a final concentration of 2 x 10⁴/well. Wells not receiving antibody or chemotactic factor received equal volumes of appropriate vehicles so that final incubation volumes were 140 µl. Experiments were performed in replicates of four or eight each, and fluorescence of scopoletin was determined immediately after addition of neutrophils and at 30-min intervals thereafter in a Titertek Fluroskan II fluorometer (Flow) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. Plates were maintained at 37°C in the absence of CO₂. Values of hydrogen peroxide production (decrease in scopoletin fluorescence compared with baseline at time zero) were plotted vs. time, and the areas under the curve for each experimental condition were determined using a digitizer pad and Sigma Scan software (Jandel Scientific, Sausalito, CA). Statistical comparisons were made by analysis of variance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To establish an experimental condition where neutrophil adhesion was dependent on LFA-1 (CD11a/CD18) but not Mac-1 (CD11b/CD18), we allowed isolated canine neutrophils to contact L-cell monolayers stably transfected with a chimeric ICAM-1 composed of canine domains 1 and 2 and human domains 3–5. In preliminary experiments, we found that canine neutrophils did not adhere to human ICAM-1, and given the observations of Diamond et al. [¹⁶ ] that LFA-1 adhesion to ICAM-1 involves domains 1 and 2, and Mac-1 adhesion to ICAM-1 involves domain 3, canine neutrophils should adhere to this chimeric ICAM-1 using only LFA-1. We observed that in the absence of chemotactic stimulation, isolated canine neutrophils exhibited a significant level of adhesion (Fig. 1 ). That this adhesion was dependent on LFA-1 was confirmed by blocking studies with mAbs against the subunits of LFA-1 and Mac-1. Antibody R7.1 (CD11a) inhibited adhesion as effectively as R15.7 (anti-CD18), while MY904 (anti-CD11b) was without effect. Confirmation that the adhesion was dependent on interaction with ICAM-1 was obtained using antibodies against canine ICAM-1. We recently analyzed the binding characteristics of mAbs CL18/1 and CL18/6 Hydrogen peroxide induces LFA-1-dependent neutrophil adherence to cardiac myocytes (H. Lu, K. Youker, C. Ballantyne, M. Entman, C. W. Smith, unpublished results). These mAbs bound to L cells expressing full-length canine ICAM-1. CL18/1 and CL18/6 did not bind to L cells expressing wild-type human ICAM-1. The binding specificities of these two antibodies were characterized by using two chimeric ICAM-1 constructs, C1,2:H3–5 and H1,2:C3–5. CL18/1 bound to L cells expressing H1,2:C3–5 but not to C1,2:H3–5, and CL18/6 bound to C1,2:H3–5 but not to H1,2:C3–5. As shown in Figure 1 , CL18/6 used in the current experiment inhibited neutrophil adhesion to chimeric ICAM-1 (C1,2:H3–5) and anti-CD11a, while CD18/1 was without effect, indicating that the LFA-1-dependent adhesion involved the chimeric ICAM-1 (C1,2:H3–5) on the L cells. Unstimulated neutrophils exhibited minimal adhesion to mock-transfected L cells.

View larger version (30K): [in this window] [in a new window]   Figure 1. Adhesion of unstimulated canine neutrophils to C1,2:H3–5 chimeric ICAM-1-transfected L cells. Coverslips (25 mm) with confluent L-cell transfectants were mounted to adhesion chambers. Freshly isolated canine neutrophils were injected into the adhesion chamber and allowed to adhere at 37°C for 15 min. Effects of adhesion molecules were evaluated in the presence of the following mAbs: 10 µg/ml R7.1 (anti-CD11a), 20 µg/ml MY904 (anti-CD11b), 20 µg/ml R15.7 (antiCD18), 20 µg/ml CL18/1 (anti-ICAM-1, nonblocking), and 20 µg/ml CL18/6 (anti-ICAM-1). Values represent mean ± SEM, n = 3, *P < 0.05.

View larger version (30K): [in this window] [in a new window]   Figure 2. Adhesion of unstimulated canine neutrophils to LPS-stimulated CJVEC monolayers. Confluent canine endothelial cell monolayers were treated with 30 ng/ml of LPS for 18–24 h, washed by dipping in PBS, and incubated with PBS (control), or 20 µg/ml CL18/1 (anti-ICAM-1, nonblocking) or CL18/6 (anti-ICAM-1). These monolayers were then placed in adhesion chambers. Isolated canine neutrophils were incubated at room temperature in PBS, 10 µg/ml R7.1 (anti-CD11a), 20 µg/ml MY904 (anti-CD11b), 20 µg/ml R15.7 (antiCD18), or R7.1 and MY904 combined, and the cell suspension containing antibody was injected into the adhesion chamber. Adherence was determined using a visual assay at room temperature. Values represent mean ± SEM, n = 5, *P < 0.05.

View larger version (19K): [in this window] [in a new window]   Figure 3. Kinetics of hydrogen peroxide production when unstimulated canine neutrophils were exposed to C1,2:H3–5 chimeric ICAM-1-transfected L cells and mock-transfected L cells. Ninety-six-well plates with confluent monolayers of C1,2:H3–5 chimeric ICAM-1-transfected L cells and mock-transfected L cells were rinsed with warm PBS three times before neutrophils were added. Neutrophils at 2 x 104/well were incubated with the L cells up to 3 h. Hydrogen peroxide production was measured by the scopoletin assay. Results were represented as values above the background. Values are mean ± SEM, n = 7, and the curves are significantly different, P < 0.01.

View larger version (29K): [in this window] [in a new window]   Figure 4. Effect of anti-CD11a, anti-CD11b, and anti-ICAM-1 on hydrogen peroxide production from unstimulated canine neutrophils exposed to C1,2:H3–5 chimeric ICAM-1-transfected L cells. Ninety-six-well plates with confluent monolayers of C1,2:H3–5 chimeric ICAM-1-transfected L cells were rinsed with warm PBS three times before neutrophils were added. Neutrophils were incubated with chimeric ICAM-1-transfected L cells in quadruplicate for 3 h in the presence of PBS (control) or F(ab')2 fragments of 10 µg/ml R7.1 (anti-CD11a), 20 µg/ml MY904 (anti-CD11b), 20 µg/ml R15.7 (anti-CD18), 20 µg/ml CL18/1 (anti-ICAM-1, nonblocking), and 20 µg/ml CL18/6 (anti-ICAM-1). Hydrogen peroxide production was measured by the scopoletin assay. Results were represented as values above L-cell basal level. Values are mean ± SEM, n = 7, *P < 0.01.

View larger version (31K): [in this window] [in a new window]   Figure 5. Blocking effect of mAb MY904 (anti-CD11b). (A) KLH-coated coverslips were mounted into adhesion chambers. Canine neutrophils stimulated with 1% ZAS were injected into adhesion chamber in the presence of PBS or F(ab')2 fragments of MY904. Adhesion of neutrophils was measured. (B) Canine neutrophils were added to KLH-coated 96-well plates in the presence of 1% ZAS and F(ab')2 fragments of MY904. Hydrogen peroxide production was measured by scopoletin assay. Values represent mean ± SEM, n = 3. Values for MY904 concentrations above 2.5 µg/ml were significantly different from controls, P < 0.01.

View larger version (28K): [in this window] [in a new window]   Figure 6. Effect of NIF on the hydrogen peroxide production from neutrophils exposed to C1,2:H3–5 chimeric ICAM-1 or KLH. KLH or L cell transfected with C1,2:H3–5 chimeric ICAM-1 was prepared in 96-well plates. Unstimulated neutrophils were added to chimeric ICAM-1 in the presence of PBS or NIF. Neutrophils added on KLH were stimulated with ZAS (1% vol) and incubated in the presence of NIF. Hydrogen peroxide production was measured for 3 h by scopoletin assay. Values represent mean ± SEM, n = 3, *P < 0.05.

View larger version (18K): [in this window] [in a new window]   Figure 8. Kinetics of hydrogen peroxide production from neutrophils exposed to C1,2:H3–5 chimeric ICAM-1 and KLH. KLH or L cell transfected with C1,2:H3–5 chimeric ICAM-1 was prepared in 96-well plates. Unstimulated neutrophils were added to chimeric ICAM-1. Neutrophils added on KLH were stimulated with ZAS (1% vol). Hydrogen peroxide production was measured for 3 h by scopoletin assay. (A) Expanded view of the first 60 min of the experiment shown in (B). Values represent mean ± SEM, n = 7; *, P < 0.01.

View larger version (18K): [in this window] [in a new window]   Figure 9. Effect of cytochalasin B or H7 on hydrogen peroxide production from neutrophils exposed to C1,2:H3–5 chimeric ICAM-1 or KLH. KLH or L cell transfected with C1,2:H3–5 chimeric ICAM-1 was prepared in 96-well plates. (A) Unstimulated neutrophils were added to chimeric ICAM-1 in the presence of PBS (control) or cytochalasin B at 2 µg/ml, 5 µg/ml, or 10 µg/ml. Neutrophils added on KLH were stimulated with ZAS (1% vol) and incubated in the presence of PBS (control) or cytochalasin B. Hydrogen peroxide production was measured after 3 h by scopoletin assay. Values represent mean ± SEM, n = 3, *P < 0.05. (B) Unstimulated neutrophils were added to chimeric ICAM-1 in the presence of PBS (control) or H7 and HA1004 at 150 µM (HA1004 as a chemical control). Neutrophils added on KLH were stimulated with ZAS (1% vol) and incubated in the presence of PBS (control) or H7 and HA1004. Hydrogen peroxide production was measured for 3 h by scopoletin assay. Values represent mean ± SEM, n = 3, *P < 0.05.

View larger version (22K): [in this window] [in a new window]   Figure 7. Effect of R7.1 F(ab')2 (anti-CD11a) on the hydrogen peroxide production from neutrophils exposed to L cells expressing C1,2:H3–5 chimeric ICAM-1 or KLH-coated plastic. KLH and L cells were prepared in 96-well plates. Unstimulated neutrophils were added to wells containing L cells in the presence of PBS or 10 µg/ml of R7.1 F(ab')2. Neutrophils added on KLH were stimulated with ZAS (1% vol). Hydrogen peroxide production was measured at 15 min, 30 min, and 60 min by scopoletin assay. Values are mean ± SEM, n = 3, *P < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
LFA-1 (CD11a/CD18) is capable of triggering cellular activation of lymphocytes and monocytes [²⁴ ]. For neutrophils, LFA-1 is known to facilitate adhesion to endothelial cells and transendothelial migration [²⁵ ]. Berton et al. [⁷ ] found that isolated human neutrophils, when trapped on albumin-coated plastic by surface-bound mAbs to CD11a, would release H₂O₂, and they concluded that LFA-1 could function as a signaling molecule, triggering the oxidative burst. Because Mac-1 is known to bind to albumin-coated surfaces [²⁶ ] and to signal release of H₂O₂ [³ ], it was not clear from the Berton et al. [⁷ ] study that LFA-1 functioned as more than a tethering mechanism potentiating the interactions of Mac-1 with the substrate. In the current study, we have demonstrated that LFA-1 expressed on unstimulated canine neutrophils can mediate adhesion to canine ICAM-1 domains D1 and D2, confirming earlier observations with human neutrophils [⁵ ] and that Mac-1 is not involved in this interaction. In addition, we have shown that this interaction results in significant release of H₂O₂ by canine neutrophils in the absence of exogenous chemotactic activation, thus supporting the conclusions of Berton et al. [⁷ ]. Our results indicate that LFA-1 can mediate adhesion and H₂O₂ production of unstimulated neutrophils interacting with ICAM-1. This conclusion is supported by the following evidence: 1) Unstimulated neutrophils adhered to chimeric ICAM-1 (C1,2:H3–5), and the adhesion was blocked by anti-CD11a but not anti-CD11b. 2) The resulting H₂O₂ production was inhibited by anti-CD11a but not anti-CD11b mAbs. 3) NIF, a specific inhibitor of Mac-1 adherence [²⁷ ], failed to inhibit this adhesion or H₂O₂ production but did block these functions of chemotactically stimulated neutrophils on protein-coated plastic, functions previously shown to be inhibited by anti-CD11b but not anti-CD11a mAbs [³ ]. 4) In addition to data from blocking studies, other evidence indicates that the H₂O₂ release following interaction of canine neutrophils with the C1,2:H3–5 chimeric ICAM-1 was distinct from that following Mac-1-dependent adhesion. The kinetics of reactive oxygen production were measurably faster than with Mac-1 adhesion, the magnitude of H₂O₂ production was significantly less, and in contrast to LFA-1-dependent secretion, that dependent on Mac-1 was inhibited by cytochalasin B and H7.

These findings are of potential interest in light of recent data showing that neutrophil LFA-1 and Mac-1 have several functional distinctions. Isolated neutrophils exhibit negligible Mac-1-dependent adhesion unless exposed to concentrations of chemotactic factors in the chemokinetic range [⁵ ]. In contrast, LFA-1-dependent adhesion to ICAM-1 can be seen without added chemotactic factors or with markedly low stimulus levels [⁵ , ¹⁰ , ²⁸ ]. Emigration, in vitro and in vivo, appears to require LFA-1-dependent adhesion much more than Mac-1-dependent adhesion. Transmigration, in vitro through endothelial monolayers stimulated with IL-1ß, TNF-, or endotoxin [^{29 30 31} ] or in response to chemotactic gradients, is more efficiently blocked by anti-LFA-1 mAbs than anti-Mac-1 mAbs [⁵ , ³² ]. Studies of mice with targeted deletions of CD11a or CD11b reveal significantly reduced emigration of neutrophils in thioglycollate-induced peritonitis in CD11a-deficient mice [³³ ] but no impairment of emigration in mice with CD11b deficiency [³⁴ ]. Rutter et al. [³⁵ ] and Graf et al. [³⁶ ] have found in rabbit models of peritonitis that mAbs blocking Mac-1 were ineffective in preventing neutrophil emigration, and mAbs blocking LFA-1 markedly reduced neutrophil emigration. In a rat model of dermal inflammation, Issekutz and Issekutz [³⁷ ] found that anti-CD11a mAb was more effective than anti-CD11b mAb in reducing neutrophil localization, and Argenbright et al. [¹¹ ] found that anti-CD11a mAb was as effective as anti-CD18 in reducing firm adhesion of leukocytes in an intravital model of inflammation in the rabbit mesentery. Similar observations in a rat model of uveitis were made by Rosenbaum and Boney [³⁸ ]. Thus, it appears that Mac-1 is relatively less important than LFA-1 for neutrophil emigration in many experimental models with canine, rat, mouse, rabbit, and human cells, although optimal transmigration requires cooperation of LFA-1 and Mac-1 [⁴ , ⁵ , ³² , ³⁷ ].

In contrast to the dominant roles of LFA-1, the cytotoxic activity of neutrophils for parenchymal cells appears to be heavily dependent on Mac-1 in vivo and in vitro [⁴ , ¹⁰ , ^{39 40 41} ], a process probably linked to secretory activity markedly augmented by Mac-1-dependent adhesion.

The functional significance of LFA-1-dependent triggering of the oxidative burst in neutrophils is obscure. Because the kinetics are more rapid than with Mac-1-dependent adhesion, oxygen radical production seems likely to immediately follow the process of transendothelial migration. For example, neutrophils contacting endothelial monolayers stimulated in vitro for 3 h with IL-1ß will undergo LFA-1-dependent transmigration in high numbers within <15 min [²⁹ ]. Individual cells attaching to the endothelial monolayer under conditions of shear, transmigrate within 1 min after forming a stable adhesion [⁴² ]. There is evidence from studies in vivo that oxidant stress can occur in cells of the vascular wall at sites of leukocyte adhesion. Using carboxydichlorofluorescein (CDCF) as a probe for intracellular oxidant stress, Suematsu et al. [⁴³ ] observed the mesenteric microcirculation in rats following administration of an inhibitor of nitric oxide synthesis, N^G-nitro-L-arginine methy ester (L-NAME). Significant increases occurred in leukocyte adhesion, a phenomenon first reported by Kubes et al. [⁴⁴ ], and in CDCF fluorescence in endothelium. Anti-ICAM-1 and anti-CD18 mAb significantly attenuated leukocyte adhesion and oxidant stress. Similar results were obtained after superfusion of normal mesenteric vessels with formyl-Met-Leu-Phe (fMLP). Although these authors did not assess the relative contributions of LFA-1 and Mac-1, our results and those of others showing that LFA-1 is heavily involved in the emigration of neutrophils raise the possibility that LFA-1-dependent oxidative burst may be occurring near endothelial cells at the time of transmigration.

	ACKNOWLEDGEMENTS

 
This work was supported by NIH grants HL42550, AI19031, and ES06091 (C.W.S.) and AHA Established Investigators Award (C.B.). The assistance of Michelle Swarthout, Lisa Thurmon, and Celetta Callaway was greatly appreciated.

Received July 21, 1999; revised January 19, 2000; accepted January 20, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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