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Section of Cardiovascular Sciences, DeBakey Heart Center and Department of Medicine, Methodist Hospital, Baylor College of Medicine, Houston, TX 77030;
Spiros P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics, Texas Children’s Hospital, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß subunits of Mac-1 heterodimer were released.
Immunoprecipitation of control PMN lysates revealed bands of 198 kDa
and 91 kDa slightly greater than those from the released Mac-1. An in
vitro model of homotypic aggregation showed a similar loss of Mac-1
from PMNs; immunoprecipitates of the supernatant demonstrated peptide
bands identical with those found in postischemic cardiac lymph. The
appearance of soluble Mac-1 in vitro was prevented by anti-CD18
mAb, R15.7, and also by protease inhibition by PMSF. Thus, in vivo and
in vitro, activated PMNs lose Mac-1 in a process that may be dependent
upon adhesion and subsequent proteolysis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In clinical trials, early reperfusion reduces infarct size, provides better ventricular repair, and improves survival (13). Even when reperfusion is initiated after a longer period of time beyond which reduction of myocardial necrosis can be expected, reperfusion improves ventricular geometry and enhances survival (14). Among the factors thought to be responsible for this improved tissue repair is the increased presence of leukocytes in the infarcted area (15, 16). Reperfusion has been associated with accelerated clearance of necrotic debris presumably as a result of increased phagocytosis by the augmented leukocyte influx (15). Other studies have shown that neutrophils that have been stimulated or transmigrated have decreased apoptosis and thus may remain in tissues for much longer times (17, 18). Thus, leukocytes may potentially increase tissue injury upon reperfusion but may also play a role in later myocardial healing.
In this manuscript we describe a time-dependent loss of Mac-1 that occurs in neutrophils infiltrating the tissue of the reperfused myocardium but not in intravascular neutrophils. Peptides immunoreactive for CD11b and CD18 (but not for CD11a) appear in the cardiac lymph after reperfusion. In vitro, activation of neutrophils with chemotactic stimulants results in loss of Mac-1 into the supernatant, which is at least partially blockable by protease inhibition. Immunoprecipitates of the supernatant of activated neutrophils precipitated by either R15.7 (anti-CD18) or MY904 (anti-CD11b) are identical with that immunoprecipitated from postischemic cardiac lymph.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy mongrel dogs (15–25 kg) of either sex were surgically instrumented as previously described (19, 20). Anesthesia was induced i.v. with 10 mg/kg methohexital sodium (Brevital; Eli Lilly, Indianapolis, IN) and maintained with the inhalational anesthetic Isoflurane (Anaquest, Madison, WI). A midline thoracotomy provided access to the heart and mediastinum, and in some experiments, the cardiac lymph duct was cannulated as previously described (19, 20). Subsequently, a hydraulically activated occluding device and a Doppler flow probe (19, 21) were secured around the circumflex coronary artery just proximal or just distal to the first branch. The animals were allowed to recover for at least 72 h before occlusion. Ischemia-reperfusion protocols were performed in awake animals as previously described (9, 19, 21, 22). Coronary artery occlusion was achieved by inflating the coronary cuff occluder until mean flow in the coronary vessel was zero, as determined by the Doppler flow probe. After 50 min of occlusion, radiolabeled microspheres (for subsequent blood flow determinations) were injected into the left atrium. At the end of 1 h of occlusion, the cuff was deflated and the myocardium was reperfused. Reperfusion intervals ranged from 1 to 24 h with cardiac lymph collected and spun at 10,000 x g for 4 min to remove cells and particulates, and protease inhibitor mixture (Complete protease inhibitor mixture; Sigma, St. Louis, MO) was added and immediately frozen in liquid nitrogen and kept at -80°C until analyzed. Upon sacrifice, samples were taken systematically from the left ventricle and ischemic blood flow was quantitated. Control (no evidence of necrosis and normal blood flow) and ischemically injured myocardium (based on methods mentioned below and on reductions in blood flow) were thus defined.
Myocardial sampling and calibration with coronary blood flow
After the reperfusion periods, hearts were stopped by the
infusion of saturated potassium chloride, removed from the chest, and
sectioned from apex to base into four transverse rings 
1 cm in
thickness. Transmural myocardial samples (1.0 g) were isolated from
myocardial rings and labeled as control (obtained from the anterior
wall) or infarcted (obtained from the posterior papillary muscle and
posterior free wall) based on anatomic location within the distribution
of the circumflex artery and on visual inspection. Myocardial samples
were then dissected into smaller pieces, with adjacent transmural
sections taken and fixed in 2% buffered paraformaldehyde for
immunostaining or used for blood flow determinations using radiolabeled
microspheres as previously described (21, 23, 24, 25).
Analysis of blood flow determinations and histopathologic examinations
of samples obtained from each experiment were later matched to ensure
that reduced blood flow occurred in appropriate samples.
Histology and immunohistochemistry
For histologic studies, samples of cardiac tissue were calibrated for the level of coronary blood flow as previously described (21, 24, 25) and fixed in 2% paraformaldehyde, 4% formalin, or B*5 (ZnCl) (26). The samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (Stat Lab, Lewisville, TX) or periodic acid schiff (PAS;3 Sigma) or immunostained as indicated. mAbs to CD11b (MY904-ATCC and CA16.3E10), CD11a (R7.1), CD18 (R15.7), and the neutrophil-specific Ab SG8H6 (28) are used in this study. SG8H6 is a mAb (27) specific for canine neutrophils that is made in our laboratory. The epitope is not known but exists on canine neutrophils at a concentration of 9.6 x 105/polymorphonuclear neutrophil (PMN). In each case, controls were treated with isotype control IgG. Ab detection utilized peroxidase-based secondary Ab detection systems using either diaminobenzidine (DAB) or aminoethylcarbonate as a substrate as indicated following the manufacturer’s protocols (Vector Laboratories, Burlingame, CA) for staining. Counterstaining was hematoxylin, eosin, Fast Green (Fisher), or none as indicated.
Slot blots
Slot blots were performed using cardiac lymph samples (250 µl) or BSA (250 µl, 10 mg/ml) and IgG as controls by filtration onto a nylon membrane in a slot blot apparatus (Bio-Rad, Richmond, CA) before a 1-ml rinse of each well with PBS. After blotting, the membranes were removed and blocked for 30 min in blocking buffer (1% nonfat dry milk and 1% normal goat serum in PBS) before Ab binding for 1 h at room temperature using mAbs at 1:100 dilution in blocking buffer. Membranes were washed in PBS twice for 30 min before detection of the Abs by a biotinylated secondary Ab and an avidin-streptavidin peroxidase-based detection system using DAB as a substrate. As above, isotype-specific IgG served as a control.
Neutrophil isolation and stimulation
Canine neutrophils were isolated from citrate anticoagulated venous blood (citrate phosphate dextrose, Sigma; 1.4 cc CPD/10 cc blood) using a previously described Ficoll-Hypaque density centrifugation technique (28). After isolation the neutrophils were resuspended to 10 million cells/ml in RPMI 1640 (Sigma) containing 5% FBS and in an antibiotic/antimycotic solution (Sigma) containing penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (250 pg/ml). For ELISA studies, 250 µl of the cell suspension was placed into capped 15-ml polypropylene tubes and incubated at 37°C in a shaking convection incubator for various times as indicated (80 cycles/minute). For immunoprecipitation studies, 1 ml of cell suspension was used. The neutrophils were exposed to 4% zymosan-activated dog serum (ZADS), a source of C5a. ZADS was prepared by incubating 10 mg of zymosan A (Sigma) per ml of dog serum for 45 min at 37°C before a 30-min incubation at 56°C, centrifugation at 7000 x g for 2 min, and then isolation of the supernatant. Additional cultures were exposed to ZADS in the presence of either 10 µg/ml R15.7 (biotinylated) or a mAb to human or canine CD18 (IgG1, provided by Dr. R. Rothlein, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT). The same biotinylated R15.7 used in the ELISA (see below) was capable of blocking CD18-dependent adhesion and was used to avoid interference with the ELISA detection. Experiments in which neutrophils were stimulated with recombinant IL-8, a final concentration of 50 ng/ml was used (Genzyme, Cambridge, MA). Experiments using PMSF had 200 µm PMSF added at time 0 (before ZADS or IL-8 addition). Controls not exposed to ZADS were included in every experiment. After 1–3 h of incubation (as indicated), the cells were centrifuged at 350 x g for 5 min before centrifugation at 100,000 x g for 10 min, and the supernatants were isolated, and protease inhibitor mixture was added (Complete protease inhibitor from Sigma) and stored in liquid nitrogen for later assay for Mac-1 release or immunoprecipitation. For native neutrophil CD11b and CD18 (canine) immunoprecipitation, freshly isolated neutrophils were stimulated with 10% ZADS for 5 min before addition of lysis buffer (150 mM NaCl, 1.0% Nonidet P-40, and 50 mM Tris (pH 8.0)) and centrifugation. The supernatants were isolated, and protease inhibitor mixture was added and frozen for later use.
Mac-1 ELISA
The Mac-1 ELISA was conducted in 96-well microtiter plates (Maxisorp Immunoplates, Nunc, Naperville, IL) coated overnight at 4°C with 100 µL of 10 µg/ml MY904 F(ab')2 fragments in PBS. Plates were washed six times with 0.01% Tween 20 (Sigma) in PBS (washing was performed in the same manner between each subsequent step) and blocked for 2 h at 37°C with 2% BSA (Sigma) in PBS. Thawed culture supernatants were diluted 1:5 in RPMI 1640 and added to the wells (100 µL/well; n = 4 wells/sample). As a positive control, a 0.1% Triton X-100 neutrophil lysate aliquot was added to each plate, which served as an interassay standard as well. The plates were incubated overnight at 4°C. Subsequently, 100 µL of 2 µg/ml biotinylated R15.7 in PBS containing 2% BSA was added and incubated for 2 h at room temperature. Next was a 45-min incubation at room temperature with 100 µL of an avidin/biotin complex conjugated with HRP (Vector Laboratories) diluted 1:100 in PBS. Substrate solution 3,3',5,5'-tetramethyl-benzidine (Sigma; 200 µL/well) was then added and incubated for 30 min at room temperature before the reaction was terminated by the addition of 100 µL of 0.5 M H2SO4. The absorbance was then read at 450 nm on a microtiter plate reader (Elx800, Bio-Tek, Burlington, VT).
Immunoprecipitation
Culture supernatants, cardiac lymph, or native neutrophil
lysates were thawed and mAb R15.7 (anti-CD18) or mAb MY904
(anti-CD11b) was added to a final concentration of 25 µg/ml.
After incubation on ice for 1 h, 100 µl of washed protein G
(fixed group C Streptococcus sp. cell suspension, Sigma) was
added and incubated on ice for 30 min. The mixture was then centrifuged
at 7000 x g for 1 min, after which the supernatant was
removed and the pellet was washed with Nonidet P-40 cell lysis buffer
(150 mM NaCl, 1.0% Nonidet P-40, and 50 mM Tris (pH 8.0)). This was
repeated two more times. The final pellet was resuspended in 40 µl of
sample buffer (1% SDS, 50% glycerol, 10 mM DTT, and 0.004%
bromphenol blue). Samples were heated at 80°C for 5 min and then
loaded onto a 6–15% gradient SDS polyacrylamide gel with a 5%
stacker and electrophoresed at 120 V for 3 h. After electrophoresis,
the gel was stained with 0.25% Coomassie blue in Weber stain (45%
methanol and 9% acetic acid) for 30 min and destained in 10% acetic
acid. Samples were also slot blotted on nylon membranes and
immunostained with directly biotinylated Abs to the opposite ligand
(MY904 immunoprecipitations were stained with R15.7 mAb and R15.7
immunoprecipitations were stained with MY904 mAb) using standard
avidin-biotin complex staining techniques. Standards, samples, and
backgrounds were scanned and intensity was quantitated in relation to
an internal standard and background subtraction using ImageTool from
the University of Texas Health Science Center in San Antonio
(TX).
Statistics
All cardiac lymph experiments were repeated with at least five separate animals at each time point. Analysis of the appearance of CD11b and CD18 both in vivo and in vitro was performed using one-way ANOVA using the software In-Stat (San Diego, CA). All values are expressed as means + SD. Statistical significance is noted where indicated in comparison to the appropriate reference point.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
, A and
B demonstrate that both intravascular and extravascular
neutrophils stained for CD11b with the mAb MY904 after 3 h of
reperfusion (solid arrows), although some extravascular neutrophils do
not appear to stain (arrowheads). In all experimental animals
(n = 5) after 1–3 h reperfusion, all intravascular
neutrophils stained positive for CD11b using the mAb MY904, whereas
extravascular neutrophils showed varying degrees of CD11b staining that
decreased with time after reperfusion. A sample taken after 5 h of
reperfusion and stained for CD11b is shown in Fig. 2. Extravascular neutrophils (open
arrows) demonstrated minimal staining for CD11b (MY904 mAb) by this
time, although staining of the amorphous extracellular matrix (filled
arrows) is seen (Fig. 2). This staining pattern was seen in all five
animals. A section stained with a mAb to CD18 (R15.7) stained both
neutrophils (filled arrows) and the extracellular matrix (open arrows)
(Fig. 3B). In contrast, the Ab
R7.1 to CD11a (Fig. 3A) demonstrated staining on all
leukocytes (filled arrows) with no staining of the extracellular
matrix. This staining pattern suggests that although CD11b staining of
the extravascular neutrophils was markedly reduced, CD11a staining of
all leukocytes was retained. CD18, the common B-subunit for both Mac-1
(CD11b/CD18) and LFA-1 (CD11a/CD18), remained associated at this time
with both the Mac-1 extracellular matrix staining and the
leukocyte-associated LFA-1.
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demonstrates serial sections of a
border zone for myocardial infarction after 24 h of reperfusion.
The viable border zone is demarcated by periodic acid schiff (Fig. 4A). Neutrophil staining using the Ab SG8H6 in five separate
animals showed that neutrophils are now predominantly in the infarct
zone (example shown in Fig. 4B). In Fig. 4C, the
extravascular neutrophils no longer stain with mAbs to CD11b. In
addition, the staining of the extracellular matrix seen in Figs. 2
and 3 was lost. As seen at 5 h of reperfusion, neutrophils retain
their CD11a and CD18 staining at 24 h (data not shown).
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To characterize the apparent loss of specific immunocytochemical
staining for CD11b in tissues, we examined cardiac lymph samples taken
before occlusion and some taken at various times after reperfusion for
evidence of Mac-1 or its fragments. Fig. 5 demonstrates the results of slot blots
from five consecutive animals demonstrating the presence of
immunoreactivity to MY904 (anti-CD11b) and R15.7 (anti-CD18)
binding at 5 h of reperfusion. No significant immunoreactivity was
detected with the anti-CD11a Ab R7.1.
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, both the
anti-CD11b and the anti-CD18 Abs precipitated identical protein
bands, a doublet with a molecular mass of 190 kDa on the lower band
and an 80-kDa band. The molecular masses of these bands are smaller
than those of intact canine CD11b (198 kDa) or CD18 (91 kDa) (Fig. 6, lane C), but presumably represent proteolytic fragments.
Because both Abs precipitate identical bands, the data suggest that
both subunits of the CD11b/CD18 heterodimer were released. Attempts at
immunoprecipitation of preischemic lymph or supernatant from
unstimulated neutrophils with both mAbs revealed no evidence of
proteolytic fragments (data not shown).
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demonstrates quantitation of Mac-1 from
five experimental animals that underwent occlusion and reperfusion with
myocardial infarcts. Mac-1 was detected in cardiac lymph by 3 h of
reperfusion (**, p < 0.001). The time course for
the presence of Mac-1 in cardiac lymph using the ELISA technique gave
findings similar to those observed using slot blot analysis (Fig. 5).
In sham control animals (occluded animals that did not develop an
infarct; n = 3), no increase was seen in Mac-1 during
the reperfusion phase.
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Neutrophils were isolated as described in Materials and
Methods to determine whether neutrophils can lose Mac-1 in vitro.
Fig. 8 demonstrates that rotational
mixing of neutrophils in the presence of 4% ZADS resulted in the
appearance of Mac-1 fragments in the supernatant within 1 h. Fig. 9 demonstrates that the addition of
biotinylated R15.7 (anti-CD18; which has previously been
demonstrated to inhibit homotypic aggregation) (8)
markedly decreased detection of Mac-1 in the supernatant. As stated in
Materials and Methods, the use of the same biotinylated
R15.7 used in the ELISA was designed to obviate potential interference
with ELISA detection of Mac-1 fragments. To be certain that this was
so, the ZADS incubation was conducted in a manner identical to that in
Fig. 8 except that at the end of incubation biotinylated R15.7 was
added to the reaction before centrifugation and ELISA. Mac-1 fragments
detected in the supernatant were not different in quantity from those
detected without the late addition of biotinylated R15.7. Thus, the
inhibition observed when R15.7 was present throughout the reaction
resulted from specific CD18 blockade. The studies in Figs. 8 and 9 were
repeated with similar results using IL-8 (50 ng/ml) as a stimulus for
homotypic aggregation (data not shown).
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shows proteins that were immunoprecipitated from neutrophil supernatant
after stimulation for 2 h with ZADS using MY904 (Fig. 6, lane D) and R15.7 (Fig. 6, lane A). Fig. 6, lane C (native neutrophils) has two bands of interest: the
198-kDa band (consistent with the molecular mass of human CD11b) and
the 91-kDa band (consistent with human CD18). Both the supernatant from
in vitro-stimulated neutrophils (Fig. 6, lanes A
and D) and the cardiac lymph (Fig. 6, lanes
B and E) show a high molecular mass doublet,
having a molecular mass of 190 kDa. The presence of this doublet
suggests proteolytic cleavage of the CD11b in both the in vitro
neutrophils’ supernatant and the cardiac lymph (Fig. 6). Similarly,
there is appearance of 80-kDa bands in samples immunprecipitated from
cardiac lymph and in the supernatant of in vitro-stimulated
neutrophils. The reduction in molecular masses (from 198 kDa to 190 kDa
and from 91 kDa to 80 kDa) is consistent with the proteolytic cleavage
of a cytoplasmic portion of the CD11b and CD18 molecules, respectively.
In Fig. 10, we demonstrated that PMSF
reduced the release of Mac-1 into the supernatant (>60%). At this
concentration, PMSF did not affect initial homotypic aggregation
measured by flow cytometry in the first 15 min (S. Simon, unpublished
observation). In contrast to the in vivo observation in Fig. 2, histologic examination of the neutrophils from rotational mixing
studies demonstrated dramatic reduction of MY904 staining; however,
significant staining persisted during the 5-h period studied. This
suggests the possibility that tissue proteases may accentuate Mac-1
loss; however, the specificity of the fragments and the specificity of
the target (Mac-1) suggests that the in vivo and in vitro studies
derive primarily from the same mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, leukocyte infiltration associated with postreperfusion injury in a number of experimental paradigms more likely evolved as an important part of a mechanism of myocardial healing (30). Reperfusing previously ischemic myocardium, with its attendant inflammatory response, enhances tissue repair (15, 31, 32). We hypothesize that neutrophil-induced inflammatory injury occurs under circumstances in which normal defenses designed to prevent injury of healthy tissue are ineffective or inadequate. In the case of postreperfusion-inflammatory responses, reperfusion of a vascular bed rich in chemotactic factors (16) might result in a very intense inflammatory response that would overwhelm such a defense system under some clinical and/or experimental circumstances.
It might be postulated that proteolysis or loss of Mac-1 represents one such defense mechanism. In the in vitro experiments, Mac-1 loss begins within an hour and is dependent on chemotactic stimulation and integrin adhesion. Loss of Mac-1 generates proteolytic fragments that are similar to those encountered in postischemic cardiac lymph. The time course of their generation after chemotactic stimulation is similar to the time course of the loss of neutrophil Mac-1 and appearance of proteolytic fragments in vivo after reperfusion (after exposure of neutrophils to a highly chemotactic environment (23, 33)).
The data suggest that the loss of Mac-1 occurs as a result of an event involving loss of about 10 kDa from both CD11b and CD18. The inhibition by the serine protease inhibitor PMSF suggests proteolysis by a neutrophil-derived protease. This loss is specific for Mac-1; we were unable to detect loss of LFA-1 either histologically or immunochemically. The fragments of CD11b and CD18 are found in the cardiac lymph or in vitro supernatants as a heterodimer that can be immunoprecipitated by Abs to either CD11b or CD18 and can be readily measured in an ELISA using an anti-CD11b (MY904) mAb as the capture Ab and an anti-CD18 (R15.7) Ab as the detection Ab. The release of soluble Mac-1 in vitro has been demonstrated before in cell supernatants after phorbol mysistate treatment of neutrophil monolayers (34). This was associated with lactoferrin release and exocytosis of full-length Mac-1 (CR3), in contrast to the proteolytized Mac-1 under our conditions and observed in vivo by us in this report.
In each case, relatively intact soluble ß2
integrins are released into the surrounding extracellular fluid. It has
been suggested in the past that soluble integrins might function as
anti-inflammatory protectants or signaling molecules
(35). Studies have shown that recombinant soluble Mac-1
heterodimer is capable of inhibiting stimulated neutrophils from
binding to cultured endothelial cells (36). We have
previously shown that the neutrophil infiltration of reperfused
myocardial infarct takes place initially in the viable border zone
where they are found over the first 5 h (22, 27).
This is also demonstrated in
Figs. 1–3 where neutrophils are
infiltrating an area of normal myocyte architecture. At 24 h (Fig. 4), the neutrophils appear to have migrated primarily to the infarcted
area where they remain for several days as demonstrated previously
(27). This expanded life span is compatible with the data
of Watson et al. (17), which demonstrate comparable delays
in apoptosis after migration of neutrophils across an endothelial
monolayer. Based on the discussion above, one might speculate that
these neutrophils play an important role in tissue repair; precise
mechanisms by which this could occur remain to be investigated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Keith A. Youker, Department of Medicine, Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498. E-mail address:
3 Abbreviations used in this paper: PAS, periodic acid schiff; PMN, polymorphonuclear neutrophil; DAB, diaminobenzidine; ZADS, zymosan-activated dog serum.
Received for publication July 1, 1999. Accepted for publication December 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated apoptosis of human neutrophils. Kidney Int. 53:84.[Medline]
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).
