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Circulation. 1997;95:684-692

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(Circulation. 1997;95:684-692.)
© 1997 American Heart Association, Inc.

Articles

Complement C5a, TGF-ß1, and MCP-1, in Sequence, Induce Migration of Monocytes Into Ischemic Canine Myocardium Within the First One to Five Hours After Reperfusion

Holly H. Birdsall, MD, PhD; David M. Green, MS; JoAnn Trial, PhD; Keith A. Youker, BA; Alan R. Burns, PhD; Charles R. MacKay, PhD; Gregory J. LaRosa, PhD; Hal K. Hawkins, MD; C. Wayne Smith, MD; Lloyd H. Michael, PhD; Mark L. Entman, MD; Roger D. Rossen, MD

the Immunology Research Laboratory and the Research Center for AIDS and HIV Infections at the Houston Veterans Affairs Medical Center (H.H.B., D.M.G., J.T., R.D.R.); Section of Cardiovascular Sciences and DeBakey Heart Center, Methodist Hospital, Houston (K.A.Y., L.H.M., M.L.E.); Speros P. Martel Laboratory of Leukocyte Biology at Texas Children's Hospital, Houston (A.R.B., C.W.S.); Department of Pathology, University of Texas Medical Branch, Galveston (H.K.H.); Immunology Department, Leuko Site, Inc, Cambridge, Mass (C.R.M., G.J.L.); and the Departments of Otorhinolaryngology (H.H.B., D.M.G.), Medicine (J.T., K.A.Y., L.H.M., M.L.E., R.D.R.), Microbiology (H.H.B., C.W.S., R.D.R.), and Pediatrics (A.R.B., C.W.S.), Baylor College of Medicine, Houston, Tex.

Correspondence to Holly H. Birdsall, MD, PhD, Bldg 109, Room 230, Veterans Affairs Medical Center, 2002 Holcombe Blvd, Houston, TX 77030. E-mail birdsall@bcm.tmc.edu.


*    Abstract
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 *Abstract
down arrowIntroduction
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 down arrowResults
 down arrowDiscussion
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Background Recent studies suggest that reperfusion promotes healing of formerly ischemic heart tissue even when myocardial salvage is no longer possible. Since monocyte-macrophage infiltration is the hallmark of the healing infarct, we have attempted to identify mechanisms that attract monocytes into the heart after reperfusion of ischemic canine myocardium.

Methods and Results Isolated autologous 99mTc-labeled mononuclear leukocytes injected into the left atrium localized preferentially in previously ischemic myocardium within the first hour after reperfusion. Histological studies revealed CD64+ monocytes in small venules and the perivascular connective tissue within the first hour after reperfusion. Flow cytometric analysis of cells in cardiac lymph showed systematically increasing numbers of neutrophils and monocytes between 1 and 4 hours after reperfusion; monocyte enrichment was eventually greater than neutrophil enrichment. Monocyte chemotactic activity in cardiac lymph collected in the first hour after reperfusion was wholly attributable to C5a. Transforming growth factor (TGF)-ß1 contributed significantly to this chemotactic activity after 60 to 180 minutes, and after 180 minutes, monocyte chemotactic activity in lymph was largely dependent on monocyte chemoattractant protein (MCP)-1 acting in concert with TGF-ß1.

Conclusions Beginning in the first 60 minutes after reperfusion, C5a, TGF-ß1, and MCP-1, acting sequentially, promote infiltration of monocytes into formerly ischemic myocardium. These events may promote the healing of myocardial injury facilitated by reperfusion.


Key Words: leukocytes • blood cells • immune system • ischemia • reperfusion


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Prompt reperfusion often results in a dramatic influx of inflammatory cells into formerly ischemic myocardium.1 Some evidence suggests that this inflammatory response is responsible for some of the myocardial damage that follows a coronary artery occlusion.2 3 4 5 Neutrophils rapidly accumulate in the microvasculature of reperfused myocardium coincidently with the appearance of complement C5a in the cardiac extracellular fluids.6 7 Thereafter, neutrophils migrate into and accumulate amidst the damaged muscle cells.8 9 10 11 The potential for these neutrophils to specifically injure cardiac myocytes has been demonstrated in vitro. Neutrophils activated by C5a preferentially adhere to and inject reactive oxygen radicals into isolated cardiac myocytes that have been induced to display ICAM-1 on their cell surfaces.12 Indeed, neutrophils may not be the only cells infiltrating formerly ischemic myocardium within the first few hours after reperfusion. Interleukin-6 has been found in extravascular lymphatic fluids from ischemic myocardium within minutes to hours after reperfusion, suggesting that monocytes and lymphocytes may also accumulate in damaged heart tissue as a result of this inflammatory cell response.13

Although it is likely that infiltrating neutrophils injure cardiac myocytes, monocytes and macrophages that enter cardiac tissue during reperfusion may have other roles, including clearance of tissue debris and the promotion of scar tissue formation.14 15 16 Consistent with the idea that infiltrating monocytes facilitate a component of the inflammatory response that promotes healing, Morita et al17 observed more macrophages in reperfused myocardium than in myocardium that was not reperfused after coronary artery occlusion. It has been recognized for some time that inflammation associated with myocardial ischemia also benefits healing, since global suppression of the inflammatory response either with corticosteroids or nonsteroidal anti-inflammatory agents results in poor scar formation and an unacceptably high incidence of ventricular aneurysm formation.18 19 20

In classic histopathology descriptions, neutrophils are noted to predominate during the first 12 to 24 hours, whereas monocytes and macrophages are found in the cardiac tissues 2 or 3 days after the ischemic event.21 22 However, C5a, the dominant chemotaxin for neutrophils, is also a chemotaxin for monocytes. Thus, it is possible that monocytes begin to enter the myocardium within the first hours after reperfusion. Monocytes mixed with large numbers of neutrophils would be difficult to identify in traditional hematoxylin-eosin–stained sections of myocardial infarcts. Monocytes infiltrating myocardial tissue would be more prominent 2 to 3 days after the infarct, when the neutrophils would have died off and the monocytes have ingested necrotic material, thereby enhancing their visibility. In these studies, we sought to determine whether monocyte localization into previously ischemic myocardium begins in the first few hours after reperfusion. The present report identifies cellular and molecular mechanisms that regulate monocyte trafficking during reperfusion of previously ischemic myocardium. In these studies, we show that three chemotactic factors, C5a, TGF-ß1, and MCP-1, appear sequentially in cardiac extracellular fluids after reperfusion of ischemic myocardium, and we provide evidence that these regulate intravascular accumulation and transendothelial migration of monocytes into the formerly ischemic myocardium. Because of the postulated role of tissue macrophages as architects of tissue remodeling after ischemic injury, we have investigated, and present in a companion article,23 further evidence concerning the tissue sources of this important chemokine and mechanisms that induce MCP-1 after cardiac ischemia-reperfusion injury. Taken as a whole, the present studies and the work submitted for publication suggest that after reperfusion, well-orchestrated mechanisms exist to promote an intense early margination of both neutrophils and monocytes in the microvasculature of formerly ischemic myocardium followed by a sustained migration of these leukocytes into the cardiac tissues.


*    Methods
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 up arrowIntroduction
 *Methods
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Animal Models
The canine models used in these experiments have been described in detail.9 24 25 In brief, a hydraulic occluder was placed just proximal to the first branch of the circumflex coronary artery, and the cardiac lymph duct was cannulated. The dog was awakened and maintained for 3 days to allow the inflammation around the lymph cannula to subside before performance of a closed-chest occlusion-reperfusion experiment under general anesthesia. Occlusion and reperfusion were verified by blood flow changes measured with a Doppler flow probe previously implanted distal to the occluder and by characteristic ST-segment changes on the ECG. Regional ventricular blood flow before, during, and after occlusion was measured from the accumulation of microspheres labeled with either 85Sr, 95Nb, or 46Sc that were injected into the left atrium.9 After the 1-hour occlusion, the occluder was released gradually to reduce the likelihood of hemorrhagic infarction and the animal was reperfused for 1 to 24 hours. In some experiments, 99mTc-labeled autologous MNLs were injected into the left atrium. These were injected 45 minutes before the end of the reperfusion period, an interval previously established as minimizing hemodynamic effects related to reperfusion hyperemia and maximizing time of access of 99mTc-labeled leukocytes to the myocardial circulation after ischemia-reperfusion injury.9 At the end of the reperfusion interval, the heart was stopped by an infusion of potassium chloride, and the left ventricle was stained with TTC to identify zones of infarcted tissue in the area at risk.6 For the studies with infusion of radiolabeled MNLs, we included data only from dogs with significant infarcts (defined as involving >20% of the left ventricle). When analyzing the cellular composition of the cardiac lymph, we compared the results from 6 dogs with significant infarcts with the results obtained from 4 dogs who showed negligible infarcts after occlusion (typically <2% of myocardium infarcted, presumably because of collateral circulation).

For radionuclide counting, the heart was cut into five transverse slices from the base (slice 1) to the apex (slice 5). Each slice was divided into transmural segments representing the anterior, anterior papillary, lateral, posterior papillary, posterior, and septal sections of the left ventricular wall and then further subdivided into 1 epicardial, 2 midmyocardial, and 1 epicardial pieces. Each weighed piece was counted for radioactivity, and blood flows for each time interval were calculated.9 26 The magnitude of ischemia was estimated by the ratio of the blood flow during occlusion to the preocclusion blood flow for each piece. The numbers of leukocytes accumulating per gram of myocardium were calculated from the known specific activity of the 99mTc-labeled leukocytes. The presence of infarcted tissue in the section, based on TTC staining, was also recorded.

Cardiac lymph emerging through the cannula was collected into polypropylene tubes containing preservative-free heparin and centrifuged. The cell-free supernatant was frozen in liquid nitrogen, and the cells were resuspended in PBS.

Isolation of MNLs
All fluid reagents and media used with leukocytes were tested for and found to be free of endotoxin to the limits of the Limulus amebocyte assay (Associates of Cape Cod). MNLs were isolated from heparinized blood of the experimental subject on the day of the occlusion-reperfusion experiment by sedimentation through Ficoll-Hypaque gradients (Organon-Teknicon). Isolated MNLs were further enriched for monocytes with a modified Recalde method.27 The preparations contained 73±2% (mean±SEM) monocytes, 18±2% lymphocytes, and 5±1% eosinophils, as estimated by Wright-Giemsa–stained cytocentrifuge preparations and independently by flow cytometric analysis using MAbs specific for monocytes, T and B lymphocytes, and neutrophils. The isolates were >95% viable by trypan blue dye exclusion and free of platelets. No further enrichment was performed because attempts to isolate canine monocytes by sedimentation over 46% Percoll, by adherence, or by negative depletion using antibody-coated magnetic beads to remove lymphocytes often resulted in activation of the monocytes, as demonstrated by shedding of cell surface L-selectin and increased expression of CD11c/CD18.

Monocyte preparations were promptly labeled with 99mTc under conditions that resulted in >=90% stable cell-associated 99mTc, as shown previously.9 These cells were also analyzed by flow cytometry for L-selectin and CD11c/CD18 to ensure that cell surface expression of these antigens was not different from those found on MNLs in unfractionated whole blood.

Immunofluorescence and Flow Cytometric Evaluation
Aliquots (0.1 mL) of whole blood or cardiac lymph MNLs at 0.5x106/mL in PBS with 30% autologous serum were placed in polypropylene tubes. Cells were stained for surface markers as previously described.28 The leukocytes were fixed with 1% paraformaldehyde, and at least 5000 leukocytes were analyzed by flow cytometry. Leukocytes were initially selected for fluorescence analysis by a combination of right- and low-angle forward-light-scatter properties. The distribution of monocytes, lymphocytes, and neutrophils measured by flow cytometry was the same as that determined by examination of Wright-Giemsa–stained cytocentrifuge preparations of the same samples. Total white blood cell counts were measured by a Coulter counter (Coulter Electronics).

Histochemical Studies of Fixed Tissues
We examined tissues from the infarcted myocardium and the adjacent border zone taken from cardiac segments with occlusion regional blood flows that were <20% of preocclusion blood flows. Control tissues were selected from areas distant from any infarct. The results of regional blood flow studies were examined to verify that these control tissues were taken from areas that had remained well perfused during the 1-hour occlusion. Tissue segments were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained as previously described.29 For some studies, sections were treated to remove paraffin, hydrated in PBS, and incubated with MAbs reactive with canine monocytes or neutrophils. This was followed by peroxidase-labeled sheep anti-mouse IgG and diaminobenzidine as a substrate, which imparts a yellow-brown color to the positive cells. To accurately distinguish monocytes and neutrophils, we often used back-to-back serial sections in which one or the other cell type is positively identified with specific Abs. Sections were also probed by in situ hybridization for mRNA of ICAM-1, as previously described, to identify the border zones surrounding the areas of myocardium showing contraction band necrosis.29 Tissues for transmission electron microscopy were fixed in 4% glutaraldehyde and processed as previously described.30

MAbs Specific for Leukocytes
Monocytes were identified with PE-conjugated anti-CD14 (clone TUK4, Dako) for flow cytometry studies and anti-CD64, the Fc{gamma}1 receptor (clone 10.1, Ancell), in tissue sections. Both of these antibodies are specific for cells of the monocyte-macrophage lineage, and their selective reactivity with canine cells was verified on cytocentrifuge preparations of blood MNLs. Anti-CD5 MAbs (clone DH3B) to identify T lymphocytes and anti-CR2 (clone F46A) to identify B lymphocytes were made against canine cells (VMRD, Inc). SG8H6 recognizes canine neutrophils,31 R6.5 recognizes CD54 (ICAM-1),32 R15.7 recognizes CD18,33 and M904 recognizes CD11b.34

Chemotaxis Assay
The chemotaxis assay was performed as previously described.35 Briefly, collagen pads are allowed to polymerize in Millicell chambers (Millipore) floored with 0.45-µm filters. The source of the chemotaxin (canine cardiac lymph diluted 1:1 with medium, or chemotactic factors diluted in medium) is added below the well and diffuses up into the collagen. MNLs added to the upper chamber migrate into the collagen pad in response to the chemotaxin. After 4 hours, free MNLs are washed away, and the pad is digested with collagenase to recover the migratory cells. The migrating monocytes were positively identified by staining with PE–anti-CD14 and enumerated with the flow cytometer. Control wells contained only medium (RPMI with 10% heat-inactivated FCS) below the collagen and provided an estimate of random leukocyte migration (eg, not induced by specific chemotactic agents). Chemotactic responsiveness was reported as the net number of monocytes migrating in response to a specific agent above the baseline of random migration.

We used blocking antibodies to identify specific chemotactic agents in canine cardiac lymph. Migration attributable to C5a was blocked by addition of 7 µL of a rabbit anti-canine C5a,7 the amount required to neutralize the chemotactic activity of 10% ZADS. ZADS, a source of complement C5a, was prepared by incubation of zymosan (10 mg/mL, Sigma) with fresh serum (45 minutes at 37°C followed by 30 minutes at 56°C) and was used within 30 minutes. TGF-ß1–mediated chemotaxis was blocked with 7 µL/well of anti–TGF-ß1 (Genzyme), a quantity that was sufficient to block the chemotactic activity of 2 pg/mL of recombinant TGF-ß1 (R&D Systems) in our assay. MCP-1–mediated migration was blocked with a novel anti–human MCP-1 MAb, LS27.10F7-2 (IgG1), prepared against recombinant human MCP-1 in the facilities of Leuko Site, Inc. This MAb reacts in ELISA with recombinant human MCP-1 but not with MCP-2 or MCP-3. It inhibits binding of recombinant human MCP-1 to THP1 cells and reacts with activated canine endothelium.

To further verify the contribution of MCP-1 as a chemotaxin in cardiac lymph, we specifically desensitized canine monocytes by incubating 1x106 MNLs with 100 ng/mL of recombinant human MCP-1 (R&D Systems) for 30 minutes at 37°C and washing the cells before adding them to the chemotaxis assay. As shown in the "Results" section, this treatment completely suppressed canine monocyte migration in response to 120 ng/mL of MCP-1 without impairing the ability to migrate in response to C5a.

Statistical Analysis
Statistical significance was estimated with Student's t test and by nonparametric analyses, including the Spearman rank correlation test and the Wilcoxon signed rank test as indicated. To estimate the influence of ischemia and infarct on MNL accumulation, we performed an ANOVA with MNL uptake (cells per gram) as the dependent variable and ischemia (ratio of occlusion blood flow to preocclusion blood flow) and infarct (present or not) as the independent variables for each segment.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Localization of Radiolabeled Monocytes in the Ischemic Left Ventricle
Autologous radiolabeled MNLs infused during the first hour of reperfusion accumulated preferentially in endocardial and midmyocardial segments that had significant regional myocardial ischemia (generally <20% of preocclusion values) (Fig 1Down). By ANOVA, the accumulation of MNLs was significantly correlated with the magnitude of the ischemia during the occlusion period plus the presence or absence of infarct in the segment (P<.0001 for each dog). Regression weights indicated that both ischemia and infarct were correlated with MNL accumulation (P<.01 for each variable in all 3 dogs). When MNLs were infused 3, 6, or 24 hours after reperfusion, their numbers accumulating in the heart were generally lower, and there was no preferential localization to particular areas of the myocardium. By ANOVA, MNL uptake was not significantly correlated with either ischemia or presence of infarct in cardiac segments from dogs infused with radiolabeled MNLs after reperfusion for 3 hours (2 dogs), 6 hours (1 dog), or 24 hours (1 dog).



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  		Figure 1. Accumulation of MNLs in infarcted vs noninfarcted myocardium. Radiolabeled MNLs were infused during first hour of reperfusion after a 1-hour occlusion period. Heart was sectioned as described in "Methods." Shaded bars, Regional blood flow for each heart segment during occlusion. *Segments that included infarcted tissue identified by TTC staining. Solid bars, Quantities of 99mTc-labeled MNLs that accumulated in same segments during reperfusion. Both types of data are normalized to highest value found in any segment (defined as 100%) for clarity of presentation. Greatest accumulation of MNLs was seen in endocardial and midmyocardial segments with lowest blood flow during occlusion. This figure is representative of results obtained in all three dogs reinfused with MNLs during first hour of reperfusion. A indicates anterior; AP, anterior papillary; L, lateral; PP, posterior papillary; P, posterior; and S, septal.

Movement of Neutrophils and Monocytes Into Cardiac Extracellular Fluids After Ischemia and Reperfusion
Within the first hour of reperfusion, we also found a significant increase in the percentages of neutrophils and monocytes in cardiac lymph compared with lymph collected before the occlusion. Fig 2Down shows the results from 6 dogs that had significant left ventricular infarcts and illustrates that the percentages of neutrophils and monocytes in the cardiac lymph tended to increase throughout the 5-hour reperfusion period. There was no change in the fraction of neutrophils or monocytes in serial lymph samples from 2 dogs with negligible myocardial infarcts after coronary occlusion (Fig 2Down, top, dotted lines). The percentage of B cells showed little change, and the T-cell fraction declined in proportion to the increase in monocytes and neutrophils in lymph from the 6 dogs with significant myocardial infarction (Fig 2Down, second panel). There were no significant changes in the percentages of lymphocytes, monocytes, or neutrophils in serial blood samples obtained from these same 6 dogs (Fig 2Down, third panel), and the total white cell count did not change significantly during the first 5 hours after reperfusion (Fig 2Down, bottom panel).



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  		Figure 2. Distribution of monocytes, neutrophils, and lymphocytes in cardiac lymph and peripheral blood during reperfusion. Top, Average percentage (±SEM) of monocytes (mono) and neutrophils (PMN) in canine cardiac lymph before occlusion (Pre) and at intervals after reperfusion of the myocardium. Results are shown for six animals with significant ischemia (solid lines) and two animals without significant ischemia [dotted lines; Mono (C) and PMN (C)]. *Values significantly greater than preocclusion sample (P<.05 by both paired t test and Wilcoxon signed rank test). Second panel, Percentage of T and B lymphocytes in cardiac lymph of same six dogs with significant infarcts. Third panel, percentages of neutrophils, lymphocytes, and monocytes in peripheral blood. Bottom, total white blood cell (WBC) counts in blood of same 6 dogs at different times after reperfusion.

Immunohistological Studies of Leukocyte Infiltration Into Reperfused Myocardium
In our study of the tissue localization of radiolabeled MNLs, the heart was sectioned according to a uniform protocol. As a result, single tissue sections often contain both infarcted tissue and previously ischemic tissue in the border zone. Thus, it was not possible to discriminate between MNLs that accumulated in infarcted versus previously ischemic myocardium. We also could not use this method to distinguish between MNLs that have marginated in the microvasculature and MNLs that have migrated into the myocardium. We therefore turned to immunohistochemical analyses using specific MAbs to identify leukocytes in tissue sections. Both neutrophils and MNLs marginated in small (10- to 70-µm-diameter) veins in the left ventricular myocardium during the first hour after reperfusion (Fig 3Down). Some of these cells appear to have migrated into the connective tissues around these veins. In Fig 3Down, neutrophils in the myocardium are positively identified with specific Abs, and monocytes are recognized by the absence of staining with the granulocyte-specific MAb and by their nuclear morphology. Monocytes were also positively identified in previously ischemic myocardium by use of the monocyte-specific MAbs CD64 (Fig 4Down).



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  		Figure 3. Migration of leukocytes into formerly ischemic tissue. Small (10- to 70-µm) vein in formerly ischemic myocardium collected after 1 hour of reperfusion. Neutrophils are stained yellow-brown with the specific MAb SG8H6, and MNLs are unstained but recognizable by their characteristic nuclear morphology. Both neutrophils and MNLs have marginated along endothelial surface and begun to migrate into extravascular connective tissue. Magnification x600.



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  		Figure 4. Migration of monocytes into formerly ischemic myocardium. After 3 hours of reperfusion, immunohistochemical staining with MAb specific for CD64 demonstrates infiltrating monocytes (brown) in a necrotic region of myocardium (A) and in adjacent viable myocardium (B). Magnification x400.

ICAM-1 mRNA expression can be used as a marker of the border zone. We have previously provided immunohistochemical data showing that >=1 hour after reperfusion, ICAM-1 mRNA was upregulated in myocardial tissue adjacent to muscle displaying characteristic contraction-band necrosis29 but not in nonischemic myocardium or in necrotic tissue. In Fig 5ADown, we see that the border zone, defined by blue-green staining for ICAM-1 mRNA, appears to contain a greater density of infiltrating leukocytes. The leukocytes in this area include both those that stain with SGH86 (ie, neutrophils) and SGH86-negative leukocytes (noted with arrows). Fig 5BDown, a higher magnification of the same cells as marked with arrows, shows that the SGH86-negative cells have blue-gray cytoplasm, indicating the presence of ICAM-1 mRNA. Fig 5CDown shows the same tissue section as presented in Fig 5BDown after it was destained and then restained with hematoxylin-eosin. We see that the ICAM-1 mRNA–positive cells from Fig 5BDown have the nuclear morphology of monocytes.



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  		Figure 5. Accumulation of leukocytes in formerly ischemic but not in well-perfused tissues. A, Low-power view (x100) of myocardium harvested after 3 hours of reperfusion. Border zone, identified by expression of myocyte ICAM-1 mRNA, is located on right and is blue-green because of its hybridization with a riboprobe for ICAM-1 mRNA. Area to left is negative for myocyte ICAM-1 mRNA and is morphologically normal, indicating that it remained well perfused during occlusion. Neutrophils, stained red-brown by the MAb SG8H6, are not seen in normal myocardium. Neutrophils are seen in border zone, in both venules and extravascular foci. In addition, there are infiltrating leukocytes that do not react with neutrophil-specific Ab (marked with arrows); these same cells are marked in B and C. B, x400 magnification of upper right region of A. This magnification illustrates blue-gray color in cytoplasm of SG8H6-negative leukocytes. This suggests that ICAM-1 mRNA may be upregulated in the cytoplasm of these leukocytes. C, Same section as B after tissue was destained and then restained with hematoxylin-eosin. This shows that cells identified with arrows in A and B have morphological characteristics of monocytes.

Ultrastructural studies (Fig 6Down) demonstrate that phagocytic monocyte-macrophages ingesting red cells and subcellular organelles from damaged or dying cells can be found in myocardial tissues harvested after 3 hours of reperfusion. Numerous phagocytic cells were identified in areas that were formerly ischemic, but no phagocytic macrophages were found in sections of normal myocardium from 4 dogs with significant infarcts.



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  		Figure 6. Phagocytic monocyte-macrophages in formerly ischemic tissue. Transmission electron micrographs of actively phagocytic MNLs with nucleus indicated (Nu) present amid collagen fibers in formerly ischemic myocardium 3 hours after reperfusion. In one case (B), macrophage has ingested a red blood cell (RBC); in the other (A), phagosomes contain membrane-delimited subcellular organelles. Inset in A shows internal membrane (white arrows) around one of these organelles juxtaposed to outer membrane of phagosome (black arrows).

Molecular Basis for the Migration of Monocytes Into Cardiac Extracellular Fluids After Ischemia and Reperfusion
Canine cardiac lymph collected during the first 24 hours after reperfusion contained significant chemotactic activity for monocytes (Fig 7Down). Addition of a blocking Ab to canine C5a neutralized the chemotactic activity of cardiac lymph collected during the first hour after reperfusion. Monocyte chemotactic activity in lymph collected during the second hour was only partially inhibited by the Ab to C5a but could be completely blocked with a combination of Abs to C5a and TGF-ß1. When added to lymph collected beyond 180 minutes of reperfusion, anti-C5a plus anti–TGF-ß1 could only partially inhibit the chemotactic activity, which suggested the appearance of a third chemotaxin.



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  		Figure 7. Monocyte chemotactic activity in postreperfusion cardiac lymph. Serial lymph samples collected after reperfusion were tested for their ability to stimulate migration of freshly isolated canine monocytes in a chemotaxis assay. Gray rectangle indicates numbers of monocytes (±2 SD) that migrated spontaneously in response to media alone. From onset of reperfusion, cardiac lymph was consistently chemotactic for monocytes, causing threefold to fourfold increase in numbers of migratory cells ({circ}). Addition of antibodies to canine C5a ({bullet}) abolished chemotactic activity of lymph samples collected between 0 and 60 minutes but had no significant effect when added to lymph collected at later time points. A combination of Abs to C5a and TGF-ß1 ({triangleup}) blocked all chemotactic activity of lymph collected through 180 minutes of reperfusion. These two antibodies caused a significant but incomplete suppression of chemotactic activity of samples collected from 180 minutes on. Data shown in this figure are representative of results obtained from 6 dogs with significant infarcts.

We postulated that the late-developing chemotactic substance found in lymph samples collected beyond 180 minutes might be a ß-chemokine such as MCP-1. We used two approaches to test this hypothesis. We preincubated canine MNLs with recombinant human MCP-1, a strategy that suppressed by 89% the subsequent chemotactic response of the monocytes to MCP-1 (Fig 8Down, top). This desensitization is selective and did not affect the monocyte response to the chemotactic substance (C5a) present in ZADS (Fig 8Down, middle). We also used an MAb to MCP-1 that blocked 62% of the chemotactic activity of recombinant human MCP-1 (Fig 8Down, top). We applied both of these strategies to cardiac lymph collected 180 to 240 minutes after reperfusion (Fig 8Down, bottom). Anti-C5a was first added to the lymph to block any potential contribution of C5a to the chemotactic activity. Prior desensitization with MCP-1 reduced the migration of canine MNLs in response to this canine cardiac lymph by 65%, whereas addition of anti–MCP-1 suppressed the chemotactic activity of this material by 53%.



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  		Figure 8. Testing canine cardiac lymph for MCP-1–like activity. Top, Recombinant human MCP-1 is chemotactic for canine monocytes. Response of MNLs can be blocked by 89% by pretreatment with MCP-1 to desensitize them to its chemotactic effects. Chemotactic activity can also be blocked by 62% by addition of an MAb to MCP-1. Middle, MNLs desensitized to chemotactic effects of MCP-1 are still able to respond to chemotactic effects of C5a to the same degree as untreated MNLs. Chemotactic activity of ZADS can be blocked (83% suppression) by inclusion of a blocking Ab to dog C5a. Bottom, Cardiac lymph collected 180 to 240 minutes after reperfusion of ischemic myocardium provides a chemotactic stimulus for canine monocytes even after addition of Abs to C5a. Monocyte chemotactic activity in this lymph is equivalent to 120 ng/mL of recombinant human MCP-1 or 10% vol/vol ZADS. Response of monocytes to chemotactic activity present in this lymph can be partially blocked by either pretreating (desensitizing) the MNLs with MCP-1 (65% suppression, P<.01, t test) or by adding an anti–MCP-1 Ab to lymph (53% suppression, P<.01). Data are representative of results obtained in two replicated experiments.

In Fig 9Down, we demonstrate that TGF-ß1 is also present in the cardiac lymph collected beyond the third hour of reperfusion. Marked monocyte chemotactic activity was seen in the lymph treated with anti-C5a alone. Further addition of antibodies to TGF-ß1 or desensitization of the monocytes by preincubation in MCP-1 reduced the chemoattractive properties of this lymph significantly, by {approx}50%. When both TGF-ß1 effects and MCP-1 effects were blocked, there was a further significant reduction of the chemotactic activity in the lymph. Indeed, elimination of C5a, MCP-1, and TGF-ß virtually abolished all of the chemotactic activity of this material.



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  		Figure 9. TGF-ß1 and MCP-1 are the principal chemotactic substances in cardiac lymph collected beyond 3 hours of reperfusion. Addition of Abs to TGF-ß1 to lymph or desensitization of MNLs with recombinant MCP-1 significantly reduced chemotactic activity of lymph collected 180 to 300 minutes after reperfusion (*P<.01, t test). Both treatments used together effectively abolished (**P<.001) chemotactic activity of this material. Anti-C5a was added uniformly to all samples to block any potential contribution from this chemotaxin. Data are representative of results obtained in two replicated experiments.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
MNLs appear to be a significant component of the inflammatory cell infiltrates that accumulate in formerly ischemic tissues within the first hours after reperfusion. By reinfusing radiolabeled MNLs in the left atrium 45 minutes before the termination of the reperfusion period, we tested the hypothesis that reperfusion of ischemic myocardium stimulates production or release of chemoattractive agents that can cause MNLs to collect selectively in these regions. We had used this approach to demonstrate that neutrophils preferentially localize in formerly ischemic myocardium initially in response to C5a, an anaphylatoxin released by complement, activated by damaged cardiac myocytes.6 7 9 26 Our present results with monocytes are very similar to those obtained with neutrophils,9 36 with reinfused monocytes accumulating in approximately threefold greater numbers in endocardial and midmyocardial segments with reduced blood flows and infarcts. This preferential localization was seen at 1 hour after reperfusion, a time course that may be earlier than has been historically recognized.21 22 MNLs infused 3 to 24 hours after reperfusion showed no preferential localization to either the previously ischemic or the infarcted areas compared with areas that remained well perfused throughout the occlusion. Monocytes may continue to preferentially enter the injured myocardium beyond the first hour of reperfusion but at a rate that is not detectable with our technique.

The early preferential localization of MNLs to previously ischemic, infarcted, or border-zone myocardium suggests that there must be a potent chemotaxin that is expressed very rapidly in those areas. Generation of C5a in the heart begins when dying myocytes extrude mitochondria. These bind C1q and activate the complement cascade.30 The production of C5a is consequently greatest in the areas in which infarcted myocardium is most prevalent, and it is therefore not surprising that these are also the areas in which leukocytes accumulate.

It is important to recognize that the initial leukocyte accumulation is primarily intravascular and perivascular, as illustrated in Fig 4Up. The major contribution of C5a may be to induce the margination of monocytes in the microvasculature during the initial reperfusion. These adherent cells become the initial MNLs that migrate into the infarct. Histological studies of ischemic myocardial tissue,21 22 together with the studies of canine cardiac lymph presented here, suggest that several factors continue to cause a steady movement of monocytes into the formerly ischemic myocardium for hours and perhaps days after reperfusion.

Cardiac lymph provides a sample of the leukocytes that have moved through the endothelium into the perivascular tissues. Our analyses of cardiac lymphatic fluids support the hypothesis that MNLs continue to move across the endothelium into the previously ischemic myocardium well beyond the first hour after reperfusion. During the first 2 hours of reperfusion after significant myocardial ischemia, CD14+ monocytes in the cardiac lymph were about half as abundant as neutrophils (Fig 2Up). Later, monocytes became an increasingly large component of the inflammatory cells in these lymphatic fluids, and by the fifth hour after reperfusion their concentration in the lymph equaled or exceeded that of neutrophils.

Analyses designed to identify the leukocyte chemotactic agents in these lymphatic fluids suggest that there is a complex, well-orchestrated mechanism for continued recruitment of both granulocytes and monocytes into the damaged myocardium. From our previous studies, we anticipated that C5a would have a powerful, albeit short-lived, influence on monocyte migration into myocardial tissues.6 7 In the present studies, we found that C5a was indeed an important chemotactic agent for monocytes during the first hour after reperfusion (Fig 7Up). In the second and third hours after reperfusion, TGF-ß1 appeared in the lymph as another monocyte chemotaxin. This is consistent with reports that preformed TGF-ß1 is released in active form after ischemic myocardial injury.37 Considering that TGF-ß1 is one of the most potent chemotactic substances known for granulocytes as well as for monocytes,15 38 active in picomolar concentrations,39 it seems very likely that this cytokine plays a prominent role in directing the migration of both monocytes and neutrophils into formerly ischemic myocardium.

A third monocyte chemotaxin, not C5a or TGF-ß1, appeared in cardiac lymph samples collected >=180 minutes after reperfusion. Considering that induction of other inflammatory mediators has been demonstrated in the reperfused border zone,29 40 41 42 43 44 we tested the lymph 180 to 300 minutes after reperfusion to evaluate whether it might contain a protein with functional properties similar to those recognized for human MCP-1. Two strategies were used to demonstrate the presence of MCP-1–like activity. The first involved selective desensitization of canine MNLs by prior exposure to recombinant human MCP-1. This desensitization effectively suppressed chemotaxis of these monocytes in response to recombinant human MCP-1 but had no effect on the response to C5a. Since cross-desensitization may occur among the peptide chemoattractants,45 we confirmed our studies with a neutralizing MAb to human MCP-1 that had demonstrable cross-reactivity with canine MCP-1. Both strategies suggested that MCP-1 was present in cardiac lymph fluids collected >=3 hours after onset of reperfusion, indicating that MCP-1 is the third chemotactic factor, besides C5a and TGF-ß1, that attracts monocytes into formerly ischemic canine myocardium.

It has been suggested that monocytes may play an important role in healing the defect created by myocardial necrosis.14 17 We observed phagocytic macrophages in the formerly ischemic myocardium as early as 3 hours after reperfusion. Activated macrophages can promote the deposition of connective tissue ground substances, stimulate fibroblast proliferation, and induce synthesis of collagen in inflamed cardiac tissue.16 38 46 47 Considering the known ability of MCP-1 to activate MNLs, it is likely that this chemokine also plays an important role in stimulating infiltrating monocytes.48

Further evidence that MCP-1 is destined to play a role in attracting MNLs to ischemic myocardium is presented in the companion article.23 This study shows that MCP-1 mRNA appears in formerly ischemic myocardium within the first hour after reperfusion. The MCP-1 mRNA reaches its highest levels by the third hour after reperfusion, at which time, with immunohistological techniques, one can demonstrate MCP-1 protein on infiltrating leukocytes and endothelial cells in areas of maximal leukocyte infiltration. These investigations further characterize factors controlling induction and synthesis of MCP-1, a chemokine that we postulate may play an important role both in recruiting monocytes to ischemic myocardial tissues and in stimulating their differentiation into tissue macrophages.


*    Selected Abbreviations and Acronyms
 
Ab = antibody
FCS = fetal calf serum
ICAM-1 = intercellular adhesion molecule-1 (CD54)
MAb = monoclonal antibody
MCP-1 = monocyte chemoattractant protein-1
MNL = mononuclear leukocyte
PBS = Dulbecco's phosphate-buffered saline
PE = phycoerythrin
TGF-ß1 = transforming growth factor-1
TTC = 2,3,5-triphenyltetrazolium chloride
ZADS = zymosan-activated dog serum


*    Acknowledgments
 
This study was supported by the Houston Veterans Affairs Medical Center, the Methodist and Texas Children's Hospital, and NIH-DHHS grants HL-42550, HL-41408, NS-32583, and AI-28071. Alan R. Burns is a recipient of a Medical Research Council of Canada fellowship.


*    Footnotes
 
Guest editor for this article was Judith L. Swain, MD, University of Pennsylvania, Philadelphia.

Received April 14, 1996; revision received September 19, 1996; accepted September 30, 1996.


*    References
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