Emigrated Rat Neutrophils Adhere to Cardiac Myocytes via α4 Integrin
Abstract Previous work has shown that neutrophils isolated from whole blood adhere to cardiac myocytes via CD18 (β2 integrin) to cause injury to the heart cells. In vitro, we have found that upon endothelial transmigration, neutrophils can also express α4β1; however, whether this contributes to neutrophil adhesion to parenchymal cells remains entirely unknown. Unstimulated and tumor necrosis factor-α–stimulated rat cardiac myocytes adherent to gelatin-coated coverslips supported N-formyl-Met-Leu-Phe (fMLP)–induced neutrophil (isolated from whole blood) adhesion entirely via CD18 (blocked with monoclonal antibody [mAb] WT-3). Emigrated neutrophils spontaneously adhered to cardiac myocytes also entirely via CD18. However, if fMLP was used to restimulate emigrated neutrophils, the adhesion to cardiac myocytes was entirely independent of CD18. Although an anti–α4 integrin antibody (mAb TA-2) alone did not reduce the emigrated neutrophil-myocyte interaction, dual administration of TA-2 and WT-3 reduced adhesion by 81%. α4 integrin was expressed in small amounts on the surface of circulating neutrophils, increased following transmigration, and then increased >5-fold after restimulation of these emigrated neutrophils. In the presence of the anti-CD18 antibody, a fibronectin fragment (FN-40) but not a vascular cell adhesion molecule-1 antibody (mAb 5F10) inhibited neutrophil-myocyte interactions by 80%. Similar results were seen when the rat chemokine CINC-gro was used instead of fMLP, suggesting that the α4-dependent adhesion was not specific to fMLP. These data demonstrate that α4 integrin can be physiologically induced to increase in number and avidity after neutrophil emigration and that this adhesion molecule can cause firm adhesion to fibronectin on parenchymal cells, including rat cardiac myocytes.
Myocardial inflammation associated with ischemia/reperfusion, sepsis, and other pathologies has been shown to have a major neutrophil component, inasmuch as prevention of neutrophil infiltration into myocardium decreases myocardial damage during the inflammatory process.1 2 3 4 Neutrophils are recruited to damaged myocardium by interacting with activated cardiac vascular endothelium in a sequential cascade of events. Selectin adhesion molecules that are expressed on both cell types initiate neutrophil tethering and rolling, whereas CD11/CD18 (a β2 integrin) on neutrophils mediates firm adhesion by interacting with various ligands on endothelium (ICAM-1, etc).5 6 7 Adherent neutrophils then migrate through the endothelium toward the affected cardiac tissue by following increasing concentrations of chemotactic stimuli, such as fMLP from bacteria or chemokines such as CINC-gro or interleukin-8 from interstitial cells.
The mechanisms involved in neutrophil-myocyte interactions have not been fully elucidated. As with adhesion to endothelium, neutrophil adhesion to cardiac myocytes has been shown to be a CD18/ICAM-1–mediated event.8 9 Moreover, the adhesion of the neutrophils to cardiac myocytes was closely associated with myocyte death.10 Although these studies were seminal in demonstrating the importance of neutrophils and adhesion molecules in cardiomyopathy, they differ critically from the physiological condition in that the neutrophils were isolated from whole blood (ie, circulating neutrophils). This latter point is not trivial, inasmuch as neutrophils that have emigrated in response to increasing concentrations of a chemotactic gradient have been shown to express a novel adhesion molecule, the α4 integrin.11 α4 integrin was previously thought to be expressed on all leukocytes, with the exception of neutrophils, and has been implicated in several disease processes, including contact hypersensitivity,12 allergic asthma,13 inflammatory bowel disease,14 and cardiac allograft failure.15 Whether the observation that emigrated neutrophils express α4 integrin can be extended to suggest that these neutrophils will bind to myocytes via α4 integrins is highly speculative. Ligands for α4 integrin include the matrix protein fibronectin and the adhesion molecule VCAM-1. Although VCAM-1 has not been detected on quiescent or activated myocytes, fibronectin is expressed on the surface of cardiac myocytes and may serve to support this adhesive interaction.16 17
The objectives of the present study were to determine whether emigrated neutrophils are able to adhere to cardiac myocytes and, if so, to examine whether the adhesive profile that underlies the neutrophil-myocyte interactions changes as a result of emigration. We have used a static adhesion assay that allowed us to visualize emigrated rat neutrophil adhesion to isolated rat cardiac myocytes and report herein that whereas circulating neutrophils bind to activated myocytes exclusively via the β2 integrin, emigrated neutrophils increase expression and avidity of the α4 integrin, particularly after a second stimulus, and firmly bind via this new adhesion molecule.
Methods and Materials
mAbs and Reagents
mAb TA-2 (anti-rat α4) and WT-3 (anti-rat CD18) were gifts from Dr Thomas Issekutz (University of Toronto, Canada). Anti-rat VCAM-1 antibody (5F10) was a gift from Dr Roy Lobb (Biogen, Inc, Cambridge Center, Mass). TNF-α was a gift from Knoll Pharmaceutical (Markum, Ontario, Canada). Purified human fibronectin fragment 40K was purchased from Chemical International Inc. CINC-gro was a gift from Dr Makato Suematsu, Keio University, Tokyo, Japan. Unless otherwise stated, all other reagents were purchased from Sigma Chemical Co. All cells used were obtained from Sprague-Dawley rats (225 to 275 g, Harlan Sprague Dawley, Inc, Indianapolis, Ind).
Neutrophil Isolation and Treatment
Circulating rat neutrophils were isolated from citrate-anticoagulated whole blood collected by cardiac puncture. Briefly, red blood cells were removed by dextran sedimentation followed by hypotonic lysis. Neutrophils were further purified by centrifugation through a Histopaque gradient and resuspended in HBSS at 2×107/mL. To obtain emigrated neutrophils, rats were injected intraperitoneally with 10 mL of 1% oyster glycogen in PBS. After 4 hours, the rats were killed, and the peritoneal fluid was collected. After centrifugation, the emigrated neutrophils were resuspended in HBSS at 2×107/mL. Neutrophils were activated by treating them with 20 μmol/L fMLP or 10 nmol/L CINC-gro just before injection into the adherence assay chambers (see below). Antibodies were added at the time of activation.
Circulating or emigrated rat neutrophils (1×106 per tube) were fixed in 1% formalin (30 minutes at 4°C) and then washed. TA-2 antibody (2 μg per tube) was added to stain for α4 integrin. After 30 minutes, the cells were washed and labeled with FITC-conjugated goat anti-mouse IgG (Serotec) and incubated at room temperature for a further 30 minutes. After washing, the level of α4 integrin expression was measured on a FACScan flow cytometer (Becton Dickinson Immunocytochemistry Systems).
Cardiac Myocyte Isolation and Treatment
Right ventricular myocytes from rats were isolated as previously described.18 Briefly, the heart was rapidly removed from decapitated animals, and Tyrode’s solution was perfused retrogradely (10 mL/min for 5 minutes) via a canula attached to the aorta. The heart was then perfused with Tyrode’s solution containing collagenase (0.02 mg/mL), protease (0.004 mg/mL), and taurine (2.4 mg/mL) for 7 minutes, after which the right ventricle was separated from the heart and minced with 10 mL of Tyrode’s solution containing collagenase (0.05 mg/mL), protease (0.1 mg/mL), taurine (2.5 mg/mL), BSA (5 mg/mL), and CaCl2 (50 mmol/L). After agitation for 10 to 20 minutes at 34°C, the supernatant (containing separated myocytes) was collected and placed in Tyrode’s solution with BSA (5 mg/mL), taurine (2.5 mg/mL), and CaCl2 (50 mmol/L). Taurine was added to prolong the viability of the myocytes during storage.
To mimic an inflammatory state, tubes containing ventricular myocyte suspensions (≈1×104 cells/mL) were dosed with TNF-α (300 U/mL) and placed on a rotating rack for 4 hours at 37°C, after which the myocytes were layered onto coverslips and incubated for 1 more hour (see below). Control myocytes were subjected to the same protocol without dosing with TNF-α.
Round glass coverslips (25 mm, Bellco Glass Inc) were pretreated with 1 mL of 1% gelatin in PBS for 60 minutes at 37°C. The coverslips were washed once in PBS, and 1 mL of myocyte suspension was gently layered onto the coverslip. The myocytes were allowed to settle and adhere to the coverslips for 60 minutes at 37°C, after which the coverslips were inserted into adherence assay chambers. The chamber is designed to hold two coverslips separated by an O-ring gasket (Bellco Glass Inc). Neutrophil suspensions (diluted to 1×106/mL in HBSS) were injected into the chamber and allowed to settle onto the cardiac myocytes for 10 minutes, after which the chambers were inverted. At this point, nonadherent neutrophils settled to the other side of the chamber. This design allowed for visualization of the cardiac myocytes (magnification, ×200) using phase-contrast microscopy with an inverted microscope (Zeiss Canada). The number of adherent neutrophils per myocyte was determined for a minimum of 16 myocytes on each coverslip, with each condition repeated three to eight times.
Circulating Neutrophils Adhere to Cardiac Myocytes via CD18
Freshly isolated neutrophils from rat blood showed minimal adherence to unstimulated rat cardiac myocytes (Fig 1⇓). When neutrophils were stimulated with fMLP, adhesion to myocytes increased significantly, an event that was totally inhibitable by the addition of an anti-CD18 antibody (mAb WT-3). Stimulation of cardiac myocytes with TNF-α increased the level of fMLP-stimulated neutrophil adhesion (Fig 1⇓), and this interaction was also inhibited by an anti-CD18 antibody. Fig 2⇓ is a photomicrograph of the system used in the present study illustrating fMLP-treated neutrophils adhering to TNF-α–stimulated myocytes.
Emigrated Neutrophils Can Adhere to Cardiac Myocytes via a CD18-Independent Mechanism
Emigrated neutrophils adhered to both unstimulated and TNF-α–stimulated cardiac myocytes (Fig 3⇓). Once again, addition of the anti-CD18 antibody inhibited these interactions. When emigrated neutrophils were exposed to a dose of chemoattractant (fMLP), the adhesion to cardiac myocytes was further increased. However, the anti-CD18 antibody was unable to inhibit the adhesion of emigrated neutrophils treated with fMLP to either unstimulated or TNF-α–stimulated cardiac myocytes.
Emigrated Neutrophils Express and Functionally Adhere via α4 Integrin
Fig 4⇓ is a flow cytometric histogram demonstrating α4 integrin expression (staining with mAb TA-2) on circulating (from whole blood) and emigrated (elicited into the peritoneal cavity) rat neutrophils. These data confirm the observations of Issekutz et al,19 who reported a low level of expression on circulating rat neutrophils. However, our data extend previous work, inasmuch as emigration of neutrophils resulted in a 2-fold increase in α4 integrin expression (mean fluorescence: circulating neutrophils, 7.0; emigrated neutrophils, 14.0). Moreover, when emigrated cells were stimulated with fMLP, a further increase in α4 integrin expression was noted (mean fluorescence, 37.9). In fact, the difference for α4 integrin expression between circulating and restimulated emigrated neutrophils was >5-fold. To determine if the CD18-independent adhesion was attributable to α4 integrin, anti–α4 integrin antibody (mAb TA-2) was added to emigrated neutrophils that had been stimulated with fMLP. Whereas neither antibody had an effect, tandem addition of TA-2 and WT-3 significantly decreased adhesion (Fig 5⇓). Similar results were seen for unstimulated cardiac myocytes (not shown).
Neutrophil α4 Integrin Binds to Fibronectin on Cardiac Myocytes to Support Adhesion
The CD18-independent adhesion of emigrated neutrophils to TNF-α–stimulated myocytes could be inhibited if, in addition to an anti-CD18 antibody, a fibronectin fragment (FN-40) was also added (Fig 6⇓). This effect was dose dependent, as a higher concentration of FN-40 (10 μg/mL) was more effective at inhibiting adhesion. Much like the anti–α4 integrin data in Fig 5⇑, addition of FN-40 alone did not inhibit neutrophil adhesion. Whereas inhibiting α4 integrin binding to fibronectin abrogated adhesion, the addition of a polyclonal anti-rat VCAM-1 antibody (5F10) had no effect (Fig 6⇓).
CINC-gro Is Able to Stimulate α4 Integrin–Dependent Adhesion of Emigrated Neutrophils to Cardiac Myocytes
To determine if other physiological agonists are able to induce CD18-independent adhesion of emigrated neutrophils, we tested the endogenous rat chemokine CINC-gro (10 nmol/L), a potent chemoattractant of rat neutrophils. As with fMLP stimulation, the adhesion of CINC-gro–stimulated emigrated neutrophils resulted in significant adhesion to TNF-α–stimulated cardiac myocytes, an event that was not inhibitable by anti-CD18 or anti–α4 integrin antibodies alone (Fig 7⇓). When anti-CD18 antibody was used in combination with the anti–α4 integrin antibody or the fibronectin fragment (10 μg/mL), significant attenuation of adhesion was observed.
In vitro, we have previously shown that emigration across endothelium induces the expression of the β1 integrin α4β1 on human neutrophils.11 The purpose of the present study was to determine whether neutrophils use α4 integrin to adhere to freshly isolated ventricular myocytes and whether this event could be influenced by the inflammatory state of the myocyte. The present study is the first to demonstrate that the physiological event of emigration alters the mechanisms by which neutrophils adhere to parenchymal cells such as cardiac myocytes. Both circulating (ie, isolated from whole blood) and emigrated rat neutrophils adhered to rat cardiac myocytes via CD18. This occurred regardless of the inflammatory state of the myocyte. Application of the chemoattractant fMLP or CINC-gro to emigrated neutrophils resulted in increased adhesion. However, the anti-CD18 antibody was unable to inhibit the adhesion of emigrated neutrophils treated with chemoattractant to either unstimulated or TNF-α–stimulated myocytes, whereas coadministration of antibodies against CD18 and α4 integrin did inhibit >80% of the adhesion. Thus, emigration induced a second adhesive mechanism on neutrophils, mediated by α4 integrin.
These data suggest that the ligand for CD18 is constitutively expressed on rat cardiac myocytes and is increased with inflammatory cytokine stimulation. Previous studies have established the importance of CD18 in neutrophil adhesion to cardiac myocytes under conditions in which both the neutrophil and the myocyte were stimulated.8 We have found significant adhesion when only the neutrophils were stimulated. This may be due to differences in the models used (rat versus canine). Whereas canine cardiac myocytes do not express ICAM-1 unless they have been stimulated with an inflammatory cytokine,9 cultured neonatal rat myocytes are known to constitutively express ICAM-1, the level of which can be increased with TNF-α stimulation.20 Our data suggest that this may also be the case for adult rat myocytes, although the expression of other potential CD18 ligands (eg, ICAM-2) cannot be excluded.
The induction of α4 integrin may strengthen the adhesive interaction between neutrophils and extravascular tissue, thus anchoring the emigrated neutrophil firmly to an inflammatory site. Alternatively, α4 integrin may be important for extravascular adhesion when ligands for CD18 are reduced or not present. Interestingly, circulating neutrophils or emigrated neutrophils that were not restimulated with chemoattractant did not demonstrate α4 integrin–dependent adhesion, despite the presence of α4 integrin in both situations. The reasons for this may be 2-fold. First, the level of α4 integrin expression may have been insufficient to support adhesion. This observation is consistent with our flow cytometric experiments, inasmuch as restimulation of emigrated neutrophils with fMLP increased α4 integrin expression 5-fold. Dalton et al21 have demonstrated that β1 integrin surface expression is lost if the integrin does not engage its ligand. It is conceivable that elicited neutrophils isolated from the peritoneal cavity are nonadherent and might be expected to therefore have reduced expression of surface integrins. Chemoattractant stimulation may function to remobilize the α4 integrin that was expressed during emigration. An alternative explanation for the need for restimulation may be that the binding affinity of the α4 integrin may be reduced. Indeed, it is well known that α4 integrin can be in a low- or high-affinity state and that perhaps glycogen-elicited neutrophils require further stimulation to activate α4 integrin. This closely mimics the pathophysiological condition; such an increase in stimulation with chemotactic agents would occur as neutrophils emigrated toward an inflamed site. Although this has not been previously described for α4 integrin, Hughes et al22 have demonstrated that newly mobilized Mac-1 (CD11b/CD18) is capable of functioning in adhesion only if the neutrophils are subsequently exposed to an increased level of stimulus.
In the present study, emigrated neutrophils had to be restimulated with an exogenous signal. Even TNF-α–stimulated myocytes were unable to deliver an activating signal to emigrated neutrophils to induce the α4 integrin–dependent adhesion. However, this may be due to the low density of myocytes in our model versus the case in vivo, where emigrated neutrophils are closely associated with a large number of inflamed cardiac myocytes and in the presence of many interstitial cells (including mast cells and fibroblasts). These cells are known to produce many proinflammatory molecules in myocardial inflammation; the complement fragment C5a has been found in cardiac lymph during reperfusion of the ischemic heart23 and in bacterial products such as fMLP in bacteria-associated myocarditis. Moreover, mast cells release many different stimuli, including platelet activating factor, leukotrienes, and TNF-α, all capable of activating neutrophils. Thus, in vivo, it is likely that emigrated neutrophils would be exposed to higher concentrations of endogenously derived agonists than are present in our experimental model, a situation that may invoke α4 integrin–mediated adhesion.
α4 integrin can support leukocyte adhesion by binding to VCAM-1 or to fibronectin. Anti–VCAM-1 antibody (5F10) had no effect on CD18-independent adhesion, suggesting that VCAM-1 is not important for adherence in this model. Fibronectin is a constituent of the cardiac extracellular matrix and is found within the transverse tubules of cardiac myocytes.17 During inflammatory episodes, immunoreactivity for fibronectin increases in affected cardiac tissue and is associated with penetration of fibronectin into the myocytes.16 The α4 integrin–dependent adhesion of neutrophils to myocytes was inhibited by the addition of FN-40 (Fig 6⇑). As with the anti–α4 integrin antibody, the inhibitory effect was revealed only in the presence of anti-CD18 antibody. Because fibronectin is constitutively present on heart tissue, the lack of requirement for protein synthesis may be significant during postischemic neutrophil influx into affected heart tissue, where emigrated neutrophils would find a readily available ligand for newly expressed α4 integrin.
Whether this newly identified adhesive pathway is physiologically relevant remains to be resolved. To date, anti-CD18 antibodies have confirmed that the β2 integrin adhesion pathway is important in, for example, myocardial ischemia/reperfusion. However, this is almost certainly due to the fact that neutrophils were prevented from adhering to the endothelium via CD-18 and therefore were unable to interact with the postischemic myocyte. In fact, it may not be technically feasible to resolve the importance of CD18 and α4 integrin as adhesive mechanisms involved in parenchymal cell adhesion in vivo. It is interesting that in some studies the protective effect of α4 integrin antibodies could be dissociated from leukocyte recruitment13 24 and begs the question of whether inhibition of adhesion to parenchymal cells can account for some of this protection. Related to this issue, Entman et al10 have demonstrated that preventing neutrophil adhesion to myocytes with antiadhesive therapy does reduce canine myocyte death. In the present study, we saw few myocytes die, even when numerous activated neutrophils were observed to adhere to myocytes. This may be related to differences between dog and rat myocytes or incubation conditions (eg, we use taurine, an antioxidant, in our isolation procedure). The lack of myocyte death following neutrophil adhesion does not preclude the possibility that more subtle dysfunction has occurred in these cells and certainly warrants further investigation.
It is apparent that the myocardial damage is due to neutrophil interaction with myocytes under various inflammatory conditions. The present study demonstrates that after emigration, CD18 continues to be important in mediating neutrophil adhesion to cardiac myocytes. However, emigration of neutrophils invokes a second adhesive mechanism, namely, α4 integrin. This emigration-dependent adhesive mechanism may require some consideration in designing drugs to inhibit neutrophil/myocyte interactions.
Selected Abbreviations and Acronyms
|FN-40||=||fibronectin fragment containing CS-1 binding motif for α4 integrin|
|ICAM||=||intercellular adhesion molecule|
|TNF||=||tumor necrosis factor|
|VCAM||=||vascular cell adhesion molecule|
The authors gratefully acknowledge support from the Canadian Medical Research Council.
- Received January 27, 1997.
- Accepted May 29, 1997.
- © 1997 American Heart Association, Inc.
Ma X, Tsao PS, Lefer AM. Antibody to CD-18 exerts endothelial and cardiac protective effects in myocardial ischemia and reperfusion. J Clin Invest. 1991;88:1237-1243.
Lefer DJ, Nakanishi K, Johnston WE, Vinten-Johansen J. Antineutrophil and myocardial protecting actions of a novel nitric oxide donor after acute myocardial ischemia and reperfusion in dogs. Circulation. 1993;88(pt 1):2337-2350.
Romson JL, Hook BG, Kunkel SL, Abrams GD, Schork A, Lucchesi BR. Reduction in the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circulation. 1983;67:1016-1023.
Albelda SM, Smith CW, Ward PA. Adhesion molecules and inflammatory injury. FASEB J. 1994;8:504-512.
Entman ML, Youker K, Shappell SB, Siegel C, Rothlein R, Dreyer WJ, Schmalstieg FC, Smith CW. Neutrophil adherence to isolated adult canine myocytes: evidence for a CD18-dependent mechanism. J Clin Invest. 1990;85:1497-1506.
Smith CW, Entman ML, Lane CL, Beaudet AL, Ty TI, Youker K, Hawkins HK, Anderson DC. Adherence of neutrophils to canine cardiac myocytes in vitro is dependent on intercellular adhesion molecule-1. J Clin Invest. 1991;88:1216-1223.
Entman ML, Youker K, Shoji T, Kukielka G, Shappell SB, Taylor AA, Smith CW. Neutrophil induced oxidative injury of cardiac myocytes. J Clin Invest. 1992;90:1335-1345.
Kubes P, Niu X-F, Smith CW, Kehrli ME Jr, Reinhardt PH, Woodman RC. A novel β1-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration. FASEB J. 1995;9:1103-1111.
Abraham WM, Sielczak MW, Ahmed A, Cortes A, Lauredo IT, Kim J, Pepinsky B, Benjamin CD, Leone DR, Lobb RR, Weller PF. α4-Integrins mediate antigen-induced late bronchial responses and prolong airway hyperresponsiveness in sheep. J Clin Invest. 1994;93:776-787.
Podolsky DK, Lobb R, King N, Benjamin CD, Pepinsky B, Sehgal P, deBeaumont M. Attenuation of colitis in the cotton-top tamarin by anti-a4 integrin monoclonal antibody. J Clin Invest. 1993;92:372-380.
Molossi S, Elices M, Arrhenius T, Diaz R, Coubler C, Rabinovitch M. Blockade of very late antigen-4 integrin binding to fibronectin with connecting segment-1 peptide reduces accelerated coronary arteriopathy in rabbit cardiac allografts. J Clin Invest. 1995;95:2601-2610.
Bouchard RA, Clark RB, Giles WR. Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes: action potential voltage-clamp measurements. Circ Res. 1995;76:790-801.
Issekutz TB, Miyasaka M, Issekutz AC. Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation. J Exp Med. 1996;183:2175-2184.
Ban K, Ikeda U, Takahashi M, Kanbe T, Kasahara T, Shimada K. Expression of intercellular adhesion molecule-1 on rat cardiac myocytes by monocyte chemoattractant protein-1. Cardiovasc Res. 1994;28:1258-1262.
Dalton SL, Scharf E, Briesewitz R, Marcantonio EE, Assoian RK. Cell adhesion to extracellular matrix regulates the life cycle of integrins. Mol Biol Cell. 1995;6:1781-1791.
Hughes BJ, Hollers JC, Crockett-Torabi E, Smith CW. Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion. J Clin Invest. 1992;90:1687-1696.
Dreyer WJ, Smith CW, Michael LH, Rossen RD, Hughes BJ, Entman ML, Anderson DC. Canine neutrophil activation by cardiac lymph obtained during reperfusion of ischemic myocardium. Circ Res. 1989;65:1751-1762.