The α_Mβ₂ integrin and its role in neutrophil function

ABSTRACT

Neutrophils are the first cell type to arrive at the injury sites and play a critical role in host defense, by virtue of its ability to adhere and transmigrate through endothelium, to phagocytose foreign pathogens, and to produce free oxygen radicals and proteolytic enzymes. Yet, inappropriate neutrophil activation causes tissue damage and various inflammatory diseases. These physiological and pathological functions of neutrophils depend on the engagement of certain surface receptors, especially α_Mβ₂, the major β₂ integrin receptor present on neutrophil surface. Understanding of the molecular mechanisms underlying ligand binding by α_Mβ₂, as well as the roles of α_Mβ-ligand interactions in neutrophil functions will enable us to regulate more precisely neutrophil activities: that is, to promote their host defense functions, and at the same time to minimize their deleterious effects on normal cells.

INTRODUCTION

Neutrophils play key roles in the host defense network against pathogens by virtue of their abilities to phagocytose microorganisms and to produce oxygen free radicals and proteolytic enzymes. Extravasation of neutrophils from the blood stream proceeds through three coordinated steps: rolling and tethering, firm adhesion, and transmigration¹. The first step depends on the selectin molecules expressed on both neutrophils and endothelial cells (EC)²,³. The second step is mediated through interactions of the β₂ integrins⁴,α_Lβ₂ and α_Mβ₂ , present on the neutrophils and their counter receptors, ICAM-1 and ICAM-2, on the EC. Neutrophil-EC interaction can also be mediated by fibrinogen (Fg)⁵. ICAMs bind directly to α_Lβ₂⁶and aMb2⁷, whereas Fg bridges neutrophils and EC by binding to α_Mβ₂ and ICAM-1⁵. Neutrophils from patients with Leukocyte Adhesion Deficiency (LAD) fail to adhere and transmigrate through EC, resulting in life-threatening bacterial and fungal infections⁸. The role of α_Mβ₂ in neutrophil adhesion and transmigration has been well demonstrated in animal models using function blocking mAbs^{9, 10, 11}, α_Mβ₂ inhibitors¹², and α_Mβ₂-deficient mice¹³,¹⁴.

Neutrophils and their associated diseases

Despite the essential role of neutrophils in host defense, inappropriate neutrophil activation has detrimental consequences¹⁵. The superoxide radicals and proteolytic enzymes produced by activated neutrophils cause ischemia/reperfusion injury and tissue damage¹⁶,¹⁷. In addition, activated neutrophils produce a multitude of cytokines¹⁸,¹⁹ which initiate and sustain the chronic inflammatory process, leading to the development of various autoimmune diseases¹⁶,²⁰. Consistent with these deleterious effects, blockade of α_Mβ₂ -mediated ligand recognition by neutrophils using mAbs or inhibitors decreases ischemia/reperfusion injury²¹,²², reduces myocardial infarction size, myocardial necrosis²³, and liver cell injuries²⁴, and diminishes neointimal thickening and restenosis after angioplasty²⁵. The α_Mβ₂ blocking mAbs are also effective in the treatment of gram-negative sepsis and hemorrhagic shock²⁶. Although therapies using these function-blocking antibodies are very promising, non-selective blockade of all leukocyte functions, such as neutrophil activation, transmigration, and phagocytosis, also leads to severe complications, such as bacteriall and fungal infections²⁷.

The α_Mβ₂ integrin recognizes multiple ligands

α_Mβ₂, a heterodimeric surface receptor, belongs to the β₂ integrin subfamily. These “leukocyte” integrins are composed of a common β₂ subunit noncovalently linked to one of four distinct yet highly homologous a subunits, αL, αM,αX, and αD²⁸,²⁹. α_Mβ₂ is expressed by neutrophils, monocytes and NK cells, and recognizes a multitude of very different protein and nonprotein ligands. These multiple interactions provide a molecular basis for the versatile roles of neutrophils and monocytes in host defense. Protein ligands for α_Mβ₂ include extracellular matrix proteins such as fibronectin, laminin, collagen and vitronectin³⁰; counter-receptors of the immunoglobulin superfamily such as ICAM-1³¹ and ICAM-2³²; blood coagulation proteins such as fibrinogen³³, factor X³⁴, and kininogen³⁵; and the complement pathway product, C3bi³⁶; as well as haptoglobin³⁷,denatured albumin³⁸, KLH³⁹, myoloperoxidase⁴⁰ and elastase⁴¹; non-protein ligands for α_Mβ₂ include LPS⁴², zymosan, β-glycans⁴³, heparin⁴⁴,⁴⁵ and oligodeoxynucleotide⁴⁶. In addition, a variety of microorganisms produce α_Mβ₂ ligands (e.g. NIF⁴⁷, WI-1⁴⁸ and gp63⁴⁹) as a means of subverting or bypassing host defense mechanisms⁵⁰. Unlike certain integrins in the β₁and 3 sub-families, where a single receptor interacts with many different proteins through the common RGD sequence⁵¹, the α_Mβ₂ ligands share few, if any, similarities or conserved sequences. The molecular structure of the α_Mβ₂ receptor that enables it to interact with many unrelated ligands is not yet clear, nor are the physiological functions of these interactions.

Structural basis of α_Mβ₂-ligand interactions

At least five structural domains exist in α_Mβ₂: the I-domain, the cation-binding repeats, and the lectin binding domain in the a subunit, and the putative I-domain and the protease resistant cysteine-rich region in the b subunit. The I-domain is a region of ∼ 200 amino acids and is found only in certain integrin a subunits. The crystal structure of the α_MI-domain was solved⁵² and was shown to contain seven helices and six b sheets connected by short surface loops. Five residues located within several of these loops form a novel cation binding site, termed the MIDAS motif⁵². Recently, structures of other I-domains were determined. a-helices/b-sheets folds similar to those of the α_MI-domain have been observed^{53, 54, 55}. The role of the I-domains in the ligand binding has been well established. Diamond, et al⁵⁶, find that binding of C3bi and ICAM-1 to α_Mβ₂ is blocked by mAbs to the aMI-domain, suggesting a spatial proximity between these ligand-binding sites. We⁵⁷ and others⁵⁸, ⁵⁹ showed that the recombinant aMI-domain interacts with NIF, ICAM-1, C3bi, and Fg. The importance of the I-domain in ligand binding has also been demonstrated in other integrins (α_Lβ₂ ,α_Xβ₂ , α₂β₁and α₁β₁)^{60, 61, 62, 63}. Given the similarity of the I-domain structures, it is not well understood how I-domains can recognize a multitude of very different proteins. Previous studies have shown that five amino acids (Asp¹⁴⁰ , Ser¹⁴² , Ser¹⁴⁴ , Asp²⁴² , and Thr²⁰⁹ ), conserved within essentially all I-domains, are critical to ligand-binding activity of all I-domain-containing integrins⁵²,^{64, 65, 66, 67, 68}, regardless of the nature of their ligands. Thus, the specificity of each integrin, which is vital to its individual in vivo roles, can not be derived from these conserved residues. To determine the molecular basis for integrin α_Mβ₂ to interact with multiple ligands, we have used the Homologue- Scanning Mutagenesis approach⁶⁹ and systematically probed the entire outer hydrated surface of the I-domain of aM. We found that overlapping but non-identical regions within the I-domain are involved in recognition of different ligands by the α_Mβ₂ receptor⁷⁰. We have further mapped the ligand binding pocket for NIF to a narrow region composed of Pro¹⁴⁷ -Arg¹⁵² , Pro²⁰¹ -Lys²¹⁷ , and Asp²⁴⁸ - Arg²⁶¹ of α_M⁷¹.

In addition to the I-domain, the cation binding repeats in integrin a subunits also have been implicated in ligand binding. D'Souza, et al, first implicated the second cation-binding repeat of aIIb in α_IIb β₃ binding of the Fg- γ chain⁷². The cation-binding repeats of α_Mβ₂ are also important in ligand binding. Altieri, et al, showed that a mAb (OKM1) recognizing an epitope in the cation-binding repeat⁵⁶, completely blocked Fg binding to α_Mβ₂⁷³. Other antibodies mapped to the cation-binding repeats have potent inhibitory effects on ICAM-1 binding to α_Mβ₂⁵⁶. In contrast, antibodies recognizing the region between the cation-binding repeats and the transmembrane region are poor inhibitors of α_Mβ₂ functions, and are presumed not to be directly involved in ligand interaction⁵⁶. The involvement of the cation-binding repeats in ligand recognition was also illustrated in α_Lβ₂ and α₂β₁using recombinnt fragments⁶¹,⁷⁴. Springer has recently proposed, based on computational analysis, that the region surrounding the cation-binding repeats folds into a β-propeller-like structure⁷⁵. Though very appealing, this model is yet to be confirmed directly with experimental data. To this end, several recent studies have provided encouraging data consistent with this model⁷⁶,⁷⁷. The ultimate test of this model will rely on structural studies using either X-ray crystallography or two-dimensional NMR.

In addition to the a subunit, binding of some ligands requires cooperation from the bsubunit as well. Similar to aIIb β₃, the homologous D¹³⁴ XSXS sequence within β₂ is also important for α_Mβ₂ binding to C3bi, Fg, and ICAM-1. Mutations of these oxygenated residues into Ala abolished α_Mβ₂ binding to these three ligands⁷⁰,⁷⁸,⁷⁹. Thus, α_Mβ₂-ligand interaction involves discrete regions within the I-domain and the β-propeller of the a subunit, as well as the β₂ subunit. Further studies are required to ascertain the exact roles of these different regions of α_Mβ₂ in ligand binding.

Conclusion

α_Mβ₂ is involved intimately in every aspects of neutrophil functions, by virtue of its ability to recognize multiple different protein and non-protein ligands. The molecular basis that confers α_Mβ₂ with such extraordinary capability is, in part, an overlapping but non-identical ligand binding pocket, and involves the cooperation between the I-domain and the β-propeller region, as well as the β-subunit. Understanding of the molecular basis of α_Mβ₂-ligand interactions will enable us to precisely control neutrophil functions, that is, to avoid the deleterious effects of neutrophil activation while keeping intact its host defense function.