Immunologic Mechanisms of Vasculitis

Chemoattractants, Cytokines, and Chemokines

Chemoattractants for neutrophils and monocytes play a central role in mediating vasculitis not only by attracting leukocytes but also by activating leukocytes and endothelial cells to participate in inflammatory events. Modulation of the expression of adhesion molecules and cytokine/chemokine molecules by endothelial cells, leukocytes in the circulation, and leukocytes in the tissue orchestrates the inflammation at sites of vasculitis. In small-vessel vasculitis, the balance is tipped toward neutrophil recruitment and activation in the acute phase, but within only one or two days the balance shifts toward recruitment of predominantly monocytes and T lymphocytes. However, if the pathogenic process that initiated the initial injury persists, as often is the case, lesions will include a mixture of acute and chronic inflammatory cells for extended periods of time.

Important chemoattractants for neutrophils and monocytes include C5a (which can be generated by activation of either the classical, lectin or alternative complement pathway), leukotriene B4, platelet-activating factor (PAF), and CXC chemokines such as interlukin-8 (IL-8, CXCL8), platelet factor 4 (PF4, CXCL4), GRO1 oncogene (GROα, CXCL1), and stromal cell-derived factor-1 (SDF-1, CXCL12). Progression of vasculitic lesions from an acute to a subacute or chronic phase is mediated primarily by a different set of chemoattractants that preferentially recruit monocytes and T lymphocytes, such as the CC chemokines macrophage inflammatory protein-1α (MIP-1α, CCL3), monocyte chemotactic protein-1 (MCP-1, CCL2), and regulated upon acitivation, normal T-cell expressed, and secreted (RANTES, CCL5). Chemoattractants not only attract leukocytes through direct engagement of ligands on the leukocytes, but also by enhancing the expression and binding of leukocyte adhesion molecules (e.g., β2 and β1 integrins) to their ligands on endothelial cells and elsewhere in the tissue.

Chemokines on the surface of endothelial cells attract leukocytes to the cells in concert with the adhesion molecules reviewed below, and provide a gradient for directional migration toward the intercellular junction (or transcellularly through the endothelial cytoplasm). Once through the endothelium, gradients of bound and free chemokines in the extracellular matrix and interstitial fluid continue to provide directional signals until the leukocyte encounters enough stimulation to undergo full activation. In physiologic diapedesis of leukocytes across postcapillary venules, full activation is not attained until after the leukocyte has migrated away from the vessel wall. In the absence of an adequate pathogenic stimulus in the extravascular compartment, a leukocyte eventually will undergo senescence and apoptosis without becoming activated. In vasculitis, leukocytes are activated as they attach to or enter the vessel wall resulting in inflammatory vascular damage. There are many etiologies for this mural activation, including the presence of infectious organisms in the vessel wall (e.g., Rickettsia rickettsii in Rocky Mountain spotted fever), immune complexes in the vessel wall (e.g., cryoglobulins in cryoglobulinemic vasculitis), or activation by antineutrophil cytoplasmic autoantibodies (e.g., in microscopic polyangiitis).

Leukocyte Recruitment

Leukocyte recruitment into the vessel wall is accomplished by the events of the leukocyte adhesion cascade. This is a multistep process that sequentially involves leukocyte tethering to endothelium followed by leukocyte rolling, activation, arrest, spreading and crawling on the endothelium, and finally paracellular or transcellular migration (diapedesis) across the endothelium ( Figure 83.4 ). Important initial events are the tethering and rolling of leukocytes on the surface of endothelial cells. This allows the leukocytes to sample the endothelial surface for chemokine signals derived from the underlying tissue and for upregulated endothelial adhesion molecules that can modulate rolling and mediate firm adherence. Engagement of these molecules allows static adhesion to the endothelium followed by crawling toward endothelial cell junctions where transmigration between the cells can be accomplished by further engagement of adhesion molecules in the junction. Transmigration through endothelial cells may also occur. Once through the vessel wall, movement into the perivascular interstitial tissue involves chemoattractant gradients and further engagement between integrins and ligands on extracellular matrix molecules.

Leukocyte Tethering and Rolling

The first step in the exit of leukocytes from flowing blood within the vessel lumen is margination toward the endothelial surface. The resulting juxtaposition of these cells facilitates capture or tethering of the leukocyte to the endothelial surface. Once tethered, shear stress pushing on the leukocyte, combined with the formation of new adhesive bonds at the downstream leukocyte edge and disruption of adhesive bonds at the upstream leukocyte edge, allows for rolling of the leukocyte along the endothelial surface. The adhesive interactions necessary for leukocyte tethering and rolling are mediated primarily by the binding of selectin adhesion molecules to their ligands. E-selectin and P-selectin are expressed on activated endothelium, and P-selectin is also expressed on activated platelets. L-selectin is constitutively expressed on most leukocytes. P-selectin is pre-formed and stored in Weibel-Palade bodies or alpha granules, respectively, and therefore is rapidly translocated to the cell surface quickly in response to mediators released at sites of acute inflammation, such as histamine and thrombin. In contrast, cell surface expression of E-selectin is dependent on RNA and protein synthesis stimulated by inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), and is therefore delayed for several hours after an inflammatory stimulus is received.

A number of selectin ligands (or counter receptors) have been described. The best characterized is P- selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 was originally described as a P-selectin ligand but is now known to function as a ligand for all three selectins. It is expressed on most leukocytes, endothelial cells, and platelets. Ligands for E-selectin also include E-selectin ligand -1 (ESL-1) and correctly glycosylated forms of CD44, both of which are expressed on leukocytes. L-selectin ligands include PSGL-1 on other leukocytes, and peripheral node addressins including glycosylation-dependent cell adhesion molecule-1 (GLYCAM-1), podocalyxin, and CD34, which are found on peripheral lymphoid tissue high endothelial venules (HEV).

Binding of the selectins to their ligands is modulated by several molecular features. High affinity selectin binding is dependent on ligand glycosylation, in part requiring the sialyl Lewis X tetrasaccharide as a recognition epitope. The final step in the synthesis of this critical binding epitope is the addition of an α(1,3)-linked fucose, mediated by α(1,3)-fucosyltransferases type VII and IV. L-selectin binding to ligands on peripheral lymph node HEV, or at sites of chronic inflammation, utilize a sulphated version of this epitope, 6-sulph-sialyl Lewis X. Lastly, high affinity binding of P-selectin or L-selectin to PSGL-1 requires sulphation of certain tyrosine residues near the N-terminus of the molecule. The molecular features of the selectins and their ligands result in binding interactions with very high on- and off-rates, allowing bonds to form and break quickly. In addition, shear stress is required and also enhances L-selectin and P-selectin binding. Combined, these binding features result in interactions called catch bonds that are particularly suited to mediating the tethering and rolling of leukocytes on the endothelium.

At sites of small-vessel vasculitis, increased expression of P-selectin and E-selectin on endothelial cells is induced by inflammatory chemokines. Subsequent binding to their ligands on leukocytes allows for and modulates primary leukocyte tethering and rolling. Rolling may also be triggered by secondary tethering. In this process, leukocytes previously captured onto the endothelium bind, via their PSGL-1, to L-selectin on other marginated leukocytes and mediate their capture and tethering to the endothelium. The physiologic significance of secondary tethering remains unclear, although it can be observed in vivo.

In addition to selectin-mediated rolling, it is known that some integrin adhesion molecules can mediate and modulate this process. For example, the β ₁ integrin very late antigen 4 (VLA4) has been shown to mediate rolling of monocytes and T cells. Similarly, the β2 integrin lymphocyte function-associated antigen 1 (LFA-1) can interact with endothelial intercellular adhesion molecule 1 (ICAM-1) to modulate slow rolling. In this process, neutrophil signaling events triggered by binding to E-selectin result in expression of an intermediate-affinity form of LFA-1 that transiently interacts with endothelial ICAM-1 to decrease rolling velocity. A second β2 integrin, macrophage-1 antigen (MAC-1) can also mediate slow rolling. Thus, the additive ligation of more than one class of adhesion molecule may be required for efficient transition from fast rolling to a state of arrest.

Leukocyte Activation and Arrest

Leukocyte transition from rolling to arrest is the next step in leukocyte transmigration. Leukocyte activation is rapidly triggered by chemokines and other cell activators, and leukocyte arrest is mediated primarily by binding of leukocyte integrin-type adhesion molecules to members of the immunoglobulin superfamily expressed on the endothelium. Leukocyte rolling facilitates activation by slowing transit time which allows for increased exposure to soluble activating factors and also exposure to endothelial-bound activating factors. Evidence suggests that chemokines that facilitate engagement of integrins and direct endothelial transmigration are bound to glycosaminoglycans (GAGs) or the Duffy antigen/receptor for chemokines (DARC) on the surface of endothelial cells. The type of chemokine that occupies these binding sites depends on the relative availability and concentration of the chemokines. These sites can engage different cytokines depending on the type of leukocyte that is being recruited for a particular physiologic or pathophysiologic process. Thus, if high levels of CXC chemokines are present in the vessel wall, CXC chemokines will occupy the endothelial binding sites and will attract predominantly neutrophils. In contrast, if high levels of CC chemokines are present in the vessel wall, CC chemokine will occupy the endothelial binding sites and will attract predominantly T lymphocytes. Chemokines can reach the luminal surface of endothelial cells by vesicular abluminal-to-luminal transcytosis, or be deposited there by circulating platelets or microparticles, or the exocytosis of intracellular granules derived from other cells.

Integrins with the β ₁ or β2 subunit are particularly important in the arrest of neutrophils and monocytes and their recruitment to sites of acute vascular inflammation. These include CD49d/CD29 (VLA-4), CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18. The avidity of certain integrins on leukocytes can be rapidly upregulated by chemokines at sites of inflammation and enhance arrest and accumulation of inflammatory cells. Ligation of specific heterotrimeric G-protein coupled receptors (GPCRs) by chemokines triggers a complex intracellular signaling cascade that results in integrin transition to intermediate and high affinity forms to promote arrest. This process, termed inside-out signaling, is incompletely understood but is thought to sequentially involve phospholipase C signaling, small GTPase activation, and induction of integrin conformational changes.

Several members of the immunoglobulin superfamily are the ligands on the surface of endothelial cells for the leukocyte integrins. Important examples are the intracellular adhesion molecules (ICAMs) that include ICAM-1, ICAM-2, and ICAM-3. Up-regulation of these ligands, especially ICAM-1, on endothelial cells at sites of vascular inflammation works in concert with the up regulation of integrins on circulating leukocytes to induce leukocyte infiltration at sites of vasculitis. Ligation of CD11a/CD18 (LFA-1) on neutrophils to ICAM-1 on endothelial cells causes adhesion and ligation of CD11b/CD18 (MAC-1) to ICAM-1, promoting arrest and diapedesis. Ligation of β2 integrins to ICAMs is particularly important for neutrophil influx at sites of acute vascular inflammation, whereas lymphocyte recruitment to sites of inflammation depends more on the binding of the β4 integrin VLA-4 (CD49d/CD29) to a different immunoglobulin, vascular cell adhesion molecule-1 (VCAM-1 [CD106]).

Many of the leukocyte adhesion cascade processes and events described thus far can contribute to the specificity of leukocyte recruitment and arrest. Specificity can be accounted for by the combinatorial effects of differential expression of integrins and their receptors, differential expression of the chemokines and their receptors, and varied intracellular signaling to regulate integrin function.

Leukocyte Crawling and Transmigration

Static leukocyte adhesion to the endothelium is followed by transmigration of the cell across the endothelial barrier. Adherent leukocytes crawl over the endothelial surface (termed lateral migration) to locate a preferred site at which to cross the endothelium. Neutrophil crawling on venular endothelium is dependent MAC-1 and its ligand ICAM-1, and disruption of these binding events results in a delay of transmigration. Crawling is likely to also occur as an integral part of the prominent leukocyte recruitment that occurs on the arterial side of the circulation in the vasculitides.

Once at an endothelial cell junction, neutrophils and monocytes are able to crawl through the junctions between endothelial cells (paracellular transmigration) by progressive engagement and disengagement of a series of adhesion molecules on the surface of the leukocytes with ligands on the junctional surfaces of the endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD31), an immunoglobulin superfamily member, is expressed on leukocytes and on endothelial cell surfaces and intercellular junctions. Homophilic PECAM-1 adhesion is particularly important for transmigration of leukocytes through intercellular junctions between endothelial cells. Transmigration of neutrophils and monocytes also involves the interactions of LFA-1, VLA4, or MAC1 on the leukocytes with immunoglobulin superfamily adhesion molecules in the endothelial intercellular junction, JAM-A (junctional adhesion molecule-A), JAM-B, or JAM-C, respectively. Distal to the PECAM-1-mediated and JAM-mediated events, homophilic binding between CD99 on leukocytes and CD99 in the intercellular junction between endothelial cells takes over to facilitate completion of diapedesis. Recent work also shows that endothelial cell-selective adhesion molecule (ESAM) at the endothelial junction facilitates transmigration of neutrophils but not lymphocytes, possibly by activation of the Rho GTPase and destabilization of the tight junction. Leukocytes may also cross the endothelial barrier by a transcellular route through the body of the endothelial cell (transcellular migration). The significance of this route in vivo , and the underlying mechanisms involved in this process are currently poorly understood.

During the process of diapedesis, there are endothelial cytoskeletal and junctional events that result in localized retraction of endothelial cytoplasm and opening of the intercellular junction. These events result from signals transduced from the ligation of endothelial adhesion molecules (e.g., ICAM-1 and PECAM-1). Endothelial stimulation by inflammatory cytokines and adhesion molecule ligation, activation of membrane bound and interstitial matrix metalloproteinases (MMPs), and release of serine proteinases at the surface of migrating leukocytes cause altered distributions and integrity of junctional adhesion molecules (JAMs), vascular endothelial cadherins, and other junctional complex components so that leukocytes can crawl between endothelial cells into the intima, muscularis, or perivascular interstitium. Once past the endothelial barrier, leukocyte migration through the underlying lamina and matrix involves progressive engagement of matrix molecules by leukocyte adhesion molecules, especially PECAM-1, β1 integrins (e.g., VLA-4, VLA-5) and carcinoembryonic antigen–related cell adhesion molecules (CEACAMs).

At sites of vasculitis, the endothelium may be absent because of inflammatory damage. Denudation of endothelium will expose surfaces that contain ligands for neutrophils, monocytes and other inflammatory cells ( Table 83.3 ). At sites of acute necrotizing vasculitis, the lumen often is immediately adjacent to zones of fibrinoid necrosis that contain fibrin that has been generated by the activation of the coagulation cascade by tissue factor and other thrombogenic materials at the site of injury. Mac-1 and other neutrophil and monocyte adhesion molecules can adhere to fibrin and begin the process of activation and migration into the inflamed tissue. Matrix molecules exposed by endothelial denudation, such as fibronectin, laminin, entactin, and collagen also present ligands for leukocyte adhesion molecules, especially β1 integrins such as VLA-4 and VLA-5. Serine proteinases (e.g., elastase) released at the surface of migrating leukocytes act synergistically with β1 integrins and upregulated leukocyte PECAM-1 to allow the leukocytes to migrate into the tissue.