Process of Leukocyte Extravasation in Inflammation

Adhesion of peripheral blood leukocytes to endothelium leads to the process of leukocyte transendothelial migration into inflammatory sites such as the arthritic synovium.^2–4 High endothelial venules (HEVs) primarily found in lymphoid organs are also present at sites of lymphoid neogenesis in the synovium.^3,29,30 Lymphocytes recirculate through HEVs during homing, and thus inflammatory leukocyte recruitment may be considered as “pathologic homing”^3,4,29 (see Figure 25-1).

During leukocyte adhesion and transendothelial migration, an early, weak adhesion termed rolling occurs first. This step involves selectins and their ligands and leads to leukocyte activation. Activation-dependent, firm adhesion involves mostly integrin-dependent interactions, as well as the secretion of numerous chemokines. Chemokines preferentially attract endothelium-bound leukocytes^2,20,31 (see Figure 25-1).

Adhesion Receptors and Ligands

CAMs have been classified into integrin, selectin, immunoglobulin, and cadherin superfamilies^4,32 (Table 25-1). E-, P-, and L-selectin contain a lectin-like extracellular N-terminal domain, an epidermal growth factor (EGF)-like motif, and two to nine moieties related to complement regulatory proteins.^33,34 E- and P-selectin are expressed by ECs, whereas L-selectin is mostly expressed by leukocytes.³⁴ During leukocyte transendothelial migration, selectins mediate the initial tethering and rolling of leukocytes.^20,34,35 E-selectin is a marker for cytokine-induced EC activation.³⁴ E-selectin ligand-1 (ESL-1) and P-selectin ligand-1 (PSGL-1) contain sialylated glycan motifs such as sialyl Lewis-X (sLe^x).³⁴ P-selectin is constitutively present on the membrane of EC Weibel-Palade bodies.³⁴ P-selectin is involved in the early phases of leukocyte-EC adhesion.³⁵ L-selectin serves as a lymphocyte homing receptor, where it mediates the physiologic recirculation of naïve lymphocytes through specialized HEV.^32,34 However, L-selectin has also been implicated in inflammatory leukocyte recruitment.^3,34 All three selectins are expressed in the arthritic synovium^3,4,36 (see Table 25-1).

Table 25-1 Relevant Members of Adhesion Molecule Superfamilies*

^* See text for abbreviations.

Integrins are αβ heterodimers. Each of the common β chains is associated with one or more α subunits.^3,32 Cell adhesion to the extracellular matrix (ECM) is mostly mediated by β₁ and β₃, whereas intercellular adhesion is assisted through β₁ and β₂ integrins.^2,32 β₁ and β₃ integrins are expressed on ECs, whereas β₂ integrins are leukocyte CAMs.³² Integrin-mediated adhesion and migration have been associated with arthritis.^3,4,37 The α₁-α₆, α_V, α_L, α_M, α_X, and β₁-β₇ integrin subunits have all been detected in the inflamed synovium.^3,4,37

The immunoglobulin superfamily of CAMs is a group of transmembrane glycoproteins containing one or more immunoglobulin-like motifs of 60 to 100 amino acids.³² Vascular cell adhesion molecule-1 (VCAM-1) is constitutively expressed on ECs; however, its expression is upregulated by proinflammatory cytokines.³⁸ There is abundant VCAM-1 expression in the inflamed synovium.^3,37,39 ICAM-1, the counterreceptor for the β₂ integrins LFA-1 (α_Lβ₂), Mac-1 (α_Mβ₂), and α_Xβ₂, is expressed on both ECs and leukocytes.^3,32,40 ICAM-1 is highly expressed on ECs in inflammatory sites such as in the RA synovium.^37,40 Among other ICAMs, ICAM-2 is constitutively expressed on ECs and may not be an activation marker.⁴⁰ ICAM-3 is a leukocyte CAM; however, it is also present on some RA synovial ECs.⁴⁰ All three ICAMs bind to β₂ integrins.^3,40 Other members of this superfamily include CD2 and LFA-3. CD2 binds to LFA-3, and both CAMs exert abundant expression in the arthritic synovium.^3,41 Platelet-endothelial adhesion molecule 1 (PECAM-1 or CD31) mediates homotypic adhesion by binding to another PECAM-1 molecule, as well as heterotypic adhesion by recognizing the α_Vβ₃ integrin.^3,32,42 PECAM-1 is a marker of endothelial activation, and it is expressed in the arthritic synovium.^42,43

Some other CAMs involved in leukocyte-EC adhesion underlying inflammation and associated with arthritis include CD44, vascular adhesion proteins (VAP-1 and VAP-2), endoglin, E- and N-cadherin, cadherin-11, and junctional adhesion molecules (JAMs).2–4,36,41,44–48 CD44 is a receptor for hyaluronate³² and is expressed on activated ECs in inflammatory sites.^36,41,49,50 VAP-1 has originally been isolated from synovial ECs. The expression of VAP-1 is increased in RA.^46,51 Endoglin (CD105) is a receptor for transforming growth factor (TGF)-β1. Endoglin is involved in EC adhesion and is expressed in synovitis.^52,53 Cadherins are primarily involved in embryogenesis; however, synovial fibroblast cadherin-11 has been implicated in arthritis as well^4,54,55 (see Table 25-1).

Chemokine Superfamilies

Most CXC chemokines chemoattract neutrophils. Many genes coding these chemokines are clustered on chromosome 4q12-13.⁵⁷ In contrast, the genes of some CXC chemokines such as CXCL4 (platelet factor 4; PF4) and CXCL10 (IFN-γ-inducible 10-kD protein; IP-10) are located on different chromosomes, and these chemokines recruit lymphocytes and monocytes.^56,57

CC chemokines stimulate monocyte chemotaxis, but some members of this subclass may also recruit lymphocytes. The genes of monocyte-chemoattracting CC chemokines have been clustered to chromosome 17q11.2. In contrast, genes of CC chemokines recruiting lymphocytes are generally located elsewhere.^9,56,57

The CX₃C chemokine family has only one member, CX₃CL1 (fractalkine).^{12,56,58–60} This chemokine is chemotactic for mononuclear cells, but it also serves as an adhesion molecule.^59,60

The C family contains two members: XCL1 (lymphotactin) and XCL2 (single C motif 1β; SCM-1β). Lymphotactin is primarily involved in the migration of T lymphocyte subsets to inflammatory sites.^61,62

Chemokine Receptors

The chemokines described earlier mediate their effects via 7-transmembrane domain receptors expressed on the target cells.^56,57 Although some receptors (e.g., CXCR2, CCR1, CCR3) have multiple chemokine ligands, others (e.g., CXCR6, CCR8, CCR9) are specific receptors for one single ligand.^9,57 Again, there may be a relationship between some chemokine receptors and the functions of their ligand(s). For example, single-ligand receptors such as CCR8 or CCR9 bind to chemokine ligands mostly exerting homeostatic functions (see later). In contrast, CXCR2, a receptor recognizing multiple CXC chemokines, plays a crucial role in inflammation and angiogenesis.^9,63 Chemokine receptors have also been associated with various types of autoimmune inflammation. For example, RA, a mostly Th0-Th1 type disease, is associated with CXCR3 and CCR5, whereas asthma, a known Th2 type disease, is rather associated with CCR3, CCR4, and CCR8 tissue expression.^12,64,65

Inflammatory and Homeostatic Chemokines: Is It a Justified Classification?

Chemokines have recently been functionally classified into these subgroups.^10,56 As many functions of these chemokines overlap, this classification may not be really justified. Numerous CXC, CC, and CX₃C chemokines implicated in the pathogenesis of arthritis are termed inflammatory chemokines. These chemokines recruit mostly effector cells including monocytes, neutrophils, and T cells into tissues.^9,10,56,66 As included in Table 25-2, there is a great body of evidence suggesting the role in RA of CXCL1 (growth-regulated oncogene α; GROα); CXCL4 (platelet factor 4; PF4); CXCL5 (epithelial-neutrophil activating protein 78; ENA-78); CXCL6 (granulocyte chemotactic protein 2; GCP-2); CXCL7 (connective tissue activating protein III; CTAP-III); CXCL8 (interleukin 8; IL-8); CXCL9 (monokine induced by interferon-γ; Mig); CXCL10 (interferon-γ-inducible 10-kD protein; IP-10); CXCL12 (stromal cell–derived factor 1; SDF-1); CXCL13 (B cell–activating chemokine 1; BCA-1); and CXCL16. Among CC chemokines, CCL2 (monocyte chemoattractant protein 1; MCP-1); CCL3 (macrophage inflammatory protein 1α; MIP-1α); CCL5 (Regulated upon Activation, Normal T cell Expressed and Secreted; RANTES); CCL19 (Epstein-Barr virus–induced gene 1 ligand chemokine; ELC); CCL20 (MIP-3α); and CCL21 (secondary lymphoid tissue chemokine; SLC) have been implicated in leukocyte recruitment underlying inflammatory synovitis. Finally, CX₃CL1 (fractalkine) and XCL1 (lymphotactin) are also considered as inflammatory chemokines.^7,9,56 Accordingly, CXCR1-CXCR6, CCR1-CCR6, and XCR1 and CX₃CR1 are involved in the pathogenesis of RA.^7,56,67

Homeostatic chemokines are constitutively produced in microenvironments of lymphoid or nonlymphoid tissues such as in the skin or mucosa. These chemokines promote lymphocyte homing into these tissues, a process associated with the physiologic function of the adaptive immune system. Lymphocyte recirculation is involved in antigen sampling and immune surveillance.^10,68,69 Among CXC chemokines, CXCL12 (SDF-1), CXCL13 (BCA-1), and CXCL16, as well as their respective receptors, CXCR4, CXCR5, and CXCR6, exert such effects.^10,68,69 Among CC chemokines, CCL17 (TARC), CCL19 (ELC), CCL21 (SLC), CCL22 (MDC), CCL25 (TECK), CCL27 (CTACK), CCL28 (MEC), as well as their receptors, CCR4, CCR7, CCR9, and CCR10, are involved in the homeostasis of lymphoid tissues.^56,68,69 As described earlier, among others, CXCL12, CXCL13, CXCL16, CCL19, and CCL21 have also been implicated in arthritis-associated inflammatory cell recruitment and synovial lymphoid neogenesis.^{7,10,56,67–71} The synovium is, in many ways, similar to mucosa-associated lymphoid tissues (MALT), which may explain the dual role of some chemokines in physiologic lymphocyte homing and inflammation.^10,68,69,71

Angiogenesis and Vasculogenesis

Angiogenesis is the formation of new capillaries from preexisting blood vessels, whereas vasculogenesis is the outgrowth of vessels from endothelial progenitor cells (EPCs).^{7,13–15,72–77} Angiogenesis may increase the total endothelial surface and thus may enable leukocyte extravasation into inflammatory sites.^14,15 The perpetuation of angiogenesis has been associated with inflammatory diseases such as RA or psoriasis and in malignancies.^14,15,77,78 The outcome of such “angiogenic diseases” is dependent on the balance or imbalance between angiogenic mediators and angiostatic factors.⁷⁷ Several cytokines, growth factors, chemokines, certain CAMs, and other mediators can modulate neovascularization in inflammation^14,15,73,77 (Table 25-3; see Figure 25-1).

Table 25-3 Angiogenic and Angiostatic Factors in Rheumatoid Arthritis*

^* See text for abbreviations. See Table 25-2 for traditional chemokine designations.

^† Mediators with both proangiogenic and antiangiogenic effects.

Angiogenic Factors

The hypoxia-vascular endothelial growth factor (VEGF)-angiopoietin system seems to be of outstanding importance in arthritis-associated angiogenesis.^73,79,80 VEGF is induced by hypoxia and hypoxia-inducible factors 1 and 2 (HIF-1, HIF-2) in RA.^{11,73,80–85} Significant hypoxia is characteristic of RA joints.^73,86 Recently, the stimulatory effect of hypoxia on the angiogenic drive of RA synovial fibroblasts has been demonstrated.^84,87 Hypoxia-inducible HIF-1 and HIF-2 are strongly expressed in the RA synovium.^81,84,85,87 However, hypoxia may also act via HIF-independent regulatory pathways including the peroxisome-proliferator-activated receptors (PPARs).⁸⁸ The angiopoietin-1 (Ang1)/Tie2 complex interacts with VEGF during the stabilization of newly formed blood vessels.⁸⁹ In contrast, Ang2, an antagonist of Ang1, inhibits vessel maturation.^80,89 Ang1 and Tie2 have been detected in the RA synovium.^90,91

Apart from VEGF, other growth factors including fibroblast growth factors (FGF-1 and FGF-2), transforming growth factor β (TGF-β), connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF) have been implicated in synovial angiogenesis.^1,7,14 Recently, the role of placenta growth factor (PIGF) in RA and inflammatory angiogenesis has been postulated. PIGF, like VEGF, binds to the flt-1/VEGF-R1 receptor. There is abundant expression of PIGF in the synovial tissue of arthritic mice.⁹²

Proinflammatory cytokines may exert direct angiogenic activity or may act indirectly via VEGF-dependent pathways.^14,93 Primarily TNF, IL-1, IL-6, IL-15, IL-17, IL-18, oncostatin M, granulocyte (G-CSF), and granulocyte-macrophage colony-stimulating factors (GM-CSFs) have been implicated in synovial angiogenesis.^14,93 Monocyte migration inhibitory factor (MIF) has been implicated in angiogenesis, as well as atherosclerosis, a major cause of death in RA patients.^94,95

Among numerous other angiogenic mediators not mentioned earlier, serum amyloid A (SAA), an important acute-phase reactant, has also been implicated in arthritis and angiogenesis. Interaction of SAA with formyl peptide receptor-like 1 (FPRL1) induces endothelial cell proliferation, migration and angiogenesis, and synovitis.^96,97 Further angiogenic factors include, without mentioning further details, endothelin 1 (ET-1), members of the cyclooxygenase-2 (COX-2)-prostaglandin E₂ network, angiogenin, angiotropin, pleiotrophin, platelet-activating factor (PAF), substance P, erythropoietin, adenosine, histamine, prolactin, thrombin, and sphingosine-1-phosphate (S1P).^1,14,15,98

The involvement of chemokines, chemokine receptors, and CAMs in angiogenesis is discussed later in context with the complex regulation of leukocyte recruitment.

Vasculogenesis in Inflammatory Conditions

EPCs are hematopoietic stem cells expressing, among other antigens, CD34, CD133, type 2 VEGF receptor (VEGFR-2 or Flk-1), and the CXCR4 chemokine receptor.15–17,99–101 During vasculogenesis, EPCs differentiate into mature endothelial cells.¹⁰¹ Vasculogenesis is involved in tissue development, vascular repair, atherosclerosis, and inflammation.^{15,17,99–102}

Several groups have described defective vasculogenesis related to impaired EPC numbers and functions in RA and scleroderma.^{1,16,17,102–105} Impaired vasculogenesis has been associated with increased cardiovascular morbidity and mortality in these disease states.^1,102,106 Effective control of inflammation using corticosteroids and anti-TNF agents may stimulate EPCs and thus may restore defective vasculogenesis.^103,107 In addition, the induction of vasculogenesis may be beneficial for patients with cardiovascular disease¹⁰⁶ and the stimulation of EPCs and vasculogenesis may also suppress premature atherosclerosis in RA.¹

Chemokines and Adhesion Receptors

The molecular mechanisms and signaling pathways of chemokine-induced CAM expression have been described. Briefly, an atypical protein kinase C, PKC-ξ, has been identified. Treatment of cells with chemokines induces PKC-ξ kinase activity through its interaction with PI3K. This leads to increased cell surface integrin expression via further signaling steps.¹

Another example for chemokine-CAM interactions is the regulation of β₃ integrin expression by CCL2 (MCP-1) through the Ets-1 transcription factor, and the ERK-1/2 cascade. This pathway is involved in both inflammation and angiogenesis.¹⁰⁸ The CCL21-CCR7 interaction results in the stimulation of LFA-1- and ICAM-1-dependent adhesion.¹⁰⁹ Stimulation of CXCR1- and CXCR2-dependent pathways in the antigen-induced arthritis (AgIA) model resulted in increased neutrophil adhesion to endothelium.¹¹⁰ Thus various chemokines and chemokine receptors are involved in driving leukocyte transendothelial migration.

Chemokines and Chemokine Receptors in Angiogenesis and Vasculogenesis

Numerous CXC and CC chemokines, fractalkine, and their receptors have been implicated in angiogenesis underlying RA.^7,12,14,111 The angiogenic nature of most CXC chemokines has been associated with the glutamyl-leucyl-arginyl (ELR) amino acid motif within their structure.⁶³ ELR-containing CXC chemokines that mediate angiogenesis include CXCL1 (GROα), CXCL5 (ENA-78), CXCL7 (CTAP-III), and CXCL8 (IL-8).^12,15,63,111 In contrast, the ELR-lacking CXCL4 (PF4), CXCL9 (Míg), and CXCL10 (IP-10) inhibit angiogenesis.^8,9,39 Interestingly, some authors suggest that the effect of VEGF on endothelial cells may be, in part, mediated by CXCL10.⁴⁴ It seems that CXCL12 (SDF-1) may play a significant role in RA-associated angiogenesis despite the fact that this chemokine lacks the ELR motif.^7,112,113 CXCL12 is also a major mediator of lymphoid neogenesis in the RA synovium.^112,113 Hypoxia stimulates CXCL12 production by RA synovial fibroblasts.¹¹² CXCL12 has even been implicated in vasculogenesis. Virtually all EPCs express CXCR4 and migrate in response to SDF-1/CXCL12.^42,43

Much less evidence is available regarding the role of CC chemokines in angiogenesis. CCL2 (MCP-1) may induce endothelial cell chemotaxis in vitro and angiogenesis in vivo.^8,47 As described earlier, CCL2-induced angiogenesis may occur via β₃ integrins.¹⁰⁸ CCL23 has been implicated in the migration of vascular endothelial cells and angiogenesis-associated matrix metalloproteinase (MMP) production.⁴⁸ In contrast, CCL21 (SLC) may exert strong angiostatic and antitumor effects.⁴⁹

CX₃CL1 (fractalkine) is involved in both angiogenesis and atherosclerosis underlying inflammatory rheumatic diseases.^8,37,38

Adhesion Receptors, Ligands, and Proteases in Angiogenesis and Vasculogenesis

Both CAM receptors and their extracellular matrix (ECM) macromolecule ligands mediate adhesive interactions during inflammatory neovascularization.^4,14,114 Among ECM components, type I and other minor collagens, fibronectin, heparin, laminin, tenascin, vitronectin, and fibrinogen promote angiogenesis.^13,114 Vasculogenesis is stimulated by the laminin matrix Matrigel.¹¹⁵ Thrombospondin-1 (TSP-1) is an angiostatic ECM component naturally produced within the RA synovium.^14,116,117

Among endothelial CAMs, soluble E-selectin; soluble P-selectin; the L-selectin ligand CD34; soluble VCAM-1; some endothelial β₁, β₃, and β₅ integrins; PECAM-1 (CD31); endoglin (CD105); and some cadherins have been implicated in angiogenesis.^{13,14,118–121} The α_Vβ₃ integrin and the ITGAV gene play critical roles in inflammatory angiogenesis. The integrin has become a major target for specific therapy.^{108,120–122} Other angiogenic factors such as chemokines may act via integrin-dependent pathways.¹⁰⁸ Recently, the role of the ITGAV allele in angiogenesis has been further analyzed in four Caucasian sample sets. The genetic association could not be confirmed in New Zealand and Oxford (UK) sample sets, suggesting that the link between ITGAV gene polymorphism and RA may be limited.¹²⁰ Focal adhesion kinases (FAKs) are involved in α_Vβ₃ integrin signaling underlying synovial inflammation and angiogenesis.¹²³ Among glycoconjugates with adhesive properties, blood group antigens Lewis-y and Lewis-H promote neovascularization.¹²⁴ JAMs (JAM-A, JAM-B, and JAM-C) have also been implicated in the adhesive processes underlying RA, as well as in synovial angiogenesis.^45,125 Vasculogenesis also involves numerous integrins including α_Vβ₃ and E-selectin.^{1,99,126–128}

MMPs promote angiogenesis by synovial matrix degradation.^{14,87,129,130} The role of hypoxia in MMP production is described earlier.⁸⁷ Some ADAM and ADAMTS proteases have also been implicated in inflammatory neovascularization.^131–133

Targeting Cell Adhesion, Chemokines, and Angiogenesis: Possible Therapeutic Approaches in Inflammatory Arthritides

Leukocyte recruitment inhibition may be a result of nonspecific anti-inflammatory therapeutic strategies. Numerous traditional and biologic disease-modifying drugs (DMARDs) and immunosuppressive agents may, in addition to other effects, suppress leukocyte recruitment, chemokine production, and angiogenesis. Inhibition of cell adhesion and migration, angiogenesis, chemokines, and chemokine receptors using specific antibodies or purified ligands has provided an important perspective on the molecular pathogenesis of RA. In addition, some of these strategies may be included in the future therapy of arthritis.*