**6. Innate immunity abnormalities in GCA**

Immune responses are initiated by the recognition of foreign molecular structures, such as invading pathogens, by the antigen presenting cells (APCs) of the innate immune system. Tissue macrophages and dendritic cells (DCs) represent the main classes of professional APCs and are characterized by the membrane expression of germ-line receptors (pattern recognition receptors, PRRs). These receptors are able to recognize specific molecular patterns of exoge‐ nous and/or endogenous foreign proteins, known as pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs).

Upon recognition of a certain PAMP or DAMP, dendritic cells become differentiated and activated and produce cytokines, which are able to recruit neutrophils and macrophages, activate the adaptive immune system and trigger the complement cascade. The physiological goal of early innate immune response is to control and demarcate infection and prevent microbe spreading and further tissue damage.

it has been shown that DCs' depletion abrogates vasculitis, thus confirming the critical role of

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The activation of vascular DCs is mediated via the ligation of their TLRs. It has been shown that certain infectious agents are able to legate to specific Toll-like receptors, such as TLR-4 (LPS) or TLR-5 (flagellin). The ligation of a PAMP (or DAMP), such as LPS or flagellin, to the extracellular portion of the TLR provokes the activation of the intracellular TRAM motif and the consequent activation of an intracellular phosphorylation cascade (second message). The final result is the activation of the NfKB, which enters the nucleus and induces certain genes. This mechanism leads to the translation of pro-inflammatory molecules with autocrine or paracrine actions, expression of co-stimulatory receptors on cell surface and production of

Additional research, in regard to the role of TLRs in GCA pathogenesis, led to some very interesting results. It is well known that GCA shows an impressive, yet unexplained, predi‐ lection for specific sites of the vasculature, such as the 2nd to 5th aortic branches. [1] Histopa‐ thologic studies demonstrated that DCs express different type of TLRs in different arteries. [30]

The distribution of TRLs in the vessel wall is highly determined by the embryological origin of the tissue. The aortic arch and its branches derive from the ectoderma, whereas the de‐ scending aorta derives from mesodermal cells. The heterogeneity of the immune response in GCA is believed to be strongly influenced by the specific type of TLRs, whose expression varies in the different blood vessels. Indeed, in an experimental study, Pryshchep et al showed that the distribution of TLRs, in various sites of the vascular tree, determine the extent and profile of the inflammatory reactions. [30] DCs, with differential surface expression of TLRs, display a marked heterogeneity in their immune-regulatory functions, providing a possible clue toward the tissue tropism of GCA. Furthermore, the immunological identity of blood vessels, as defined by the expression of a vessel-specific profile of TLRs, has been considered to determine the nature of the inflammatory reaction in various types of vasculitides. [21]

In this context, it has been shown that TLR-4, abundantly expressed on adventitial DCs, recognize LPS from bacterial pathogens. Upon recognition, IFN-γ is produced in large amounts and leads the subsequent mononuclear infiltration in all layers of the arterial wall. This all-layer inflammation characterizes panarteritis, with granuloma formation, which is

On the contrary, when TLR-5 recognizes flagellin, the elicited inflammatory response is characterized by the sole infiltration of the adventitia (periarteritis). In this case, disruption of the elastic lamina and subsequent luminal occlusion is typically lacking. [31] Clinical obser‐ vations have suggested that periarteritis rarely accompanies systematic inflammatory proc‐ esses, such as aneurysm formation. Subtle alterations in inflammatory reactions guided by DCs with TLR-4 and/or TLR-5 overexpression may explain the differences in the clinical

typically found in biopsies of the temporal artery in GCA. [31]

phenotype of giant cell arteritis.

activated DCs in sustaining wall inflammation. [29]

antimicrobial substances. [26]

Recently, DCs were shown to initiate the immune response in GCA. [23] These cells lay dormant, in a ring-like structure around the adventitia-media border. It is suggested that, in normal arteries, DCs are sentinels that form a part of the first line immune defense of the vessel wall. [24]

The population of the immune cells in the adventitia of the large-vessel wall is mainly consisted of immature myeloid DCs, with a characteristically high threshold of activation. [25] In contrast to mature APCs that induce adaptive immunity, immature DCs do not express co-stimulatory molecules on their surface, such as CD80 and CD86. This condition is primarily responsible for maintaining an anergic state for T cells. In normal arteries, immature APCs are tolerogenic, thus supporting T-cell unresponsiveness. [21] They have been found to be positive for the S-100 protein and express the chemokine receptor CCR6. [26] As guardians of the immunoprivileged arterial wall, DCs are committed to protect the structural integrity of these vital and nonregenerative tissue structures.

However, in susceptible individuals, such as those bearing the HLA-DR4 allele or in older persons (immune-aging), an unknown instigator or a persistent stimulus activates DCs and initiates an innate immune response. In this context, certain antigens (derived from pathogens or locally formatted by tissue calcification) are considered to infiltrate the vessel wall adven‐ titia, through vasa vasorum, and activate immature DCs.

Physiologically, dendritic cells subsequently migrate to the local lymph nodes and clear the antigens, without triggering inflammation. [26] In GCA, however, for yet ill-defined reasons, the activated DCs remain *in situ* and mature in the vessel wall. [27] Existing evidence supports that the maturation of DCs is a very early step in the initiation of the vasculitic process and occurs long before the chronic phase of wall inflammation. In biopsy studies from patients with polymyalgia rheumatica (PMR), mature DCs, already expressing co-stimulatory mole‐ cules, were found in their temporal arteries, despite the absence of any clinically apparent sign of inflammation. [25] These observations are closely correlated with the fact that a great proportion of PMR patients will eventually develop giant cell arteritis.

The principal role of DCs in GCA pathogenesis lies, not only in initiating the inflammatory process, but, also, in perpetuating immune reactions. Dendritic cells, found in vasculitic lesions, are able to produce high amounts of IL-12 and IL-18 and up-regulate the release of IFN-γ from T cells. [22] In addition, dendritic cells, in inflamed arteries, can release the homing chemokines CCL19 and CCL21, which bind to the receptor CCR7. The expression of CCR7 results in the local arrest of activated DCs, which are no longer able to leave the tissue. Instead, they are trapped in the arterial wall and enforce an aberrant T-cell response. [28] Furthermore, it has been shown that DCs' depletion abrogates vasculitis, thus confirming the critical role of activated DCs in sustaining wall inflammation. [29]

activate the adaptive immune system and trigger the complement cascade. The physiological goal of early innate immune response is to control and demarcate infection and prevent

Recently, DCs were shown to initiate the immune response in GCA. [23] These cells lay dormant, in a ring-like structure around the adventitia-media border. It is suggested that, in normal arteries, DCs are sentinels that form a part of the first line immune defense of the vessel

The population of the immune cells in the adventitia of the large-vessel wall is mainly consisted of immature myeloid DCs, with a characteristically high threshold of activation. [25] In contrast to mature APCs that induce adaptive immunity, immature DCs do not express co-stimulatory molecules on their surface, such as CD80 and CD86. This condition is primarily responsible for maintaining an anergic state for T cells. In normal arteries, immature APCs are tolerogenic, thus supporting T-cell unresponsiveness. [21] They have been found to be positive for the S-100 protein and express the chemokine receptor CCR6. [26] As guardians of the immunoprivileged arterial wall, DCs are committed to protect the structural integrity of these vital and non-

However, in susceptible individuals, such as those bearing the HLA-DR4 allele or in older persons (immune-aging), an unknown instigator or a persistent stimulus activates DCs and initiates an innate immune response. In this context, certain antigens (derived from pathogens or locally formatted by tissue calcification) are considered to infiltrate the vessel wall adven‐

Physiologically, dendritic cells subsequently migrate to the local lymph nodes and clear the antigens, without triggering inflammation. [26] In GCA, however, for yet ill-defined reasons, the activated DCs remain *in situ* and mature in the vessel wall. [27] Existing evidence supports that the maturation of DCs is a very early step in the initiation of the vasculitic process and occurs long before the chronic phase of wall inflammation. In biopsy studies from patients with polymyalgia rheumatica (PMR), mature DCs, already expressing co-stimulatory mole‐ cules, were found in their temporal arteries, despite the absence of any clinically apparent sign of inflammation. [25] These observations are closely correlated with the fact that a great

The principal role of DCs in GCA pathogenesis lies, not only in initiating the inflammatory process, but, also, in perpetuating immune reactions. Dendritic cells, found in vasculitic lesions, are able to produce high amounts of IL-12 and IL-18 and up-regulate the release of IFN-γ from T cells. [22] In addition, dendritic cells, in inflamed arteries, can release the homing chemokines CCL19 and CCL21, which bind to the receptor CCR7. The expression of CCR7 results in the local arrest of activated DCs, which are no longer able to leave the tissue. Instead, they are trapped in the arterial wall and enforce an aberrant T-cell response. [28] Furthermore,

microbe spreading and further tissue damage.

96 Updates in the Diagnosis and Treatment of Vasculitis

wall. [24]

regenerative tissue structures.

titia, through vasa vasorum, and activate immature DCs.

proportion of PMR patients will eventually develop giant cell arteritis.

The activation of vascular DCs is mediated via the ligation of their TLRs. It has been shown that certain infectious agents are able to legate to specific Toll-like receptors, such as TLR-4 (LPS) or TLR-5 (flagellin). The ligation of a PAMP (or DAMP), such as LPS or flagellin, to the extracellular portion of the TLR provokes the activation of the intracellular TRAM motif and the consequent activation of an intracellular phosphorylation cascade (second message). The final result is the activation of the NfKB, which enters the nucleus and induces certain genes. This mechanism leads to the translation of pro-inflammatory molecules with autocrine or paracrine actions, expression of co-stimulatory receptors on cell surface and production of antimicrobial substances. [26]

Additional research, in regard to the role of TLRs in GCA pathogenesis, led to some very interesting results. It is well known that GCA shows an impressive, yet unexplained, predi‐ lection for specific sites of the vasculature, such as the 2nd to 5th aortic branches. [1] Histopa‐ thologic studies demonstrated that DCs express different type of TLRs in different arteries. [30]

The distribution of TRLs in the vessel wall is highly determined by the embryological origin of the tissue. The aortic arch and its branches derive from the ectoderma, whereas the de‐ scending aorta derives from mesodermal cells. The heterogeneity of the immune response in GCA is believed to be strongly influenced by the specific type of TLRs, whose expression varies in the different blood vessels. Indeed, in an experimental study, Pryshchep et al showed that the distribution of TLRs, in various sites of the vascular tree, determine the extent and profile of the inflammatory reactions. [30] DCs, with differential surface expression of TLRs, display a marked heterogeneity in their immune-regulatory functions, providing a possible clue toward the tissue tropism of GCA. Furthermore, the immunological identity of blood vessels, as defined by the expression of a vessel-specific profile of TLRs, has been considered to determine the nature of the inflammatory reaction in various types of vasculitides. [21]

In this context, it has been shown that TLR-4, abundantly expressed on adventitial DCs, recognize LPS from bacterial pathogens. Upon recognition, IFN-γ is produced in large amounts and leads the subsequent mononuclear infiltration in all layers of the arterial wall. This all-layer inflammation characterizes panarteritis, with granuloma formation, which is typically found in biopsies of the temporal artery in GCA. [31]

On the contrary, when TLR-5 recognizes flagellin, the elicited inflammatory response is characterized by the sole infiltration of the adventitia (periarteritis). In this case, disruption of the elastic lamina and subsequent luminal occlusion is typically lacking. [31] Clinical obser‐ vations have suggested that periarteritis rarely accompanies systematic inflammatory proc‐ esses, such as aneurysm formation. Subtle alterations in inflammatory reactions guided by DCs with TLR-4 and/or TLR-5 overexpression may explain the differences in the clinical phenotype of giant cell arteritis.

Nevertheless, independently of the mode of the initial stimulation, DCs become activated and subsequently produce cytokines with redundant and pleiotropic actions. In inflamed temporal arteries, DCs secrete pro-inflammatory cytokines, mainly IL-2, IL-6 and IFN-γ, which, in turn, mediate the recruitment of inflammatory cells, inhibition of cell migration, enhancement of T cell proliferation and stimulation of T and B cells. [32] The net result is further amplification of the immune response, through positive feedback loops.

**8. Th1 cells in GCA**

pro-inflammatory environment.

treated patients. [38]

destruction. [21]

**9. Th17 cells in GCA**

inflammatory bowel disease. [41]

Th1 cells represent the dominant cell population in the intramural lesions and the periphery of patients with untreated GCA. [37] These cells produce IFN-γ, as their signature cytokine, which, physiologically, has a critical role against viral and intracellular bacterial infections. Once called macrophage activating factor, IFN-γ target macrophages and provide a substantial

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IFN-γ committed T cells are considered to account for >20% of circulating CD4+ T cells, an almost 100% increase compared to age-matched healthy controls. [38] Corticosteroid therapy cannot affect the expansion of this subpopulation, indicating continuous signaling from the respective DCs. The underlying mechanism of this resistance involves the triggering of APCs that continue to release IL-12. Actually, both in the blood and the temporal arteries of GCA patients, IL-12 production continued unabated during the chronic phase of the disease in

At the tissue level, cytokine profiling in GCA temporal arteries has demonstrated robust expression of IFN-γ and an association with a defined disease phenotype. [32] In particular, high tissue IFN-γ levels are typical for patients with ischaemic complications, implicating its crucial participation in the process leading to luminal occlusion. Pathophysiological studies have correlated increased IFN-γ levels with the production of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF), which are molecules implicated in the intimal response that leads to lumen stenosis. [39, 40] VEGF may, in turn, promote IFN-γ production, thus leading to a vicious cycle of inflammation and structural stenosis. [39]

It is currently unknown which aspects of the granulomatous inflammation depend upon IFNγ. The ability of this cytokine to activate monocytes and macrophages certainly has a role in promoting the differentiation of lesional histiocytes. However, the profound differences in the clinical presentation of treated and untreated GCA patients suggest that IFN-γ is less relevant to the systemic manifestations of the disease and, instead, the major mediator of vessel wall

Th17 cells play an important role in antimicrobial immunity where they regulate the recruit‐ ment of neutrophils and facilitate protection against extracellular bacteria and fungi. Far beyond their role in host defense, Th17 cells have been implicated in the pathogenesis of several autoimmune and inflammatory disorders, such as rheumatoid arthritis, multiple sclerosis and

In untreated GCA patients, the frequency of Th17 cells is 10-fold elevated in the peripheral blood and they accumulate in the vascular infiltrates. [37] In healthy individuals, Th17 cells are infrequent and account for less than 0.3% of the circulating CD4+ T cells. In newly diag‐
