Paper : 10 Module :19 Leukocyte Activation and Migration

Development Team

Principal Investigator: Prof. Neeta Sehgal Department of Zoology, University of Delhi

Co-Principal Investigator: Prof. D.K. Singh Department of Zoology, University of Delhi

Paper Coordinator: Prof. Shibnath Majumder Paper Coordinator : Department of Zoology, University of Delhi Department of Zoology, University of Delhi Content Writer: Dr. Anita Kamra Kirori Mal College, University of Delhi

Content Reviewer: Prof. Sukhmahendra Singh Content Reviewer : Banaras Hindu University

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ZOOLOGY Immunology Leukocyte Activation and Migration

Description of Module

Subject Name ZOOLOGY

Paper Name Zool 010: Immunology

Module Name/Title Regulation of Immune Responses

Module ID M19 Leukocyte Activation and Migration

Keywords Leukocyte, , , ,

Contents 1. Learning Outcomes 2. Introduction 2.1. Why do leukocytes migrate to the tissue? 2.2. Importance of molecules 2.3. Structurally CAM can be classified into four families 2.3.1. Few Selected CAMs belonging to each family 2.4 facilitate both activation and migration 3 “Multi-step model” of Leukocyte/ Recruitment 3.1 The neutrophils move slowly 3.2 Rolling 3.3 Activation 3.4 Firm Adhesion 3.5 Interaction of leukocyte with the vascular 3.6 Leukocyte migration requires polarization 3.7 Transendothelial migration of leukocytes 3.8 Recruitment to non-inflamed tissues 4. T Lymphocyte Recirculation via a multi-step pathway 4.1 Transmigration 4.2 Extravasation of Naïve T 4.3 Selective Homing or Trafficking 2

ZOOLOGY Immunology Leukocyte Activation and Migration

4.4 Lymphocyte rolling 4.5 Signaling 4.6 Firm adhesion 4.8 Transmigration. 4.8 Tissue-specificity of T lymphocyte transmigration 4.9 The control of T lymphocyte motility 5. Monocyte Trafficking 5.1 Transient activation of the endothelium 5.2 PSGL-1 expression 5.3 Inflammatory response 5.4 Intraluminal crawling 5.5 There are two phases of monocyte transmigration 5.6 Three types of migration of 5.7 Extravasation 6. Conclusions

1. Learning Outcomes

By the end of this lecture, students will be able to appreciate that migration of leukocytes is a critical component of both innate and adaptive immunity as the immune responses primarily depend on the capability of leukocytes to traffic from the circulation into the tissues. The foremost objective is to discuss the mechanisms, profile and consequences of i)neutrophil recruitment, ii) lymphocyte re-circulation, iii) monocyte trafficking, all of which require adhesion and transmigration through blood-vessel wall that has beenstimulated. The pathways that help facilitate the movement through the early phases of leukocyte or endothelial cell when it comes in-contact with the lumen of vessels will be discussed especially about migration across the peri-vascular basement membrane, a hurdle that is rarely explored in the milieu of leukocyte emigration in vivo.

2. Introduction

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ZOOLOGY Immunology Leukocyte Activation and Migration

The phagocytic activity of leukocytes together with its ability to differentiate into - presenting cells (APCs) and monocytes contribute in innate and adaptive immune responses. Leukocytes are derived from the progenitor cells in the bone marrow, and are in blood circulation as monocytes but segregate as dendritic cells of the myeloid origin in the periphery and in the tissues as . Therefore, a critical component of both innate and adaptive immunity is the migration of leukocytes to inflamed/injured sites because the immune responses depend primarily on the ability of the leukocytes to pass from the blood circulation to the tissue. The threat signals triggered by /injury stimulate the resident cells of the i.e the mast cells, macrophages and dendritic cells that cause secretion of and release of other pro-inflammatory mediators to stimulate neighboring endothelial cells in the blood vessels, hence forth, a cascade of events is elicited that enables the circulating leukocytes to distinguish the vascular endothelium in the inflamed tissue and to interact with the blood vessel wall. To achieve this, precisely co- ordinated pathways are already present that recruit intravascular leukocytes and assist them to infiltrate the vascular wall allowing them to migrate to the injury sites or to the infection site without causing any observable damage to the vessels while emigrating. The foremost aim is to discuss the profile, mechanisms and the consequences of i) recruitment of neutrophil, ii) lymphocyte re-circulation, iii) monocyte trafficking, all of which necessitate adhesion and transmigration through stimulated blood-vessel wall. The mechanisms that facilitate the initial stages of leukocyte/endothelial cell interactions within the blood-vessel lumen will be discussed in particular, migration through the perivascular basement membrane, a barrier that is rarely investigated in the context of leukocyte emigration in vivo.

2.1 Why do leukocytes migrate to the tissue?

Leukocytes originate from the bone marrow progenitor cells, found in circulation in the blood as monocytes and differentiate as myeloid dendritic cells in the periphery and into the tissues as macrophages. Cells of the immune system constantly traffic throughout the body to justify their function of immune scrutiny leading to pathogen elimination, (eg blood leukocytes,

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ZOOLOGY Immunology Leukocyte Activation and Migration

which comprise lymphocytes, monocytes, dendritic cells and neutrophils), cross the blood barrier and travel to the infection site. • The phagocytic activity of leukocytes, coupled with their ability to differentiate into APCs contributes to the innate and adaptive immune system. • The threat signals caused by injury/inflammation trigger the innate arm of the immune system - Macrophages, mast cells and dendritic cells, secrete pro-inflammatory cytokines to stimulate the endothelial cells. • An important component of both innate and adaptive immune system is the migration of leukocytes from circulation to the tissues. 2.2 Importance of cell adhesion molecules - CAMs are required to ensure that circulating leukocytes migrate to the inflamed tissue or to the peripheral lymphoid organs, and it is important that the cells adhere to and cross-over the endothelial cells liningof the blood vessels - extravasation. - Endothelial cells express leukocyte-specific cell-adhesion molecules (CAM) - CAMs present on the leukocytes help them to attach to the endothelial cells of the blood vessels. To enhance the strength of the interactions among the cells of the immune system, e.g., T - APC, T - B, CTL - target cells. H H 2.3 Structurally CAM can be classified into four families:  Mucin like CAM which express sialylated aldehyde side chains  An integrin family which is a heterodimer consisting of alpha and beta polyeptide  , wherein, L- is expressed on the leukocyte and P and E selectins are expressed on the inflamed endothelium that expresses CD62 that ends in a lectin domain.

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ZOOLOGY Immunology Leukocyte Activation and Migration

Sialylated CHO moiety

L-selectin (CD62L) P-selectin (CD62P) E-selectin (CD62E)

Fig1: Structural classification of the Cell-Adhesion Molecules (CAM)

2.3.1. Few Selected CAMs belonging to each family

Mucin-like CAMs: Selectins:

GlyCAM-1 (on endothelium) L-Selectin (on Leukocyte) CD34 (on endothelium) P-Selectin (On inflamed endothelium) PSGL-1 (on neutrophils) E-Selectin MAdCAM-1 Ig-superfamily CAMs: (αβ heterodimers) ICAM-1, -2,-3 (CD54, CD102, CD50) : VCAM-1 α4β1 (VLA-4, LPAM-2) LFA-2 (CD2) Leukocyte Function Associated α4β7 (LPAM-1) LFA-3 (CD53) (Lymphocytes Peyer’s α6β1 (VLA-6) MAdCAM-1 Patches adhesion molecule-1) (on mucosal epithelium, αLβ2 (LFA-1) has both mucin-like αMβ2 (Mac-1) and Ig- like domains) αXβ2 (CR4, p150/95)

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2.4 Chemokines facilitate both activation and migration

1. Are small polypeptides, mostly made up of 90 -130 AA residues 2. Modulate the , adhesion, and activation of leukocytes – regulators of leukocyte trafficking. 3. Few are principally involved in inflammatory response, some are constitutively expressed and have an important homeostatic or developmental roles. 4. -mediated effects may not restricted to the immune system. 5. The pro-inflammatory chemokines are induced in response to infection and are capable of recruiting and lymphocytes to the inflammatory sites. 6. Four classes: CXC, CC, C, CXXXC (or CX C) 3 7. Ligands: e.g., CXCL8, Receptors: e.g., CXCR1

“Multi-step model” of Leukocyte/Neutrophil Recruitment “This “multi-step” model of leukocyte transmigration (Fig.3) includes tethering to, slow rolling and regulated rolling of leukocytes across the vascular endothelium. Graphic representation of the sequential steps involved in leukocyte migration through the stimulated vessel walls, indicating the 1. Capture for transitory adhesion 2. Rolling 3. Slow rolling 4. Firm adhesion 5. Diapedesis / Transmigration across the endothelium

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ZOOLOGY Immunology Leukocyte Activation and Migration

Fig 3: “Multi-step mode” of Leukocyte Recruitment The adhesion cascade includes capture, slow rolling, arrest, adhesion strengthening, intraluminal crawling that involves spreading, para-cellular and trans-cellular diapdesis, and migration through the basement membrane. Under normal physiological conditions, the lining of the endothelial cells of the blood vessels exhibit an anti-adhesive and anti-thrombogenic surface that ensures blood flow along with the blood components to maintain homeostasis. 3.1 The neutrophils move slowly rather rolls over the endothelium unlike the other blood components that move along blood flow with a high velocity. But at the injury site or the place of infection, or inflammation, presence of endotoxins, expression of certain cytokines and other secreted molecules renders adhesive properties to these circulating leukocytes thereby initiating a cascade of leukocyte emigration.

Fig 4: Slow movement of the Neutrophils in the blood vessel

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ZOOLOGY Immunology Leukocyte Activation and Migration

3.2 Rolling: These responses are generally mediated by weak and revocable molecular interactions between the selectins (P-selectin, E-selectin, and L-selectin) present on the endothelium and their counter ligands (PSGL-1) present on the leukocyte (Fig 3). The interaction between selectins and PSGL-1 facilitates rolling.

Fig 5: Rolling mechanisms facilitated by selectins and their counter ligands 3.3Integrin Activation The active up-regulation of P-selectin in inflamed endothelial cells reduces the velocity of rolling leukocytes. In addition, certain integrins such as VLA-4, can also mediate leukocyte rolling under conditions of flow. The slowing down of leukocytes during rolling facilitates the ligation of specific and high affinity G-protein coupled receptors by endothelial cells of lumen associated activating factors such as chemokines. This interaction augments the ligand binding profile of integrins that is achieved by increased affinity and avidity of the molecules. Integrin activation plays a critical role in facilitating the adhesion of leukocytes firmly to the endothelial cells leading to flattening of the leukocytes over the endothelium thereby ensuring the two crucial steps in leukocyte emigration. β2 and β1 family of integrins are the major integrins involved in this step of leukocyte or endothelial cell interaction.

Fig 6: Integrin activation facilitating firm adhesion of leukocytes to endothelial surface

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ZOOLOGY Immunology Leukocyte Activation and Migration

3.4 Firm Adhesion. At the sites of inflammation, interleukin-8 [IL-8] molecules are secreted by the endothelial cells and remain in the periphery of these cells bound to heparin sulfate proteoglycan. This leads to conformational changes that result in the formation of high- affinity interactions between the ICAM molecules on the inflamed endothelium cells of the vessels. These strong interactions assist the leukocytes to firmly adhere to the surface of the endothelial cell.

Fig 7: Firm adhesion 3.5Interaction of leukocyte with the vascular endothelium is a highly, coordinated process, that includes the arrest of free-flowing leukocytes in the blood with subsequent leukocyte rolling arrest, firm adhesion and ensuing diapedesis. These interactions happen only under high shear stresses within the venules and depend on multiple families of adhesion molecules. As a response to infection mediators, leukocyte gathering is considered to be crucial for an adequate defense of the host organism to any kind of injury or infection. Activation of endothelium contributes considerably to the systemic inflammatory response to septicemia and enhanced expression. Within this setup, leukocyte transmigration through vessel walls of post-capillary venules is the concluding stage of a cascade of responses of leukocyte mediated by a sequence of molecular interactions that primarily mediate the slowing down of velocity leukocyte rolling followed by firm adhesion of leukocytes to the endothelium and finally transmigration through the vessel wall Leukocyte transmigration acts as a means of guiding the emigration of leukocytes from the lumen of vessels to the extravascular tissue thereby playing a crucial role in monitoring the phenotype of the emigrated cells in such a way that responsiveness of behaviour of leukocyte to chemo-attractants, directs the migration and interactions with

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ZOOLOGY Immunology Leukocyte Activation and Migration

molecules of the or extravascular tissue may be further regulated. Therefore, such a mechanism clearly contributes to the capability of the immune system to elicit an appropriate, optimal and localized tissue response to a variety of extravascular stimuli without triggering any non-specific damage to the vasculature. 3.6 Leukocyte migration requires polarization. Leukocytes that adhere tightly to the vascular endothelium need to breach this vascular barrier to reach the underlying inflamed tissue. This can be achieved by active, integrin mediated cell migration. Migration requires polarization of the cell body and the formation of a lamellipodium. It also requires the formation of a UROPOD at the tail of the leukocyte. The formation of these structures is driven by integrins and the . Nonetheless, in situations of uncontrolled infiltration of leukocyte that may exceed either in duration and/or magnitude, or incongruous in its location, transmigration-induced alteration in the phenotype of leukocytes may lead to stimulation or exacerbation of deleterious inflammatory responses in the host. 3.7 Transendothelial migration of leukocytes Formally, the basic steps for intravasation should include interstitial migration towards the vessel, migration across the basement membrane and endothelial barriers (diapedesis), and, ultimately, release of lumenal leukocytes into the circulation. It is now clear appreciated that diapedesis, if it occurs during intravasation or extravasation, can ensue by two distinct routes or pathways: either via disassembly of the intercellular junction to form a paracellular gap (paracellular diapedesis) or via the formation of a transcellular pore directly through an individual endothelial cell (transcellular diapedesis). The concept of one cell passing through or, in effect, entering another cell (as occurs in transcellular diapedesis) might seem a bizarre and unlikely one. The complete migration of the leukocytes through the vessel wall into the surrounding connective tissue is called the Transendothelial migration

Fig 8: Transendothelial migration through endothelial barrier - Diapedesis

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ZOOLOGY Immunology Leukocyte Activation and Migration

When integrins are activated and the cell-migration machinery is functionally polarized, the leukocyte traverses along the luminal side of the vascular endothelial cells and then transmigrates, primarily by transiting between neighbouring endothelial cells.

PECAM-1

Fig 9: Activation of PECAM (CD-31) facilitates the movement through the endothelial junctions that is subject to constant remodeling to enable leukocyte trafficking

Nevertheless, leukocytes have the ability to pass through the body of the endothelial cells too. Although, there is a prevailing transcytotic pathway for extravasation, it may not be the crucial route often taken by leukocytes. A leukocyte taking the conventional paracellular route faces the vascular cells connected by a large array of endothelial junctions, which need to be penetrated. These junctions preserve the integrity of the endothelium and monitor vascular permeability. Three types of endothelial junction have been described: tight junctions, adherens junctions and gap junctions. The endothelial junctions resemble those found in epithelium, but their three-dimensional organization is often dis-ordered, indicating the perpetual remodelling that commences from leukocyte trafficking. Undeniably, transmigration is a vibrant process in which the tight junctional, gap junctional and adherens proteins facilitate the final step of leukocyte extravasation, functioning as liquid engulfing transmigrating cells, rather than as a gate.

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ZOOLOGY Immunology Leukocyte Activation and Migration

Fig 10: Regular trafficking molecules involved in Multistep adhesion cascades. Four adhesion pathways that leukocytes definitely undertake to accumulate in the blood vessel. The lower panel indicates the predominantly expressed trafficking molecules

3.8 Recruitment to non-inflamed tissues. When there is a lack of an inflammatory trigger, the adhesion molecules along with chemokines that are involved in collection of macrophages and DCs in peripheral tissues should be constitutively present. The DCs and macrophages originate from the bone marrow and hence are required to be recruited from the circulating precursors in the blood. Amongst the constitutively expressed chemotactic molecules that act on circulating monocytes, are CXC-chemokine CXCL14 (commonly identified as breast and kidney-expressed chemokine, BRAK) has been projected to be involved in the constitutive trafficking of monocytes. However, the role of CXCL14 or other chemo-attractants, such as DEFENSINS, has not been explored with regards to the recruitment of monocytes to non-inflammatory tissues.

3. T lymphocyte Recirculation occurs via a multi-step pathway

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ZOOLOGY Immunology Leukocyte Activation and Migration

The maximum exploratory behaviour among cells is displayed by T lymphocytes. They are capable of migrating throughout the body and are able to explore most of the tissues. Beside large-scale displacements, T lymphocytes continuously probe their micro- environment in a mode preferring recognition of specific at the surface of Antigen presenting cells. Mobility is a characteristic property of T lymphocytes that promises timely and optimal recognition of extraneous antigens at the site of the antigenic challenge. After stimulation by chemokines, adhesion to the specialized endothelium will occur only when up-regulated integrins interact with ICAM molecules expressed on the endothelial cell. In the lymphoid tissues, the CD 34 displayed transiently at the surface of the endothelial cells forms a momentary interaction with L-selectins. 4.1 Transmigration: When T lymphocytes travel across venular blood vessel walls to enter various tissues and organs, it is called transmigration or diapedesis. The ability to migrate is crucial for the T lymphocyte at all the life stages, from entry of T-cell precursors into the thymus for T-cell expansion to entry of naïve T cells into lymph nodes for activation, migration of effector T cells into tissues to combat infections. Similarly, transmigration remains a multi-step cascade that comprises lymphocyte rolling, signaling, firm adhesion and transmigration (Figure 6). Each step is constantly regulated by interactions among adhesion molecules and chemokine receptors that are expressed on the surface of lymphocytes and their ligands that are correspondingly expressed on the endothelial cells lining the vascular wall.

Figure 11: Transmigration in T-Lymphocytes 14

ZOOLOGY Immunology Leukocyte Activation and Migration

Fig 12: Extravasation of Naïve T lymphocytes Lymphocyte circulation is quite similar to the leukocyte extravasation. But, ‘Homing receptors’ direct the migration of lymphocyte subsets to particular lymphoid tissues or inflamed endothelium.

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Fig 13: Selective Homing or Trafficking

4.4 Lymphocyte rolling: The contact between lymphocyte and the endothelium is initiated by Lymphocyte rolling that is mediated by repeated binding-release events between selectins and their ligands. During the process of rolling, chemokines are displayed on the surface of the endothelial cell that can interact with chemokine receptors on the lymphocyte surface. 4.5 Signaling. Simultaneously, interface between a chemokine and its receptor results in a cascade of intracellular signaling, that may further activate the adhesion molecules of the integrin family to translate the signals. 4.6 Firm adhesion. Once the integrins are activated the lymphocytes display high affinity binding sites that interact with the cell adhesion molecules (CAMs) on the vascular endothelial wall, resulting in slowing of lymphocyte, its arrest and strong adhesion.

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4.7 Transmigration. The method of transmigration is not very well characterized as compared to the other steps, especially in terms of T lymphocytes.

4.8 Tissue-specificity of T lymphocyte transmigration: Transmigration does not occur indiscriminately, but is highly regulated to enable the T lymphocytes to precisely enter the specific tissues. Since, the blood venules present in different tissues exhibit discrete arrangements of adhesion molecules and their corresponding chemokine ligands, only T cells that express an accurate combination of chemokine receptors and adhesion molecules will be allowed to enter into the specific tissue. For example, the chemokine CCL25 and the CAM MAdCAM-1 are involved in recruitment of T cells that express the chemokine receptor CCR9 and the integrin a4b7 to the small intestinal mucosa. T cell migration is generally mediated by chemokine- integrin activation, which directs the chemokines to provide critical guiding signals to direct T cell migration to specific tissues within the body. Evolving substantiation supports that activated T cells migrate to certain vascularized tissues, without chemokine receptors or with the inhibition of chemokine receptors. The chemokine- independent mechanism generally involves LFA-1 through which T cells further activate integrins, induce adhesion and subsequent cell migration.

4.9 The control of T lymphocyte motility is fundamental to the multistep method of extravasation from the blood stream into tissues. The mechanisms of tethering, rolling, firm adhesion, and transendothelial migration has been discussed. Remarkably, on interaction with endothelial cells, T lymphocytes crawl with an amoeboid motility. The shear stress exerted by blood flow contributes to the creation of LFA-1-dependent adhesions. The migration of T lymphocytes is oriented against blood flow as they interact with the innermost surface of blood vessels. The mechano-taxis of upstream flow relies on passive self-steering mechanism. Lymphocytes exhibit dynamic protrusions during transendothelial migration. The exploratory structures rich in actin and are viewed as or filopodia, depending on the state of activation of lymphocyte. Apparently, they allow the lymphocytes to scan the endothelial cells and to ascertain areas that may be favourable for transcellular diapedesis. The micro-environment of the endothelium also determines the various requirements of the T lymphocytes. This is clearly shown by the role of uropod contractility in transendothelial migration to access the lymph node but does not reach the bone marrow. 17

ZOOLOGY Immunology Leukocyte Activation and Migration

4. Monocyte Trafficking

Monocytes play a fundamental role in tissue homeostasis, defensive immunity, and both enhancing and reducing inflammation. Several of the functions of monocytes are carried outside the vascular compartment; hence, migration and trafficking are necessary.

Fig: Monocyte recruitment

Recruitment of blood monocytes to the site of injury or infection and their diapedesis through the endothelium (also called extravasation) are critical events at the onset of inflammation, followed by differentiation and successive downstream cascade of the inflammatory response. A tightly regulated, multistep process, consisting of a series of interactions between endothelial cells and the cells of the immune system, precedes the actual transmigration step. For monocytes, this progression is basically believed to track the ‘cascade’ paradigm initially described for neutrophils.

5.1 Transient activation of the endothelium is initiated by inflammatory cytokines such as tumour necrosis factor-a or IL-1b, that originate from the tissue macrophages. After chemokine stimulation, the adhesion of monocytes to the endothelium occurs only when activated integrins interact with ICAMs. They elicit a quick expression of adhesion molecules such as E- and P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell 18

ZOOLOGY Immunology Leukocyte Activation and Migration

adhesion molecule-1 (VCAM-1), and the presentation of bound chemokines on the luminal surface of the endothelium. The selectins interact with the O-glycosylated carbohydrate ligands that are displayed on P-selectin glycoprotein ligand-1 (PSGL-1) and are constitutively expressed on all monocytes. This interface allows the monocytes to roll slowly on the endothelium and balance the high shear stress applied by the flow of blood in the vessels.

5.2 PSGL-1 expression Is significantly higher in inflammatory monocytes -Ly6Chi than by resident patrolling monocytes-Ly6Clo that facilitates their adhesion to atherosclerotic lesions.Ly6Chi monocytes extravasate into blood in a CCR2–CCL2 dependent manner. Ly6Clo monocytes emigrate into blood via CX3CR1–CX3CL1 signaling. Ly6Chi in blood can perform different functions – they can phagocytose microbial pathogens and migrate to inflamed tissue sites. Atherosclerosis is characterized by an up-regulation of endothelial adhesion molecules like E- and P-selectin and VCAM-1 in lesion-prone areas. Deficiency in P- and E-selectins, as well as in ICAM considerably reduces the size of artherosclerotic lesions in apoE2/2 mice individually.

5.3 In conditions of inflammatory response, monocyte rolling largely depends on monocyte-expressed very late antigen-4 (VLA-4 a4b1 integrin) and CD44. The endothelial adhesion molecule VCAM-1 binds to VLA-4 and facilitates slow rolling on - activated endothelium, thereby ensuring the switch between rolling and firm arrest. The firm adhesion of monocyte to the endothelium is synchronized by C-C and C-X-C chemokines eg CCL2 and IL-8. Monocytes may also use VLA-4 for firm adhesion. Monocyte arrest is followed by a directional chemotactic and mechanotactic step, in which the monocyte spreads, polarizes, and consequently locomote alongside the endothelium to find desired sites for extravasation.

5.4 This intraluminal crawling depends on the leucocyte integrins, lymphocyte function- associated antigen-1 (LFA-1) and -1-antigen (Mac-1) as well as the endothelial ligands ICAM-1 and ICAM-2. By blocking these adhesion molecules, the crawling and the subsequent transmigration were restricted. This step involves probing the apical surface of the endothelium with the monocyte membrane protrusions called lamellipodia. It is important to understand that this crawling is markedly distinct from patrolling, which is definitely

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intravascular and does not lead to extravasation in steady state. Finally, the monocytes have to negotiate with the endothelium to exit the blood vessel, the lamina basalis and the embedded pericytes (Figure). Transmigration seems to be the critical event in the monocyte adhesion cascade, as is the only step that is never reversed. After extravastion, the monocytes differentiate into macrophages.

5.5 There are two phases of monocyte transmigration across the vascular endothelium. The thin monolayer of endothelial cells (cell thickness 0.1 in the periphery to 1 mm over the nucleus) constitutes the primary physical barrier between blood and tissue. Endothelial cells have no true tight junctions. Adherens junctions between the cells and integrin and cadherin anchors to the basement membrane (BM) form a network that tightly regulates vascular homeostasis and restrains leucocyte transendothelial migration.

5.6 Three types of migration of monocytes have been observed.

Fig : Types of Migration in monocytes

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- Monocytes prefer transcellular migration in only 10-30% of events. - Strong activating stimuli increase the overall occurrence of transcellular transendothelial migration in monocytes. - Live cell visualization in vitro and live imaging through confocal intra-vital microscopy have greatly advanced our understanding in this field. 5.7 Extravasation The predominant paracellular route that leads monocytes through the junctions between endothelial cells and requires junctional remodelling, a different route directly through fusing vesicles in the endothelial cell cytoplasm constitutes an established mode of monocyte transmigration.

5. Conclusion

Over the past twenty years, it has become increasingly evident that the innate immune system

has a central role in polarizing the adaptive immune system towards T helper 1 (TH1)-cell,

TH2-cell or humoral responses. APCs determine the specific recruitment and positioning of T and B cells in the body. We are improving our knowledge of the recruitment of monocytes from the blood to inflammatory tissues and the capacity of monocytes to differentiate into DCs. For example, new molecules (such as RAP1, its ligand PYK2 and atypical PKC) have been added to the chemokine induced signal-transduction cascade that leads to integrin activation and leukocyte migration. Another important discovery has been the involvement of protein complexes in the establishment of cell polarity, which is essential for the directed migration of leukocytes. Furthermore, new adhesion molecules (such as the JAMs and CD99) seem to be involved in the processes that occur at vascular junctions and function as counter-receptors in leukocyte adhesion. Recently described monocyte subpopulations also contribute differentially to mount an efficient specific immune response. However, despite considerable progress, several questions remain to be answered. Does the constitutive migration of monocytes contribute to the establishment of peripheral DCs? Is a ‘danger’ signal required for this recruitment? Is the level of integrin activation crucial for the differentiation and migration of

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monocytes? What are the micro-environmental factors that control the differentiation of monocytes into DCs or into macrophages? Answering these questions will help us to understand the plasticity of the innate immune system and its impact on the maintenance of tolerance.

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