Signal Transduction
Fig 16.9 Chemokine-mediated activation of integrins in leukocytes.Binding of the chemokines causes activation of Gi. The subunits activate PLC giving rise to diacylglycerol and IP3, both of which are involved in the activation of CalDAG-GEF, which activates Rap1. This binds its effector RapL responsible for activation of integrins (for instance L 2 (LFA1)).
CalDAG-GEF, also known as RapGRP, is a Ca2 and diacylglycerol activated guanine nucleotide exchange factor. It contains a C1 domain, first identified as a conserved region in members of the PKC family, through which it binds diacylglycerol.
It is worth noting that there are numerous proteins containing C1 domains and so potential targets of the action of diacylglycerol or phorbol esters (see page 234). It also contains an EF-hand motif, which confers Ca2 sensitivity (see page 779).
Depending on the type of leukocyte, different integrins participate
in the firm arrest to endothelium. For instance, in monocytesM 2 is predominantly involved in arrest, whereas in neutrophils this role is taken by L 2. Lymphocytes rely mainly on 4 1 (see Figure 13.6, page 384).
problem that chemokine-induced integrin activation is not prevented when PKC is inhibited.61 The stimulatory effect of phorbol ester can also be explained through its direct effect on the exchange factor CalDAG-GEF causing activation of the GTPase Rap1b62 (Figure 16.9). Rap1,63 although
initially considered as an antagonist of Ras64,65 (see page 251), gained further physiological significance with the realization that it activates integrins in a number of blood-borne cells, including platelets.66–69 Although the pathway has yet to be fully elucidated, activated Rap1 interacts either with RapL or Riam70 and this causes both activation and clustering of integrins, thereby augmenting their affinity and avidity for ligands.71,72 Riam interacts with the cytoskeletal protein talin, and this in turn binds integrins and thus modifies their configuration (see also Figure 13.14, page 398).73,74 Integrin activation causes arrest of the leukocytes on endothelial cells that possess the ligands VCAM and ICAM-1, both of which are highly expressed due to the presence of TNF- (and other inflammatory cytokines). In this way, floating leukocytes take on the characteristics of adherent cells (Figure 16.13).
Transendothelial migration
The leukocytes flatten, then migrate to the endothelial cellular junctions in a complex process involving reorganization of the cytoskeleton. subunits
derived from Gi interact with the p110 subunit of class-I PI 3-kinase inducing the generation of PI(3,4,5)P3 (see page 546). This engages GEFs that activate both Rac and Cdc42 (Rho family of GTPases)75 (Figure 16.11). In particular, Rac1 is a key player in the initiation of a branched network of actin filaments and in orchestrating the formation of lamellipodia (protrusions) which cause
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Traffic of White Blood Cells
Fig 16.10 Domain architecture of components of the integrin activation pathway in leukocytes.
The N-terminal segment of the G 1 subunit binds PLC 2, leading to production of diacylglycerol and IP3. IP3 liberates Ca2 from intracellular stores. Together, Ca2 and diacylglycerol recruit CalDAGGEF to the membrane and cause its activation, leading to GTP-loading
of Rap1. Rap1-GTP binds to the Rasassociation domain of RapL and this plays a role in the activation process of integrins.
Fig 16.11 Members of the Rho family are instrumental in the control of cell migration.
Chemokine receptors are linked to heterotrimeric GTP-binding proteins. With respect to the formation of protrusions (at the leading edge), it is the-subunits that are of importance. These bind PI 3-kinase- resulting in the formation of PI(3)P. This attracts and activates the Rac1-guanine nucleotide exchange factor Tiam. GTP-loaded Rac1 binds Sra1, a component of the WAVE2 complex, and this interacts with ARP2/3. This binds first to existing actin filaments, followed by the nucleation of new filaments. The elongating actin filaments push the membrane forward. With respect to retraction at the rear of the cell, chemokine receptors mediate their effect through G 13 which binds p115RhoGEF. GTP-loaded RhoA interacts with the serine/threonine kinase ROCK1 which, in turn, phosphorylates MLC. This enables ATP hydrolysis at the head of myosin II, resulting in movement and sliding of the actin filaments (contracting stress fibres).
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Signal Transduction
Other roles of chemokines. Dendritic cells also produce chemokines, in particular CCL5 (RANTES), CCL10, CCL17, CCL18, CCL19, and CCL25. This suggests a role for chemokines in the initiation of immune responses through possible adjuvant properties. It may enable dendritic cells
to attract T cell subsets. Some chemokines
are constitutively expressed and may have a homeostatic role rather than an inflammatory. Mice that lack CXCL12 (SDF-1) die before birth and show a defect in B cell lymphopoiesis resulting from a disorganized bone marrow stromal
environment.78 Failure to express CCL21 in lymph nodes causes depletion of T cells, possibly due to a loss of their homing capacity.79
The protrusions have different names: laemellipodia and, in the case of neutrophils, pseudopods.
cell spreading.76 Rac1 is activated by a number of GEFs of which two, Tiam1 and DOCK1, qualify in the context of leukocyte migration. Activated Rac1 binds to Sra1, part of the WAVE2 complex77 that is enabled to bind to ARP2/3. This complex of actin-like proteins binds to existing actin filaments and catalyses the further polymerization of actin, thus initiating new filaments that press the membrane forward.76
Migration within the tissue
Having traversed the endothelial intercellular junction and then the basal membrane, leukocytes search for sites of infection, migrating up the gradient of chemokines (such as formylmethionyl peptides) released by bacteria. This movement is characterized by the assembly of actin fibres into protrusions at the leading edge of the cell, and by formation of contractile actin–myosin complexes at the rear and along the sides. Both these processes are initiated following receptor activation of the heterotrimeric G proteins, Gi controlling forwardness and G12/13 controlling backwardness.84
Directionality is not necessary for leukocyte binding to endothelial cells or for transendothelial cell migration. What matters is that the activated integrins are guided to the intercellular junctions. This occurs through binding of all three types of integrin involved in the arrest of leukocytes to the adhesion molecules (JAMs) that constitute the tight junctions. LFA1 ( L 2) shifts its attention from ICAM-1 towards JAM-A,80 VLA-4 ( 4 1) shifts its attention from VCAM1 towards JAM-
B,81 and Mac-1 ( M 2) shifts its attention from ICAM1 or -2 towards JAM-C.82,83 Integrin binding occurs at the membrane-proximal immunoglobin domain of the JAMs. This is thought to guide the leukocyte across the intercelluar junctions. It may also destabilize the numerous cadherin junctions that form the zonula adherens. Blocking of JAMs prevents leukocyte recruitment in cerebrospinal fluid.
As already related, subunits derived from Gi induce formation of PI(3,4,5)P3. This occurs at the point of the cell closest to the source of the chemokine, in this way positioning the leading edge (Figures 16.12 and 16.13). The GTPase Rac1 focuses membrane protrusions uniquely at the leading edge, while the role of Cdc42 is to ensure that there is no formation of secondary protrusions elsewhere.57 Retraction of the cell body at the rear is mediated through activation of the -subunits of G12 or G13.85 This occurs through the formation of actin stress fibres formed by antiparallel filaments of actin intercalated with myosin II (see Figure 16.11). Activation of the myosin II occurs through phosphorylation of the myosin light chain (MLC) in a pathway involving p115RhoGEF, RhoA and ROCK.
In the tissue, the activated integrins augment neutrophil responses such as phagocytosis, exocytosis, and generation of reactive oxygen metabolites, necessary for the killing and clearing of microorganisms. They also determine the lifespan of the leukocyte in the tissues.86
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