Organization Of Inflammation13

The inflammation in the graft is analogous in some respects to the delayed type hypersensitivity reaction (DTH), exemplified by the classic skin reaction to tuberculin. DTH is an in vivo phenomenon with no single in vitro correlate. It is manifest histologically as a heterogeneous nonspecific inflammation with edema, fibrin accumulation, T-cell infiltration (both specific and nonspecific), B cells, numerous macrophages, and lesser numbers of other leukocytes, and endothelial changes. The key events in DTH are cytokine production (especially TNF-a and p, IFN-y and IL-1), altered expression and function of adhesion molecules, and nonspecific activation of many bone marrow-derived cells, particularly macrophages. Although usually ascribed to CD4+ T cells, DTH reactions mediated by CD8 T cells have been described. The result is graft inflammation.

The Adhesion Molecules

These sets of molecules, which are involved in all levels of the immune response and inflammation, are classified into three groups: the Ig superfamily; the integrins; and the selectins.

Adhesion Molecules of the Ig Superfamily

The principal members are ICAM-1, ICAM-2, VCAM, CD2, CD58, CD28, CTLA4, B7-1, B7-2. These tend to be involved in signalling as well as adhesion. Their expression is increased by pro-inflammatory TH1 cytokines. Ig superfamily members generally interact with other Ig superfamily members or with integrins.

ICAM-1 is a chain of five Ig domains with a membrane anchor and an intracytoplasmic region. Its N terminal domain binds the integrin LFA1. The N terminal V domain of ICAM-1 uses the CDR2-like loop to interact with LFA-1.168 The interactions of CD2-LFA3; CD28-B7, as well as Ig domain interactions with integrins, may follow these principles. Detailed modelling of the interactions involving the Ig superfamily will permit the design of better monoclonal antibodies or other antagonists.

Integrins

Integrins are heterodimers of an a chain and a p chain. The integrins are classified on the basis of the p chain they employ as p1, p2, or p3 integrins. Each p chain can potentially be combined with many different a chains. p1 integrins are important markers of memory and recirculation in T cells (the VLA group). p2 integrins are important in leukocyte adherence reactions (LFA-1, Mac-1).169 Both p1 and p2 integrins are activation-dependent with low avidity in the unactivated state, but high avidity following T-cell activation. Integrins are also associated with diapedesis and intracellular signalling.

Selectins

The selectins are large molecules with three characteristics: lectins (sugar residues with the ability to bind to sugars on other molecules), epidermal growth factor-like motifs, and short consensus repeats (2-9). Each also possesses intracy-toplasmic domains. The name selectin helps us to remember these features: S (short consensus repeats), e (epidermal growth factor-motif), and lectins.

There are three members, named for the cells that express them. E (endothelial)-selectin, is induced by IL-1 and TNF. Its ligand is L(leukocyte)-selectin, which is important for both endothelial binding during inflammation and as a recirculation receptor. L-selectin also binds to P(platelet)-selectin, which is stored in granules of platelets and endothelial cells and is released in response to clotting cascade products.

Selectin interactions are weak under flow conditions and serve as first step adhesion receptors. By slowing leukocyte passage, they expose the leukocytes to the local environment and other endothelial surface molecules. Selectins are involved in all types of tissue injury and may be important mediators of reperfusion injury in transplanted organs. Antibody against P-selectin has been used to ameliorate reperfusion injury of lungs, presumably by inhibiting the interaction of neutrophils with injured endothelium.170

The Roles of Adhesion Reactions

The leukocyte interacts with endothelium through interactions between the selectins. The result is loose binding permitting the leukocyte to roll along the endothelium. This permits the integrins and Ig superfamily members to interact, which causes tight binding and flattening. This will occur only in areas where the endothelium has been activated by injury, infection or immune activity to increase the expression and activity of the adhesion molecules.

The Accumulation of the Interstitial Infiltrate in a Transplant

The first entry of T lymphocytes into the allograft probably occurs by a combination of nonspecific and specific interactions with endothelial cells. Antigen nonspecific cells interact poorly with nonactivated endothelium but may be attracted by endothelium activated by nonimmune injury from the transplant donor, the surgery, or the preservation. Antigen specific T cells probably interact with donor APC in the organ or in the host for their primary stimulation and begin the process of activating the endothelium. Sensitized lymphocytes, primed by antigen plus APC in lymphoid organs, or from a previous encounter, can then interact with the allogeneic endothelium both to infiltrate the tissue and eventually to damage the endothelium.

The T-cell response may be initiated in the central lymphoid organs such as spleen and lymphoid tissues with homing to the graft through a combination of antigen specific and adhesion molecule interactions. Inflammation is characterized by changes in vascular flow and permeability and the influx of leukocytes to the area of injury. Classic signs of inflammation—redness, edema, heat, and loss of function—are present in an acutely rejecting graft. The immune response in the interstitial areas of the graft alters the endothelium of the graft to recruit inflammatory cells. Once there, some leukocytes undergo proliferation within the graft, particularly the clones of lymphocytes which encounter their cognate antigens. Others, such as macrophages, undergo activation and immobilization in response to the products of activated T cells.

Endothelial Cells (EC)

Far from being inert lining cells, EC can act as antigen presenting cells171 and can respond to many stimuli. EC respond to cytokines in a variety of ways, ranging from selective induction of increased MHC class II and class I expression to a generalized increase in the function and expression of many adhesion molecules to generalized activation and even proliferation.172 They interact with leukocytes through their adhesion molecules, including E-selectin, P-selectin, ICAM-1, ICAM-2 and VCAM-1. IFN-y, IL-1 and TNF all induce expression of adhesion molecules. Other regulated responses include changes in hemostasis, vascular tone and permeability. Hemostasis is altered in the direction of promoting thrombosis and fibrin formation through synthesis of thromboplastin and suppression of thrombomodulin/protein C. Platelet activating factor (PAF) has been demonstrated in the EC plasma membranes and may act locally on adhering leukocytes.

Vascular tone is regulated by EC through local release of endothelin, a potent vasoconstrictor,173 and nitric oxide (NO), an endothelium-derived relaxing factor.174 Nitric oxide synthase (NOS) exists in two principal forms: a calcium-activated constitutive form in endothelium and in many other cell types and a cytokine inducible form in macrophages.175,176 Other influences include eicosanoids such as the vasodilator prostacyclin (PGI2) or the vasoconstrictor thromboxane. Cytokines affect vasomotion: IL-1 can induce the synthesis of endothelin by EC, and TNF induces both endothelin and NO production in bovine aortic EC.177 It is likely that the balance between these two forces contributes to the complex vasomotor changes such as arterial vasoconstriction and capillary leak in acute rejection. IL-1 and TNF alter vascular permeability in vivo, probably through intermediate actions on neutrophils, and could thus play a role in the edema characteristic of acute rejection.178

In addition to NO, PGI2, thromboxanes, and endothelin, EC respond to the inflammatory cytokines IL-1 and TNF by producing other soluble factors.172 These include IL-1a, IL-6, PAF, various chemokines (see below), M-, G- and GM-CSF. IL-1a activity appears predominantly associated with the EC plasma membrane and may provide co-stimulation to bound T cells during antigen activation. IL-6, particularly in the presence of IL-4, is abundantly secreted by EC. PAF, like IL-1a, may be predominantly membrane bound and its effects may be very localized.

The Chemokines

Chemokines are recently described as a family of cytokines, 8-10 kD in size, with activity in inflammation and tissue repair, such as attracting inflammatory cells. Members include IL-8, Groa, monocyte chemotactic protein (MCP), and RANTES. The cDNA for these cytokines have been recognized by their characteristic gene structure, typical signal sequences in the 5' region, AT rich sequences in their 3' untranslated regions, and rapidly inducible mRNA expression.179,180 All the chemokines have cysteine residues which form disulphide bridges. These cytokines appear to play a key role in inflammation and immune responses by their chemo-tactic activities and their ability to attract and activate neutrophils, monocytes, T cells, eosinophils and basophils 181 (Table 1.5). Antigen specific T cells activated by APC express new chemokine receptors, which are 7-pass receptors which activate G proteins. These antigen-activated T cells, now capable of directional migration into an inflammatory site in response to chemokines released in the inflamed site, reenter the circulation. T-cell infiltration into the challenged area probably involves a process of sequential endothelial adhesion and then release of T cells, followed by adherence to extracellular matrix via integrin molecules. One current hypothesis is that MIP-1a, MIP-1p, or RANTES participate in attracting the appropriate T cell subsets to an inflammatory site.182

IL-8 and Gro-a are chemoattractant for neutrophils and contribute to extravasation of neutrophils. Neutrophils can produce several polypeptides mediators of inflammation, including IL-1, IL-6, IL-8 and TNF. At the site of injury neutro-phils promote tissue damage by release of lysosomal enzymes and superoxide anions. Lung reperfusion injury and neutrophil infiltration can be prevented experimentally by a monoclonal antibody (mAb) against IL-8,183 raising the possibility that organ preservation as well as immune activity could be improved through manipulation of chemokines.

a Subfamily

Chromosome location 4

Table 1.5. Characteristics of chemokines

ß Subfamily 17

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