Natural Immune Cellular Activation

Macrophages are a heterogeneous group of cells including circulating monocytes and tissue resident macrophages. They are phagocytic, performing a major function contributing to the innate immune response by ingesting microbes and killing them intracellularly. Several different structural families contribute receptors which participate in phagocytosis [121,122]. Endocytosis has been demonstrated for FcRs (IgSF), CR3 (integrin) and the mannose-receptor (lectin), while CR1 (lectin) and CR4 (integrin), scavenger receptors MARCO and SR-A, leucine-rich repeat bearing CD 14, and additional integrins recognizing fibronectin/vitronectin-opsonized particles, bind and increase the efficiency of particle internalization [86], Phagocytosis of microbes is usually accompanied by the generation of an inflammatory response and the release of antimicrobial peptides. Some phagocytic receptors also stimulate inflammation such as FcRs. However, binding receptors such as TLRs which are not endocytic but localize to the phagosomes during phagocytosis, contribute to the regulation of the activation of inflammation. Trem-1 (IgSF) amplifies TLR-initiated responses to microbes and augments inflammatory chemokine and cytokine secretion [56], A variety of cell surface receptors which participate in the phagocytosis of apoptotic cells have been identified on the professional phagocytes, macrophages and neutrophils, including FcRs, CR3, SR-A, CD14 and the vitronectin receptor as above, plus the class B scavenger receptor CD36, the glycoprotein macrosialin CD68, the ATP-binding cassette transporter 1, ABC1, the phosphatidylserine-receptor (PS-R), the Mer receptor tyrosine kinase and a protein complex of Clq and MBL with cell surface calreticulin and CD91 [reviewed in 123],

Dendritic cells are located in most tissues but particularly in the interface with the environment are considered to play a dual role in the generation of innate responses and in the initiation of adaptive immune responses [reviewed in 124], Monocyte-derived, immature human DC express chemokine receptors CCR1, CCR2, CCR5 and CXCRI which would provide for DCs to follow concentration gradients of inflammatory chemokines such as MlP-la, RANTES and MCP-1 to inflammatory sites. In peripheral tissue, immature DCs express cell surface molecules that mediate endocytosis including FcyRI, FcyRII, FcyRIII, CD 14, mannose receptors (col-lectins), CR3 (Mac-1, integrin), MARCO (scavengerR), DEC205 and DC-SIGN [125] (multi-c-type lectins), which is linked with the induction of the inflammatory response to which TLR2/4 binding contributes. DC interaction with pathogens either directly through their PAMPs or the PAMP receptors leads to activation with production of inflammatory cytokines and chemokines attracting leukocytes, including granulocytes, macrophages, immature DCs and NK cells to the site of inflammation. DCs have been implicated in the activation of NK cells possibly through

DC production of IL-2 after microbial activation [126], With the down regulation of CXCR1, CCR1 and CCR5 and upregulation of CCR7 expression, activated DCs migrate to the regional lymph nodes where they complete their maturation process, upregulate their costimulatory molecules and act as the APCs for priming T cells.

Neutrophils, polymorphonuclear cells (PMNs), the most abundant leukocyte in the circulation, originates from a myeloid progenitor. They are short-lived, 3^1 days, granulocytic and respond to many stimuli including those of bacterial, endogenous and immunologic origin, Several chemoattractants have been shown to arrest PMNs including IL-8, C5a, formyl peptides from bacteria, leukotriene LTB4 and platelet activating factor (PAF) [reviewed in 127], Since high levels of chemoattractants can stop PMN rolling immediately [122], their extravasation into inflammatory tissues would be encouraged. Evidence of signalling in PMNs, through the G-protein-coupled chemokine receptors, Fc receptors, L-selectin and inflammatory adhesion molecules, (37 integrins like Mac-1 and LFA-1 on PMNs which possibly bind ICAM-1 and P-selectin glycoprotein ligand for P-selectin and E-selectin on endothelial cells, suggests they are all important for activation. Consistent with the multiple activation pathways for conversion of rolling to adherent PMNs, the lack or only partial effect of blockade of each individual pathway supports the proposal that integration of multiple interactions at the cell surface provides a strategy for appropriate activation [127], In further activation processes which would be important after extravasation, purified peripheral blood PMNs increase IL-8 mRNA within an hour after exposure to LPS in vitro consistent with their expression of TLR4, and they generate additional responses in the presence of contaminating monocytes [75], PMNs can be activated through TLRs. They have Fey receptors for IgG and complement CR1 receptors for C3b and C4b which are important for their phagocytic function. Thus, PMNs exhibit several activation processes associated with their different functions.

Mast cells originate from a myeloid progenitor. They are found in a variety of tissues where they mature, including vascularized connective tissue and the submucosa. They are long-lived with a life span of months to years [reviewed in 45] and are capable of proliferation. They have been shown capable of phagocytosis in vitro through binding of CD48, a mannose-contain-ing GPI-linked receptor binding to Fim-H, a mannose-binding lectin on fimbriae of bacteria [45,128,129], Binding of complement components to their C3a/C4a receptors and C5a receptors leads to the release of active agents including preformed histamine, heparin and serine proteases, and newly synthesized cytokines and chemokines leading to the development of an inflammatory response. They have receptors for opsonins including CR3 for complement, Fc R and FceRI. Mast cells can be activated directly through TLRs 2 and 4 [130], Cross linking of tiie high affinity FceRI by antigen binding to IgE of the adaptive response already bound to the FcR can also activate mast cells contributing to specific allergic responses.

Basophils are found in the circulation, have a short life span of hours to days and like other granulocytes and unlike mast cells they do not proliferate. Like mast cells they release proinflammatory mediators, although the cytokine repertoire is more limited [reviewed in 45,131]. Similar to mast cells, they express the cell surface CR3, FcyR and FcsRI. They recognize bacteria via IgE. Basophils express TLR2 and TLR4 but not CD 14 and did not respond to LPS [75].

Eosinophils are located in the tissue of the lung, gastrointestinal and lower genitourinary tracts. They can release proinflammatory cytokines, chemokines, lipid mediators and toxic granule proteins including major basic protein [reviewed in 132]. Eosinophils did not express TLR2, TLR4 or CD 14 and did not respond to LPS [75],

Epithelial cells provide a barrier that most microbial pathogens must breach. While the skin exhibits a more complex structure, the tubular mucosal surfaces of the gastrointestinal, respira-

tory and urogenital tract have only a single layer of epithelial cells to separate them from the external environment including commensal microbes [reviewed in 133], raising the question of how commensal and pathogenic microbes are distinguished. Although haematopoietic cells are the traditional mediators of innate immunity, a role for epithelial cells is clear from the evidence that activated epithelial cells rapidly secrete proinflammatory cytokines and chemokines after exposure to bacteria [reviewed in 130], in addition to their production of antimicrobial molecules. Microbes exhibit specific pathogen-epithelial interactions with a variety of receptors expressed on the apical (luminal) and/or the basolateral (BL) (facing basement membrane) surfaces of these polarized cells to facilitate their direct colonization or entry into epithelial cells, for transcytosing an epithelial cell, or for accessing directly the BL surface through cytolysis or by traversing tight junctions (TJs) for example, after inducing PMN transmigration [reviewed in 134], Apical receptors which have been implicated include, the cystic fibrosis transmembrane receptor (CFTR), the phosphorylcholine-binding platelet activating factor receptor (PAFR), CD66 (for gonococcal opacity, Opa proteins) [135], CD46 (for gonococcal PilCl) and the polymeric immunolglobulin receptor (plgR) (for ligand choline-binding protein A which binds bacterial phosphorylcholine) which is detected on apical and BL surfaces. Receptors on the BL surface include heparan sulfate proteoglycans (HSPGs) (for Opa proteins), and the integrins which are engaged by vitronectin and fibronectin (as some Opa proteins bind these extracellular matrix proteins), (31-integrins on BL and TJ (for Inv and YadA of Yersinia and for intimin of E. coli, although the typical receptor is bacterially encoded and secreted into the host cell membrane by the bacterium), E-cadherins, (for leucine-rich repeat (LRR) family member internalin, Inl A of Listeria) [136], and the fibronectin receptor and CD44 have been implicated in binding of Ipa-A complexes and IpaB complexes respectively of Shigella. InB 1 (LRR family) of Listeria has three receptors, gClqR, the receptor for complement Clq which is detected subcellularly, glycosaminoglycans and Met or the hepatocyte growth factor receptor (HGFR) [137] which is found predominantly on the BL but detected in low levels on the apical domain.

TLR2 and TLR4 are located on the apical surface of differentiated intestinal epithelial cells (IELs) optimally situated to survey the luminal contents [reviewed in 137], However, TLR2 and TLR4 are also detected on the BL surface in immature IELs and TLR5 appears to be preferentially expressed on the BL surface [reviewed in 137], Considering the efficiency of TLR in inducing inflammatory responses, it has been proposed that microbial stimulation of receptors on the BL side of epithelial cells would distinguish between commensal and pathogenic varieties. However, commensals may reduce the synthesis of inflammatory molecules by preventing IkB-oc degradation and thus inhibiting the IkB/NK-kB signalling pathway, constant exposure to LPS may induce "cross-tolerance" against other signalling pathways for example those of TLR2 and TLR4, restricted apoptosis may contribute to this tolerance and the activation of cell survival mechanisms may all contribute to distinguish the response to commensals preventing a pathogenic induction of inflammation [reviewed in 137],

B1 cells, characterized by CD5 expression are localized to the peritoneal and pleural cavities. They can be activated by LPS, less so by anti-CD40 and not by anti-IgM [reviewed in 138] and the LPS activation was inhibited by anti-CD 14 in a murine B1 cell line [139]. Activation of B cells by a variety of agents has increased CD5 expression suggesting that CD5 is a marker of B cell activation and not a marker for a distinct B cell subset, [reviewed in 140]. B1 cells have shown extensive reactivity against self including Ig, DNA, cytoskeletal molecules, phosphatidylcholine and cell surface proteins on red blood cells and thymocytes in addition to reactivity against microbial antigens including phosphorylcholine [141]. The B1 specificities are thought to be associated with their location, and with BCR-mediated signals from microenvironmental-

antigen considered essential for survival of all B cells this suggests that the CD5 expressing B1 cells may provide an activated, self-renewing effector poised to respond to changes in the microenvironment or to pathogens. As such, B1 cells could be considered as part of a "natural memory" component of lymphocytes together with marginal zone B cells, NKT and y8 T cells, which can react within hours of exposure to antigen, after the earliest innate responses and well before the initiation of the adaptive response [138],

NK cells are important producers of cytokines, in particular INF-gamma, early during infections. Activation is initiated through members of the NKG2 C-type lectin-like family (NKG2D), the Ly49 C-type lectin family (Ly49D, LY49H) in mice, the killer-cell Ig-like receptor (KIR) family in humans (KIR2DS2) [53], and additional Ig superfamily receptors NKp46, NKp44 and NKp30 [reviewed in 11,25,54], The last three are considered to be the main activation receptors for NK cells [25], NKp46 interacts with sialic acid-binding haemagglutinin on virus-infected cells based on NKp46 bearing sialic acid [11], Inhibitory receptors include the Ly49 C-type lectin family in mice, KIR Ig-like receptors in humans [53], the CD94/NKG2 heterodimer of C-type lectins in mice and primates LI 1 ] and the human leukocyte immunoglobulin-like receptor (LIR) (or ILT) family (LIR1 and LIR2), and the homologous, murine paired Ig-like receptors (PIR) (PIR-B) [11], KIRs are a diverse, rapidly evolving receptor family which recognizes polymorphic determinants on MHC-class-I molecules [reviewed in 53], CD94/NKG2A in humans recognizes a conserved class-I leader sequence peptide presented by HLA-E [reviewed in 53], While different families of NK receptors bind different aspects of their MHC class I or MHC class I homologous ligands, their combinatorial expression provides an effective mechanism for surveying host cells for changes associated with infection or oncogenic transformation [25], The balance of inhibitory signals from MHC Class-I specific receptors and signals from activating receptors with other specificities, determines the outcome of each NK-target cell interaction [reviewed in 25], yS T cells arise early in ontogeny with waves of cells expressing different Vy and VS T cell receptor gene segments appearing in the murine foetal thymus and thought to home sequentially to the epidermis, reproductive epithelium and lymphoid organs, while intestinal epithelium y8 T cells arise distinctly [142], In humans y5 T cells are also found in the epithelial-rich tissues of the intestinal and reproductive tracts, while comprising a small proportion of lymphocytes in the skin, blood and lymphoid organs [143], While the Ig-superfamily Vy and V5 receptors of cells in the lymphoid tissue exhibit diversity, a lack of diversity is detected in the y5 T cell receptors of cells in the same epithelial site. In contrast with a(3 T cell receptors, the y8 receptors do not recognize antigen as peptide/MHC complexes. y5 T cells are extensively self-reactive. Typical ligands are derived from local epithelium following infection, intact proteins including stress or heat-shock proteins, MHC class IB, and non-peptide phosphoantigens including nucleotide-conjugates and phospholipids which have been detected in association with microbes. NKG2D an NK C-type lectin receptor binds MHC-like molecules MIC A and MIC B expressed on intestinal epithelial cells in response to injury or stress. The constitutive expression of transcripts of genes associated with cytotoxic and regulatory activity and the rapid inducibility of cytotoxicity in intestinal intraepithelial y8 T cells, has raised the possibility that they may be primed effector cells capable of rapid responses [144]. Further, this state of activation may be controlled by inhibitory NK receptors [144].

NKT cells [145], provide an early antibacterial response and an early cytokine source leading to the differentiation to Th 1 and Th2 and suppression of autoimmunity. Their T cell receptors exhibit limited diversity recognizing CD1, not MHC. CD1 [90], a nonpolymorphic antigen presenting molecule is related structurally and functionally to MHC. However, it does not present peptide antigens but binds and presents lipids and glycolipids including structures from pathogenic mycobacteria. The CD1 binding groove is thought to bind the hydrophobic ligand components presenting the polar head groups for contact with the T cell receptors.

5. ACUTE PHASE RESPONSE

The acute phase response can be initiated rapidly through the release of soluble mediators into the circulation following stimulation of hepatocytes in the liver by the endogenous pyrogens IL-1, IL-6 and TNF-alpha released by activated macrophages. Thus the production of pro-inflammatory mediators from a local inflammatory response can lead to a systemic acute phase inflammatory response associated with fever and the increased production of acute phase proteins (APPs) in the liver. APPs include C-reactive protein (CRP) and serum amyloid P component (SAP), a-2-macroglobulin, mannose-binding lectin (MBL) and complement components.

a-2 macroglobulins, the universal protease inhibitors, have an intrachain thioester bond which becomes activated when the molecule is attacked by a protease. The protease is covalently bound and enfolded into the a-2 macroglobulin revealing a ligand for cell surface receptors which leads to internalization and degradation of the complex [reviewed in 11],

Thus, the acute phase response which is considered to be a systematic all-out last host defence against an invader provides a flood of diverse but in some cases homologous molecules which can react with invaders, facilitating their activation of natural immune cells directly or through activation of complement.

6. COMMON THEMES AND QUESTIONS

6.1. Repeating epitopes and polyvalent recognition

The affinity of many individual NAb interactions is considered weak. However, the binding of polyclonal natural antibodies to the multiple similar or different epitopes on the same cell surface molecule, or cross-linking of different molecules through binding of similar epitopes, plus subsequent crosslinking of bound NAb through anti-Ig reactivity of NAb can generate high functional binding activity. The oligomeric structures of ficolins and collectins, humoral lectins of the innate immune system, the latter typified by MBL, provides multiple binding receptors, which increase the potential for functional binding similar to IgM and CIq. Similarly, while the galectin binding to individual lactosamine moieties is a low affinity interaction, the avidity of galectin binding is increased for polylactosamines where the lactosamine units are repeated in chains [38],

6.2. Redundancy

Redundancy is a major aspect of the innate immune system, protecting against potentially harmful effects of individual molecular or cellular defects. Multiple solutions to self-nonself discrimination are evident in the different receptor/ligand systems. Many evolutionarily ancient structural families are employed in numerous roles in innate system recognition/activation and modulation of cell function suggesting the generation of diversity in the responses to the many challenges during evolution. Furthermore, the many structural families which provide receptors for activation also provide receptors for inhibition. The inhibition may be linked with ITIM motifs located on the cytoplasmic tail of the receptor, whereas the activating forms may bear ITAMs or have no tyrosine-based activation or inhibition motifs but interact with ITAM-bearing molecules.

6.3. Pre-activated state

Elements of the innate immune system can react very quickly employing constitutively expressed receptors and preformed molecules. Cells of the innate immune system also exhibit characteristics of activated cells. The asialo-GMl marker which initially characterized the spontaneously cytotoxic NK cell was also detected on activated cells including activated macrophages and T cells, proliferating thymoblasts and functionally mature thymocytes [146], Asialo-GMl was proposed as a marker of early activation of resting cells. An N-linked carbohydrate marker was detected on resting neutrophils, PHA- or Con-A-activated CD45RO+ lymphocytes and on neuraminidase treated T and NK cells L147J. These data raise questions regarding sialic acid in regulating binding and thus signalling. Is activation associated with desialylation ? Do the asialo residues act as targets for down regulation by the innate immune system for example by NAbs ? The relatively short lifespan of neutrophils and NK cells, approximately 4 days and two weeks respectively, would be consistent with a requirement to be present in an already activated stage.

6.4. Self-nonself discrimination

The basis of self/nonself discrimination is the major question regarding the activation and function of the natural immune system. Many strategies contribute. The theme of inhibition associated with self recognition is emerging. The expression of classical MHC Class I or sialic acid considered to distinguish mammalian cells from microbes, are the basis of two different strategies for inhibition of NK receptors and complement respectively. Recognition of self MHC by inhibitory NK cell receptors can be based on either conserved or polymorphic epitopes. The expression of multiple different epitopes on microbes and multiple receptors with different specificities on the cells of the host innate system provide for specificity. Quantitative expression of epitopes would contribute to achieving a threshold level of receptor stimulation for activation. Cooperation between TLRs for recognition of microbes provides for differential signalling which could tailor the inflammatory response to different pathogens [148], Apoptotic cells generated through many essential physiological processes are clearly a major target of the innate immune system, taken up and disposed of by phagocytes without generating the inflammatory response which results from phagocytosis of pathogens. Are other categories of cells likely targets of innate immunity? The identification of receptors which bind exogenous and endogenous epitopes suggests the coevolutionarily conserved nature of these receptor/epitope systems. This raises a question regarding the relationship of the phenotypes of the epitope-expressing cells, microbial and mammalian cells. Is it an 'activated' phenotype? The expression of activation markers on susceptible host cells and the targeting of activation markers (CD45RA, MICA, MICB) suggests that activated cells would be regulated by the natural immune system. Cells undergoing activation with sufficient targets for susceptibility should be a minor population and the targeting of such markers for negative regulation should increase the specificity of a developing response. This could be important for regulating the development of a highly specific adaptive immune response. If the cells of the innate immune system are pre-activated and the innate response targets activated cells, then the innate immune system should also be self-regulating, contributing to the generalized homeostasis of the organism by the natural immune system. Thus, the natural immune system participates in the physiological homeostasis of the organism and provides a first and last line (APR) of defence against invading pathogens.

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