Innate immunity

ARTICLE

Immune functions within the cell

To speak of immunity at single cell level may seem at first paradoxical but it allows the broadening of the definition of defence functions. Each cell can be subjected to different stresses; metabolic (nutritional deficiency), toxic, physical (hyperthermia, irradation by ultraviolet, X or gamma rays), or infections by a virus. The various responses brought into action by such stresses all consist of systems for detecting the anomaly, for transduction of the signal and of effector mechanisms using pre-existing or rapidly synthetized molecules as a response to the stress. A first example of these defense mechanisms is the stress proteins (heat shock proteins) which combine with damaged proteins exposing hydrophobic sites, and which carry them to the degradation sites (proteasome). Other examples are the synthesis of p450 cytochromes, and the opening of transmembrane channels of the ABC family (ATP binding cassettes) among which is the MDR molecule (multidrug resistance). The DNA alterations are detected and trigger a coordinated response which involves arrest of the cell cycle, DNA repair, and control of the quality of this repair, leading to the survival of the cell or to its apoptosis [3]. The genes implicated in ataxia telangiectasia (ATM) or in certain cancers (p53, BRCA1) play an essential role in these responses [4]. During oxidative stress the cell uses the pre-existing anti-radical systems (superoxide dismutases, catalase, glutathion peroxidase) and the stress proteins. Free radicals, like different environmental nucleophilic molecules, can form adducts (for example: aldehyde derivatives fixed on DNA).

Immune functions within the organism

In its most simple form, an organism is an aggregate of cells which bind to each other and to the extracelluar matrix through adherence and signalling membrane molecules. Survival, multiplication and apoptosis in each cell depends on a genetic programme of differentiation and external molecular signals: cell death can be provoked by a deficiency in a factor necessary for survival (hormone, cytokine, growth factor) or by a death signal (membrane receptor activating an apoptosis cascade or the insertion of a channel allowing the introduction of apoptosis-activating enzymes). Phagocytic cells (macrophages) absorb and degrade the dead cells. Cytotoxicity allows the destruction of cells infected by a pathogen and of cells which are foreign to the organism.

Natural or innate immunity exists in all pluricellular organisms, including plants and insects [5]. Acquired immunity, which has developed in vertebrates, completes natural immunity by using the same effector mechanisms throughout the whole body. On exposure to an infectious agent, the organism summons first the innate immune responses and then later the specific immune responses [6].

Two strategies for distinguishing self from non-self

Acquired immunity is defined as the specific interaction of an antibody (by its paratope) with the antigen or by the interaction of a TCR receptor and a peptide associated with an MHC molecule. BCR and TCR receptors are produced by the rearrangement of gene segments during the differentiation of lymphocytes. The combinatorial and junctional diversities allow the production of a very large number of different receptors so that any antigenic epitope (self or non-self) can be matched with a receptor paratope in the immune system. Thus a very diverse list of receptors is produced and on exposure to an antigen only the lymphocytes possessing the useful receptor multiply (clonal expansion) and differentiate. This strategy brings the risk of a reaction directed against the body's own antigens (auto-immunity).

Innate immunity summons receptors coded by genes which have not been rearranged and which have been selected during the course of a species' evolution in contact with an infectious environment. These receptors, in solution in the biological fluids or on the cellular membranes, interact with structures which are not part of the host and which are common to a large number of pathogens, which are given the acronym PAMP (pathogen-associated molecular patterns). These molecular structures are generally invariable and often indispensible for the survival of or functioning of the infectious agent [7]. The best described so far are the lipopolysaccharides (LPS or endotoxin from Gram negative bacteria), the peptidoglycans, the lipotechoic acids, the mannans, the bacterial DNA and notably the CpG demethylated nucleotide sequences, double strand RNA and the formyl peptides possessing an N-formylmethionin (fMLP) (Table I).

Amplifying cascades, coordinated actions and redundancy

The signals provoked by the interaction of the PAMPs with their receptors in the biological fluids or on the host cells have common characteristics: immediate activation (pre-existing receptors) and signalling cascades by binding of proteins, expression of biological activities by proteolytic cleavage (proenzymes), liberation of mediators bound to the cellular receptors on phagocytes to induce migration signals (chemotactism), activation (sythesis of cytokines and lipid mediators like prostaglandins, leukotrienes or the PAF acether), phagocytosis of opsonised bacteria and oxygen-dependent or -independent bactericidy. As the pathogen is generally localised in tissues outside the intravascular system, an essential stage in the inflammatory reaction is the interaction between leukocytes and the endothelium and the migration of phagocytes to the tissues.

One can illustrate these characteristics with the example of collectins, members of the lectin family, which bind to the terminal mannose residues of bacterial oligosaccharides. The surfactant proteins SPA and SPD opsonize bacteria in the respiratory tract. In serum, the mannose binding protein or mannose binding lectin (MBP or MBL) activates the serine proteases MASP1 and 2 (MBL-associated protases) which cleave the complement proteins C4 and then C2 to form the convertase C4b2a [8]. This same convertase can also be formed by activation of the classical complement pathway with formation of the C1 complex by direct action of the lipid A of LPS, or by attachment of the C reactive protein (CRP) to phosphorylcholines in the bacterial cell walls. CRP, like MBL, are acute phase proteins synthesized by hepatocytes under the influence of inflammation cytokines (IL-6).

Complement activation by proteolytic cleavage of C4, C3 and C5 leads to the liberation of chemotactic and pro-inflammatory peptides (anaphylatoxins C4a, C3a and C5a). It enables the opsonization of micro-organisms to occur, owing to the covalent fixation of numerous C4b and C3b molecules (inactivated in iC3b) which bind to the CR3 phagocyte receptor (CD11b/CD18). Finally it initiates the formation of the cytolytic membrane attack complex (C5b-C9).

The exclusion of a pathogen can also be ensured by the endocytosis receptors which do not induce an inflammatory reaction. This is the case with the phagocyte mannose receptor (member of the lectin family, dependent on calcium) [8] and with the scavenger receptor (assuring the elimination of waste products) [9].

The Toll family of receptors

The drosophila Toll gene controls the dorso-ventral polarisation during embryonic development [10]. This gene codes, by its intracellular region, for a homologue protein to IL-1 receptors. A dozen Toll type receptors have been characterised in the drosophila and some of them are implicated in the synthesis of anti-microbial peptides under the control of the transcription factor NFkappaB [11]. In mammals, the Toll family receptors (notably TLR2 and TLR4) initiate intercellular signalling cascades which overlap with those triggered by the pro-inflammatory cytokines IL-1 and TNFalpha (Fig. 1) and which induce gene transcription under the control of NFkappaB (for example IL-8, IL-6, GM-CSF, G-CSF, M-CSF, TNFalpha, TNFbeta, IL-2, etc.) [12].

The LPS molecule consists of a saccharide chain, which varies according to the bacterial serotype (chain carrying the epitopes recognised by the antibody), a nucleus and a hydrophobic region, the lipid A which is constituted of a diphosphoglucosamine connected to 4 to 6 fatty acids (Fig.1). The molecule combines in the form of micelles. The hydrophobic region interacts with the acute phase protein LBP (LPS binding protein) which permits the binding of LPS to CD14 on the phagocytes (mainly the monocytes). The CD14-LPS complex interacts with a soluble protein, MD2, and this complex binds to TLR4 [13, 14]. The TLR receptors are themselves in a homo- or heterodimeric form. The sCD14-LPS complexes bind to the endothelium and induce endothelial activation, chemokine synthesis and the binding of leukocytes.

Mice which are deficient in the TLR2 gene do not respond to peptidoglycans nor to lipoproteins. Mice deficient in TLR4 are very susceptible to infection by Gram negative bacteria but are resistant to endotoxin shocks [15]. It could be supposed that the allelic polymorphism of TLR genes might be accompanied by variations in the efficiency of the pro-inflammatory signals which depend on the products of these genes and, therefore, by a diversity of response to the pathogens [16].

Innate immunity controls and directs the acquired immune response

The function of antigen-presenting cells (APC) is to provide the specific information or first signal (presentation of the peptide associated with the MHC molecules) and then a second signal, which is necessary for the activation of T cells. This second signal consists on the one hand of costimulation membrane molecules (CD80, CD86 and ICOS ligands) and on the other hand of cytokines which will participate in the polarisation of the T cell response. For example, certain APCs when stimulated by demethylated CpG sequences [17] or by mycobacteria [18], produce IL-12, which induces the synthesis of IFNgamma by NK and T lymphocytes which in turn lead to a type I response, indispensable for immunity against most of the intracellular microorganisms. By contrast, other APCs, like mast cells or dendritic cells, in the absence of Toll receptors, can induce a type II response (IL-4, IL-5 and IL-13), which promotes the synthesis of IgE antibodies and antiparasite defences, using basophils and eosinophils. Other APCs can favour the emergence of a regulatory T cell response with the production of IL-10 and TGFbeta.

The influence of the location and of the type of initial inflammatory reaction on the specific immune response has been illustrated by the "danger concept" proposed by P. Matzinger [19] and confirms earlier observations on the role of adjuvants in the specific immune response. This field of research is very important for the development of new preventive or therapeutic vaccines.

At the boundary between natural and acquired immunity

Between innate and acquired immunity, there is a whole range of intermediary immune reactions which have slowly been identified, establishing a continuum between these different responses.

Antibody molecules can interact with B superantigens through a site which is outside the paratope, just as the TCR alphabeta can interact with the bacterial superantigens bound to MHC class II molecules.

Natural antibodies of the IgM class (and IgG in smaller quantities) are characterised by their cross-reactions (notably with different epitopes of pathogens). Their weak affinity is compensated for by the bonus effect of multivalent attachments and by their ability to activate the classical complement pathway by the interaction of CIq with a single IgM molecule. More specific antibodies, with an increasing affinity, are produced during the course of the immune response.

The cytotoxic NK lymphocytes receive activating or blocking signals from different membrane receptors, some of them interacting with MHC class I molecules. Tgammadelta lymphocytes recognise the phosphoantigens of different pathogens and their receptors are not very diverse. alphabeta T lymphocytes, devoid of CD4 and CD8 co-receptors, interact with microorganism glycolipids associated with the CD1d molecule. There are thus many situations where the characteristics of the immune response are intermediary between innate immunity and acquired immunity.

CONCLUSION

Natural immunity, which has been neglected for too long, is at the centre of the anti-infection defence process. The progressive identification of the principal genes implicated in this form of immunity will lead to the definition of new pathologies, whether they be immune deficiencies or chronic inflammatory diseases [20, 21]. There is already a long list of genetic deficiencies which have a bearing on natural immunity (complement, cytokines and receptors, NADPH oxidase, integrins). It would no doubt be interesting to see if certain allelic polymorphisms are likely to represent risk factors and if further studies in this area could lead to new therapies.

Acknowledgements

We are indebted to Jenny Messenger for translating this article.

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