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INNATE IMMUNITY
     Studies of the human immune system often focus on adaptive immune responses which involve B lymphocytes and their antibodies, T lymphocytes and their receptors.  These reactions are incredibly complex and rely on so many interdependent factors, that it might be difficult to imagine how such complex reactions could evolve.  Much of the answer seems to reside in the innate immune system.  The complex reactions of adaptive responses were supplements to ancestral innate responses and co-opted many innate mechanisms for new purposes.  Thus, an understanding of the molecular responses of adaptive immunity begins with innate immunity.
BLOOD BLOOD
BLOOD
TUNICATE

      Phagocytic cells evolved long before the complex immune reactions of higher vertebrates.  Vertebrate white blood cells (the blue-purple cells of the fish, frog, and turtle blood in the preceding images) are not unique in their ability to consume microbes through phagocytosis.  This ability is even possessed by many microorganisms, such as such as amoeba.  All animals possess amoeba-like cells (similar to white blood cells which perform phagocytosis) which float in the fluid around the body cells.  Such phagocytes are even known in the most primitive animal groups, such as sponges and starfish, and are present in invertebrates which lack a true circulatory system (Hoar, 1983).  These ameobocytes, like white blood cells, may be full of inclusions, some of which are phagosomes (Harrison, Vol. 2, p.47).  A number of invertebrates, including tunicates, possess cytotoxic cells which have been compared to natural killer cells (Parrinello, 1996).

     In hemichordates, blood flows through spaces in connective tissue in which amoeba-like blood cells travel.  The amoebocyte blood cells typically possess kidney-shaped nuclei. (Benito, form Harrison 1997, p. 61).  Echinoderms have a variety of immune cells in their coelomic cavity, including cells which resemble macrophages (Hibino, 2006). Ascidian blood (such as that in the tunicate larva pictured to the right) includes macrophages, different kinds of granular amoebocytes with odd-shaped nuclei which perform phagocytosis, cytotoxic cells, and a number of other cell types (Parrinello, 1996;Burighel, from Harrison, 1997, p. 269). 

     Vertebrate white blood cells are classified in two categories, granulocytes and agranulocytes, each of which contains additional subclasses of cells (neutrophils, eosinophils, and basophils are granulocytes; monocytes and lymphocytes are agranulocytes).   These leukocytes and the specific molecules they use to function are not unique to humans.  Neutrophils are typically the most common white blood cell in vertebrates and basophils the least common (Torrey). 

 

NEUTROPHIL:

neutrophil

EOSINOPHIL:

EOSINOPHIL

LYMPHOCYTE:

LYMPHOCYTE

MONOCYTE:

MONOCYTE

DAPHNIA

     Higher invertebrates involve a number of complex mechanisms into their immune responses including clotting, opsonization, the synthesis of new blood cells, phagocytosis, and a variety of antimicrobial chemicals (Jiravanichpaisal, 2006).

Although invertebrates lack the adaptive immunity mechanisms found in vertebrates, some do show some increased response to second exposure to antigens (Arala-Chaves, 2000).  Social insects have some form of adaptive immunity in that they show enhanced reactions to an antigen after other members of colony have been exposed and the arthropod Daphnia has been shown to transfer immunity between mothers and offspring (Little, 2003). In invertebrates, the Down syndrome cell adhesion molecule (Dscam) can be spliced into more than 31,000 alternate forms to allow for the recognition of diverse microbes ranging from bacteria to eukaryotes like Plasmodium. Exposure to microbes determines which of the splice forms are over- or under-represented. Loss of Dscam lowers survival of the host in response to infection (Kurtz, 2006).

Mollusks and urochordates also possess mechanisms for producing variable immune receptors with immunoglobulin domains. Apart from these ways of producing diversity by somatic recombination, there are other organisms which have expanded the gene families which produce immune proteins to achieve immune receptor diversity (such as the 200 genes of the NLR receptor family or 26 genes of the Toll-like receptor gene family in sea urchins) (Flajnik, 2007; Litman, 2007).

Lampreys produce variable lymphocyte receptors which, unlike the immunoglobulins of jawed vertebrates which utilize RAG for recombination, are members of the leucine-rich repeat (LRR) family (as are the toll-like receptors) which utilize a somatic recombination mechanism which is not related to RAG (Flajnik, 2007; Litman, 2007).

About 5% of sea urchin genes function in immunity and contribute to the great longevity of many sea urchins (up to 200 years). Although echinoderms lack an adaptive immune system, their innate immune defenses have been augmented by gene duplications which have produced more than 200 members in each of the following gene families which act in pattern recognition: Toll-like receptors, NACHT domain and leucine-rich repeat (NLR) proteins, and Scavenger receptor cysteine-rich (SRCR). (The totals of genes in the first two families are 10 times higher than that of any other known animal.) (Hibino, 2006; Cameron, 2007).

 

 

     Adaptive and innate immune systems are actually integrated and should not be treated separately (Dixon, 2001). The adaptive immune system was superimposed upon the innate system which had already existed in ancestral invertebrates.  The complement system and the Toll-like cascade were primitively part of innate responses but which became required for adaptive responses (Du Pasquier, 2004).  MHC and natural killer cells are parts of the innate which interact with mechanisms of adaptive immunity (Dixon, 2001).  In both mice and humans, it seems that NK cells of innate defenses and T cells of acquired defenses develop from a common precursor cell type.   NK cells can lyse tumor cells and cells infected with certain viruses, such as herpes viruses and adenoviruses.  They can also respond to antigens which have bound IgG.  The cytokines and interferon which they produce mediate the responses of leukocytes and the development of inflammation (Moretta, 2002).

 

ANTIGEN PRESENTING CELLS

     The human body can create millions of different kinds of B and T lymphocytes, each capable of recognizing specific antigens.  However, lymphocyte reactions typically have to be activated (somewhere in the pathway) by antigen presenting cells (APCs).  Although APCs lack the diversity of antibodies and T cell receptors found on lymphocytes, they do possess a diversity of receptors which allow them to recognize microbes.  The macrophages which develop from monocytes (pictured below) are important APCs.

MONOCYTE

      Antigen presenting cells can possess a variety of innate receptors including lectins (MMR, DC-SIGN, Dectin-1), immunoglobulin superfamily members (TREM1, TREM2, Siglec1), helical collagenous proteins (SR-AI/II and MARCO), integrin CR3, TLR, CD1, CD14, CD91, and CD163.  These receptors enable them to bind lipopolysaccharides, lipoproteins, and carbohydrates of bacteria and viruses.  They also bind to proteins produced by the multicellular host and help to remove this debris from blood, such as lysosomal hydrolases, protease-inhibitor complexes, haptoglobin-hemoglobin complexes, apoptotic cells, and denatured molecules.  Thus many of the innate immune mechanisms also function in homeostasis (Gordon, 2004).  T cell receptors detect processed antigens which Antigen-Presenting cells have recognized without the great diversity of TCR receptors.  APCs recognize microbial carbohydrates, proteins, lipids, or nucleic acids as foreign based on PAMPs, or pathogen-associated molecular patterns.  APCs utilize innate receptors such as lectins and Toll-like receptors to recognize these PAMPs (Lu, 2002).

Plasmacytoid dendritic cells (pDC) not only function as antigen presenting cells in adaptive immune mechanisms, they function in innate antiviral responses with their production of interferon and stimulation of NK cells (Tai, 2007).

 

NATURAL KILLER CELLS

     Although the best known molecules which recognize foreign molecules are antibodies and T cell receptors utilized by adaptive immune responses, the receptors which function in innate immunity can recognize foreign molecules as well.  Innate immunity is triggered by molecules which are common to a variety of microbes ranging from bacterial lipopolysaccharides to viral double stranded RNA (Dixon, 2001).

     Natural killer cells are the first line of defense against intracellular pathogens in the innate immune system.  They interact with MHC proteins and some cells present to them (Dixon, 2001; Sambrook, 2007).

NK cells have inhibitory receptors which prevent them from attacking cells which express MHC proteins.  NK receptors interact with 2 separate sites on MHC proteins, including bound oligosaccharides.  Cells which reduce the number of MHC proteins, or lose their expression altogether, are more vulnerable to destruction by NK cells (Parham, 2000; Sambrook, 2007).  Many tumor and virally-infected cells decrease the number of MHC class I proteins they express.  As a result, NK cells can attack some of the same cells which are recognized by T cells. (Moretta, 2002).

     Natural killer cells can express a variety of non-homologous receptor molecules.  NK receptors include the C-type lectins (CD94/NKG2, Ly49 family), KIRs, LIRS, and LAIRS to distinguish between self and nonself (Martin, 2004). Natural killer cell receptors are encoded in a leukocyte receptor cluster region (LRC) of mammalian chromosomes.  Zebrafish NITR genes (novel immune type receptors) share homology with them (Yoder, 2001).

NK CELLS
NK CELLS

Killer Cell Immunoglobulin-Like Receptors

     Natural killer cells depend on KIR receptor function.  Humans possess a family of killer Ig-like receptors (KIRs) on chromosome 19 which interact with class I MHC proteins and a family of CD94/NKG2A proteins on chromosome 12.  The first group is highly polymorphic in both the number of genes and the number of alleles while the second group is highly conserved. 

    NK cells depend on KIRs and CD94/nKG2A for inhibition and are activated by natural cytoxicity receptors (NCR) which mediate most lytic activity of tumor cells.  The coreceptors 2B4 and NTB-A may amplify the effect of other NK receptors (Moretta, 2002).  The KIR cluster of chromosome 19q13.4 varies in the number of KIR genes and types of KIR genes it contains (Martin, 2004).

     The KIR receptors are involved in the recognition of MHC proteins by cytotoxic T cells.  Some have 2 Ig domains and others have 3.  This family includes the genes KIR2DL1-2, KIR2DS1-5, and KIR3D1-5.  The KIR and LIR genes seem to have resulted from the duplications followed by duplication and deletion of exons in certain genes (Hughes, 2002).  KIR2 receptors can interact with MHC class I HLA-C while KIR3 proteins can interact with HLA-A, HLA-B, and perhaps HLA-G (Hughes, 2002).  There are at least 11 KIR loci on chromosome 19 in addition to molecules similar to KIRs which are expressed on monocytes, B cells, dendritic cells, and NK cells called LIRs (leukocyte immunoglobulin-like receptors) (OMIM).

     MHC proteins seem to evolve quickly as do the KIR families which bind to them.  In humans and chimps, this rapid evolution means that the similarities between the MHC and KIR proteins in the two species is less than the 98.4% average for other proteins (Khakoo, 2000).

Human and chimp recognition systems are clearly similar in that NK receptors of one species can recognize the MHC proteins of the other (Khakoo, 2000).

     Human genomes can vary in the number of KIR genes they possess.  Both the KIR and MHC genes (whose proteins bind KIR receptors) seem to have undergone a great deal of gene duplication with many of the modern human genes originating recently (Wilson, 2000a).  Rodents seem to lack KIR genes, but they possess a variety of Ly49 genes which perform the same function.  Humans possess a Ly49 pseudogene on chromosome 12 (Wilson, 2000a).

 

Leukocyte Immunoglobulin-Like Receptors LILR

The leukocyte immunoglobulin-like receptors are expressed primarily on B cells and monocytes(present in the lymph node section below), although they are also present on dendritic cells and natural killer cells.  The A subfamily members (3 genes are known) can stimulate cytotoxic cells (such as natural killer cells) while the B subfamily members (5 genes are known) can inhibit immune reactions.

--LILRA3 is probably a soluble receptor since it lacks a transmembrane region.

NK CELLS

Leukocyte Associated Immunoglobulin-Like Receptors (LAIR)

--LAIR1 is expressed in the intestines where it prevents excessive inflammation in response to antigens there. 

--LAIR2 is probably a secreted protein.  It mediates the interactions between LAIR1 and natural killer cells.

 

 

KLR—Killer Cell-Lectin Like Receptors

 Natural killer (NK) cells distinguish between self and nonself through the recognition of MHC I proteins by their receptors.  Some of these receptors are similar to immunoglobulins while others are lectins, such as CD94 (which is considered a marker for NK cells in vertebrates).    Some blood cells of urochordates express a CD94-like protein and urochordates can reject foreign transplants (a reaction which involves natural killer cells in vertebrates) (Khalurin, 2003; Dehal, 2002).

     Teleost fish possess homologs of mammalian natural killer cell receptors (C-type lectin receptors) that are located in a homologous gene cluster (Sato, 2003).  In mice, the receptors of NK cells belong to the C-type lectin family Ly49 unlike humans in which they are KIRs.  Different mammalian orders seem to have expanded different gene families to accomplish the same function.  Although the single human Ly49 gene seems to be a pseudogene, baboons possess a functional Ly49 gene in addition to multiple KIRs (Mager, 2001). 

IMMUNITY IN THE BRAIN

Innate immune mechanisms represent the primary defense of the brain. Although microglia (myeloid cells produced in the bone marrow) are the primary glial cell type which functions in immunity, astrocytes express toll receptors, scavenger receptors, mannose receptors, and complement proteins which allow them to function in innate immune responses. Astrocytes can secrete chemokines and interleukins to influence inflammation and regeneration. Astrocyte signals influence the movement of immune cells from the blood to the perivascular space and the proliferation and activation of microglia (Farina, 2007). Toll-like receptors are significant components of the microglia responses in the innate immunity of the brain (Glexer, 2007).

SIGNALS IN INNATE IMMUNITY

      When cells are in trouble, they do not keep this a secret.  There are a variety of signals which are released in response to infection and tissue damage.  A number of local hormones are involved in human immune reactions and homologs of these signals exist in simpler animals.  Starfish possess molecules similar to interleukin-1.  Worms and tunicates possess interleukin-1 and TNF; hornworms produce interleukin-1 and interleukin-6.  Three kinds of cytokines are known from invertebrates.  Mammal leukocytes are more similar to those of lungfish than to teleosts (Hine, 1990).  A cytokine-like molecule known in sponges which is similar to an enzyme functional in inflammation in mammals (Muller, 2001).  

   Chemokines are signals which induce inflammation and can attract leukocytes to the inflamed area.  They are classified by whether the first two cysteine residues are separated by another amino acid (CXC cytokines) or not (CC cytokines). Tunicates possess distant relatives of interferon receptors, the only Class 2 cytokine receptors known in invertebrates. In vertebrates, cytokines and their receptors were amplified and diversified. Many function in innate immune defenses and some evolved a signaling function in adaptive immune responses. While most cytokines function in combating viruses and regulating immune responses, the cytokine tissue factor is functional in the clotting of blood (Krause, 2005). CC cytokines, CC receptors, and CXC receptors are known in bony fish (Magor, 2001).  Teleost blood is depicted below.

BLOOD

Interleukin-6 is only known from mammals (Magor, 2001).  Two cytokines are known in sponges (Muller, 2001).   

     In mammals, the members of the TNFα superfamily include TNFα, lymphotoxin-β, FasL, CD-40L, and CD30-L.  TNFα is known in bony fish and there are some reports of its presence in protostomes and primitive deuterostomes (Magor, 2001).  Three isoforms of TGFβ are known in mammals, two additional forms are known in birds and amphibians (Magor, 2001).  Interferons are known from amniotes.  Possible homologs have also been identified in fish (Magor, 2001).

 

OTHER IMMUNE MOLECULES

     Other innate immunity elements of primitive animals are similar to those found in mammals.  In sponges, immunoglobulin domains exist in receptor tyrosine kinases and the sponge adhesion molecule (SAM) (Muller, 2001; Gamulin, 1994).Of the known signaling cascades of the immune system, the MAPK cascade may represent the oldest, having evolved in early eukaryotes. Other innate immunity cascades were present by bilaterans, given the nematode utilization of Toll-like receptors, TGF β, insulin-like receptors, apoptosis, and lectins in their innate immunity (Schulenburg, 2008).

  The enzyme eosinophil peroxidase is not known in jawless fish, its activity is weak or absent in cartilaginous fish, and is present in at least some of the members of all higher groups.  An eosinophil is depicted below.

EOSINOPHIL
The enzyme alkaline phosphatase found in neutrophils is known from jawless fish and all higher groups (Hine, 1990).
NEUTROPHIL

     Regulatory proteins are needed to prevent complement proteins from damaging host cells.  One gene family in humans, the RCA family (Regulators of Complement Activation) include the membrane proteins CR1 (CD35), MCP (CD46) and DAF (CD55) and the plasma proteins factor H and c4-binding protein (C4bp).  The gene family members share short consensus repeats (SCRs).  Factor H is known in bony fish (Zarkadis, 2001).

 

     In mammals, MIF is expressed in a variety of tissues and it thought to have a number of functions, including an anti-inflammatory action.  Hagfish, lampreys and jawed fish possess homologs of the macrophage migration inhibitory factor (MIF).  MIF is similar to a number of other enzymes such as D-dopachrome tautomerase (DDT), -carboxymethyl-2-hydroxymuconate isomerase, 4-oxalocrotonate tautomerase, chorismate mutase, and glutathione S-transferase (GST) of the mu-class.  DDT and GST are linked to the human MIF gene and related enzymes are known in nematodes (Sato, 2003a).

 

     In mammals, the MAGE (melanoma associated antigen) family of genes are expressed in fetal tissues, adult germline tissues, and in a number of tumors.  The expression of MAGE in tumor cells causes the presentation of peptides derived from these proteins on MHC proteins which can activate the anti-tumor responses of cytotoxic T cells.  Although the only known function of MAGE genes in mammals is the activation of adaptive immune responses, MAGE proteins are known in flies and thus predate the evolution of adaptive immunity (Pold, 2000). 

 

     Apopotosis may have originally been part of immune response (Kasahara, 2004).  Vertebrates possess two types of immunoglobulin which are thought to be similar to the ancestral immunoglobulin which gave rise to antibodies and TCR genes, the JAM/CTX and the nectin/poliovirus receptor (PVR) families.  JAM can activate apoptosis through the NF-κB pathway  (Du Pasquier, 2004).

The ribonuclease (RNase) superfamily is unique to vertebrates and a significant expansion in the family occurred in the ancestors of mammals. While RNase1 (pancreatic ribonuclease) digests RNA from food sources, angiogenin promotes angiogenesis, and several of the family members (RNase 2,3, and 7) inhibit bacteria or viruses in innate immune responses (Cho, 2006).

     Heat shock proteins can chaperone peptides in the MHC I presentation pathway (Robert, 2003).

 

     In horseshoe crabs, α2M is the only protease inhibitor known in the hemolymph where it is the third most abundant protein (Magor, 2001).

 

     C. elegans uses a number of pathways in its responses to pathogens including the p38 MAP kinase pathway, apoptotic pathways, TGF-β pathways, and DAF-2 insulin/IGF-I like pathway (Nicholas, 2004).  In C. elegans, apoptosis involving caspases and Bcl-2 funcitons in responses to infections (by Salmonella, for example) (Nicholas, 2004). Programmed cell death as a response to infection evolved in early eukaryotes prior to multicellularity, given homologous immune responses in diverse eukaryotes such as plants, algae, fungi, and animals (Staal, 2007).

 TOLL-LIKE RECEPTORS

 

One of the most significant discoveries in modern immunology is that of the Toll receptors used by flies in response to fungal functions and the family of Toll-like receptors in vertebrates (Glexer, 2007). The TIR domain (a component of Toll-like receptors) evolved in early eukaryotes and is functional in immune responses of plants (Staal, 2007). Drosophila uses Toll-like receptors which, after binding microbes, induces the expression of antimicrobial peptides.  C. elegans possesses one Toll-like receptor which seems to affect its reaction to microbes (Nicholas, 2004).  Toll-like receptors and a basic complement pathway are present in cnidarians (Hemmrich, 2007). Cnidarians and sponges possess homologs of Toll-like receptors and the proteins with which it associates in its immune function (Hemmrich, 2007). One of the genes in the Toll-like receptor family [Sterile-alpha and Armadillo motif containing protein (SARM)] is conserved in bilateran animals. Its function, however, apparently changed in early vertebrate evolution given that its role in down-regulating immune signaling in coelomates as diverse as humans and arthropods is different from that of homologs in nematodes where it up-regulates immune signaling (Belinda, 2008).

The discovery of Toll receptors in flies has led to the appreciation of Toll-like receptors in human innate defenses (Liu, 2001).  Toll functions in axis formation in both Drosophila and Xenopus (Prothman, 2000). When activated, toll-like receptors (TLRs) and other Pattern Recognition receptors (PRR) signal cells to release cytokines which promote both innate and adaptive immune mechanisms. In addition, reactive oxygen species and nitric oxide are released to inhibit bacteria. Thirteen TLRs are known in mammals. In humans, TLRs are important features in the defenses against a variety of pathogens including tuberulosis, salmonella, and Staphylococcus (Gerold, 2007). Specific toll-like receptors have specialized in recognizing bacteria, fungi, protists, and even viral nucleic acids (Barton, 2007).

 

 

    In higher vertebrates, cytotoxic T cells can kill infected cells with secreted molecules called perforins which generate holes in the cell membrane.  Natural killer cells also secrete perforin-like molecules.  Perforins are homologous to two proteins in mammals, one of which (Mpeg1) is expressed by macrophages and prion-infected brain cells and the other (Epcs50) is expressed in the placenta.  Mollusks possess a homolog of Mpeg1 (Mah, 2004).

 

Invertebrate innate immune reactions include the increased production of immune proteins in coelomic fluid (Kauschke, 2007).

Eicosanoids signal the migration of white blood cells/hemocytes in the immune reactions of vertebrates and arthropods respectively (Merchant, 2008).

A family of antibacterial proteins known as defensins evolved in ancestral eukaryotes, given its distribution in animals, plants, and fungi. Fungi and animals share homologs not found in plants, supporting the Ophisthokonta clade (in which fungi and animals shared a common ancestor more recently than either shared with plants) (Zhu, 2008).

The prevention of excessive immune reactions is mediated by a variety of proteins, including the suppressors of cytokine signaling (SOCS) family which includes eight known gene members in mammals. A gene duplication in this family had already produced two genes in ancestral coelomates and additional duplications in early vertebrates produced additional gene members (Jin, 2008).

Cells functioning in innate immunity (such as some phagocytes, dendritic cells, fibroblasts, and endothelial cells) produce pentraxins in response to inflammatory signals. Pentraxins in turn enhance opsonization and moderate inflammation and the distinction between self and nonself (Bottazzi, 2006).

RNAi (RNA interference) functions in innate immunity in both plants and animals (Obbard, 2006). Plants and invertebrates utilize siRNAs to cleave viral RNAs as part of an innate immunity. In mammals, it seems that the importance of siRNAs in immunity was reduced as the interferon mechanisms became more prominent (Sioud, 2007).