Teleost IgSF immunoregulatory receptors

In all animals innate immunity is the first line of immune defense from invading pathogens. The prototypical innate cellular responses such as phagocytosis, degranulation, and cellular cytotoxicity are elicited by leukocytes in a diverse range of animals including fish, amphibians, birds and mammals reinforcing the importance of such primordial defense mechanisms. In mammals, these responses are intricately controlled and coordinated at the cellular level by distinct subsets of immunoregulatory receptors. Many of these surface proteins belong to the immunoglobulin superfamily and in mammals elaborate immunoregulatory receptor networks play a major role in the control of infectious diseases. Recent examination of teleost immunity has begun to further illustrate the complexities of these receptor networks in lower vertebrates. However, little is known about the mechanisms that control how immunoregulatory receptors influence cellular decision making in ectothermic vertebrates. This review focuses on several families of recently discovered immunoglobulin superfamily members in fish that share structural, phylogenetic and in some cases functional relationships with mammalian immunoregulatory receptors. Further characterization of these teleost innate immune receptor families will provide detailed information regarding the conservation and importance of innate immune defense strategies throughout vertebrate evolution.     

Examination of the stimulatory signaling potential of a channel catfish leukocyte immune-type receptor and associated adaptor

Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.     

Chicken immunoregulatory Ig-like receptor families: An overview and expression details on ggTREM-A1

Paired immunoregulatory receptors facilitate the coordination of the immune response at the cellular level. In recent years, our group characterized chicken homologues to mammalian immunoregulatory Ig-like receptor families. The first part of this review focuses on the current progress on chicken immunoregulatory Ig-like receptor families. One of these receptors is gallus gallus TREM-A1, which was described as the only member of the chicken TREM family with activating potential. The second part of this review presents a study initiated to further characterize ggTREM-A1 expression. For this purpose we established real-time RT-PCR and generated a specific mab to analyze the expression profile of ggTREM-A1 on mRNA and protein level, respectively. GgTREM-A1 mRNA was predominantly expressed in macrophages, but was also detected in brain, bone marrow, bursa, thymus, spleen and PBMC. Analyzing ggTREM-A1 surface expression by mab staining validated the expression on macrophages. Additionally, we showed high expression on blood monocytes, heterophils and NK cells and on monocytes isolated from bone marrow. Moreover, we detected ggTREM-A1 protein also on thrombocytes, B and T cell subsets, but antigen expression seemed to be lower and more variable in these cells. Immunohistochemistry of chicken brain tissue, combining ggTREM-A1 mab and various markers specific for various brain cell subsets showed expression of ggTREM-A1 on microglial cells, but also on neurons, astrocytes and oligodendrocytes. In conclusion, ggTREM-A1 is expressed on a variety of cells, relevant for the immune system, possibly combining physiological function of different mammalian TREM.     

An extended family of Fc receptor relatives

A surprising number of Fc receptor (FcR) relatives have been recognized recently with the potential capacity to modulate innate and adaptive immune responses. The six human FcR homologs (FcRH1-6), which belong to a phylogenetically conserved gene family, have variable numbers of extracellular immunoglobulin domains of five different subtypes. FcRH immunoregulatory potential is implicated by the presence of consensus tyrosine-based activation or inhibition motifs in their cytoplasmic tails. All but one of these new receptors, FcRH6, are expressed on B cells at different stages in differentiation. Their ligands, function, and prospective roles as diagnostic B cell markers and therapeutic targets are topics of intense interest.     

Multigene families of immunoglobulin domain-containing innate immune receptors in zebrafish: Deciphering the differences

Five large multigene families encoding innate-type immune receptors that are comprised of immunoglobulin domains have been identified in bony fish, of which four do not possess definable mammalian orthologs. The members of some of the multigene families exhibit unusually extensive patterns of divergence and the individual family members demonstrate marked variation in interspecific comparisons. As a group, the gene families reveal striking differences in domain type and content, mechanisms of intracellular signaling, basic structural features, haplotype and allelic variation and ligand binding. The potential functional roles of these innate immune receptors, their relationships to immune genes in higher vertebrate species and the basis for their adaptive evolution are of broad interest. Ongoing investigations are expected to provide new insight into alternative mechanisms of immunity.     

The relationship between major histocompatibility receptors and innate immunity in teleost fish

Studies of the innate immune system have recently shown that, in addition to its role in producing the primary response that slows down pathogens, it may also play an important role in initiating and directing the type of response that the adaptive immune system makes. These discoveries have shown a complex web of control containing new roles for the innate immune system in organizing responses of T-cell to antigens being presented by major histocompatibility receptors, as well as new roles for those receptors in innate immune responses. Both of these activities are managed through feedback networks involving elements of both the innate and adaptive immune system. This paper will discuss these newly discovered interactions and how they are influencing current theories regarding the initiation of adaptive immune responses. In particular, it will highlight the recent progress that is being made towards understanding these relationships in the immune systems of teleost fish.     

Inhibitory ITAMs as novel regulators of immunity

Summary:  Immune homeostasis is regulated by a finely tuned network of positive–negative regulatory mechanisms that guarantees proper surveillance avoiding hyperactivity that would lead to autoimmunity and inflammatory diseases. Immune responses involve the activation of immunoreceptors that contain tyrosine-based activation motifs (ITAMs). One arm of control involves immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors, which upon co-aggregation initiate an inhibitory signal through recruitment of signal-aborting phosphatases. Recently, a new immunoregulatory function has been ascribed to ITAMs, which represent in fact dual function modules that, under specific configurations termed inhibitory ITAM (ITAMi), can propagate inhibitory signals. One paradigm is the immunoglobulin A (IgA) Fc receptor (FcαRI), which, upon interaction with IgA monomers in the absence of antigen, initiates a powerful inhibitory signal involving Src homology 2 domain-containing phosphatase 1 (SHP-1) recruitment that suppresses cell activation launched by a whole variety of heterologous receptors without co-aggregation. This explains the long known function of IgA as an anti-inflammatory isotype. The importance of this control mechanism in immune homeostasis is underlined by the high incidence of autoimmune and allergic diseases in IgA-deficient patients. ITAMi is now described for an increasing number of immunoreceptors with multiple roles in immunity. ITAMi signaling is also exploited by Escherichia coli to achieve immune evasion during sepsis. Here, we review our current understanding of ITAMi regulatory mechanisms in immune responses and discuss its role in immune homeostasis.     

Antimicrobial mechanisms of fish leukocytes

Early activation and coordination of innate defenses are critical for effective responses against infiltrating pathogens. Rapid engagement of immune cells provides a critical first line of defense soon after pathogen infiltration. Activation leads to a well-orchestrated set of events that sees the induction and regulation of intracellular and extracellular antimicrobial defenses. An array of regulatory mediators, highly toxic soluble molecules, degradative enzymes and antimicrobial peptides provides maximal protection against a wide range of pathogens while limiting endogenous damage to host tissues. In this review we highlight recent advances in our understanding of innate cellular antimicrobial responses of teleost fish and discuss their implications to cell survival, immunomodulation and death. The evolutionary conservation of these responses is a testament to their effectiveness against pathogen infiltration and their commitment to effective maintenance of host homeostasis. Importantly, recent developments in teleost fish systems have identified novel host defense strategies that may be unique to this lower vertebrate group or may point to previously unknown innate mechanisms that also play a significant role in higher vertebrate host immunity.     

Clinical Association of a Soluble Triggering Receptor Expressed on Myeloid Cells-1 (sTREM-1) in Patients with Systemic Lupus Erythematosus

A triggering receptor expressed on myeloid cells-1 (TREM-1) is a member of the immunoglobulin superfamily with an established role in innate and adaptive immune response. We aimed to determine the plasma concentrations and clinical association of sTREM-1 in Systemic Lupus Erythematosus (SLE) patients. Plasma from 79 SLE patients and 35 normal healthy subjects were assayed for sTREM-1 and IL-6 levels using Enzyme Linked Immunosorbant Assay (ELISA). The clinical disease characteristics and serological data were prospectively assessed. Disease activity was scored using the SLE disease activity index. We detected significantly higher levels of sTREM-1 in plasma of SLE patients than the healthy control group. We also detected high sTREM-1 levels in subgroups of patients with neuropsychiatric manifestations (NPLE) and patients with the total high disease activity and NPLE activity. In addition, sTREM-l levels were significantly correlated with parameters of disease activity, i.e. SLEDAI score, IL-6, hypoalbuminemia. On the other hand, we did not find significant differences in sTREM-1 levels in relation to age, disease duration, medications, ESR, other organ system involvement, or the presence of anti-dsDNA. Our preliminary data indicated that sTREM-1 levels may be an additional useful marker of disease activity in SLE. It also highlights its importance in patients with NPLE. An additional prospective longitudinal study should be carried out to support these findings.     

Evolution and diversity of the complement system of poikilothermic vertebrates

Summary: In mammals the complement system plays an important role in innate and acquired host defense mechanisms against infection and in various immunoregulatory processes. The complement system is an ancient defense mechanism that is already present in the invertebrate deuterostomes. In these species as well as in agnathans (the most primitive vertebrate species), both the alternative and lectin pathway of complement activation are already present, and the complement system appears to be involved mainly in opsonization of foreign material. With the emergence of immunoglobulins in cartilaginous fish, the classical and lytic pathways first appear. The rest of the poikilothermic species, from teleosts to reptilians, appear to contain a well-developed complement system resembling that of homeothermic vertebrates. However, important differences remain. Unlike homeotherms, several species of poikilotherms have recently been shown to possess multiple forms of complement components (C3 and factor B) that are structurally and functionally more diverse than those of higher vertebrates. It is noteworthy that the multiple forms of C3 that have been characterized in several teleost fish are able to hind with varying efficiencies to various complement-activating surfaces. We hypothesize that this diversity has allowed these animals to expand their innate capacity for immune recognition.     

Evolution of complement as an effector system in innate and adaptive immunity

For a long time, the complement system in mammals has been regarded as a biological system that plays an essential role in innate immunity. More recently, it has been recognized that the complement system contributes heavily to the generation and development of an acquired immune response. In fact, this ancient mechanism of defense has evolved from a primitive mechanism of innate immune recognition in invertebrate species to that of an effector system that bridges the innate with the adaptive immune response in vertebrate species. When and how did complement evolve into a shared effector system between innate and adaptive immunity? To answer this question, our group is interested in understanding the role of complement in innate and adaptive immune responses in an evolutionary relevant species: the teleost fish. The attractiveness of this species as an animal model is based on two important facts. First, teleost fish are one of the oldest animal species to have developed an adaptive immune response. Second, the complement system of teleost fish offers a unique feature, which is the structural and functional diversity of its main effector protein, C3, the third component of the complement system.     

Characterization of the CCR3 and CCR9 genes in miiuy croaker and different selection pressures imposed on different domains between mammals and teleosts

The innate immune system can recognize non-self through pattern recognition receptors and provides a first line of antimicrobial host defense. Thus innate immunity plays a very important role in resistance against major bacterial disease in vertebrates. In the innate immune responses, the chemokine receptors act as the main receptors of chemokines which are released at the sites of infection, inflammation and injury. In this study, the Miichthys miiuy CCR3 and CCR9 genes were cloned and characterized, showing that MIMI-CCR3 possesses a highly conserved DRYLA motif similar to that of other fishes. MIMI-CCR3 and CCR9 were ubiquitously expressed in all tested tissues and the expressions were significantly up-regulated after infection with Vibrio anguillarum except that of CCR9 in spleen. Evolutionary analysis showed that all the ancestral lineages of CCR3 and CCR9 in mammals and teleosts underwent positive selection, indicating that the ancestor of terrestrial animals further evolved to adapt to terrestrial environments and the continuous intrusion of microbes stimulated the evolution of CCR genes in the ancestor of teleost. Multiple ML methods were used to detect the robust candidates for sites under positive selection. In total, 11 and 8 positively selected sites were found in the subsets of current mammal and teleost CCR3 genes, and 3 and 2 sites were detected in the subsets of current mammals and teleosts in CCR9. Interestingly, for mammal CCR3 and CCR9 genes, the robust candidates of positively selected sites were mainly located in the extracellular domains which were the ligand binding and pathogen interaction regions. For teleost CCR3 and CCR9 genes, the positively selected sites were not only located in the extracellular domains but also in the C-terminal and intracellular domains, indicating mammals and teleosts experienced different selection pressures upon their N-terminus, C-terminus and intracellular loops of CCRs.     

Putting the natural killer cell in its place

Natural killer (NK) cells were originally described as 'null' lymphocytes, but we have increasing evidence of their role in recognizing pathogen, and our knowledge of NK cell receptors continues to expand exponentially. Human NK cells have many receptors for human leucocyte antigen (HLA) class I. These killer immunoglobulin-like receptors (KIRs) and CD94/NKG2 receptors can signal in both positive and negative ways to regulate NK cell functions. The inhibitory receptors are the best characterized, but even in these cases much of their functional biology remains elusive. In this review, some recent advances in terms of the three-immunoglobulin (3Ig)-domain KIRs are discussed. Natural cytotoxicity receptors (NCRs) are among the activatory receptors found on NK cells. While pathogen ligands for these receptors have been described, endogenous ligands remain elusive. NCRs and NKG2D, a receptor for stress-induced antigens, appear to play complementary functional roles in terms of NK cell activation. More recently described on NK cells are the Toll-like receptors. In particular, these receptors of the innate immune system allow NK cells to directly sense pathogen, and their ligation on accessory cells indirectly activates NK cells through cytokine production. It is becoming clear that none of these receptor systems functions in isolation and that it is the sum of the signals (which will reflect the pathogenic situation), in addition to the cytokine milieu, that will direct NK cell activation. The resulting cytotoxicity, cytokine production and direct cell-cell regulatory interactions with other cells of the immune system, for example dendritic cells, ultimately determine the role of the NK cell in the context of an overall immune response.     

DAP10- and DAP12-associated receptors in innate immunity

Summary:  The DAP10 and DAP12 signaling subunits are highly conserved in evolution and associate with a large family of receptors in hematopoietic cells, including dendritic cells, plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, natural killer cells, and some B and T cells. Some receptors are able to associate with either DAP10 or DAP12, which contribute unique intracellular signaling functions. Studies of humans and mice deficient in these signaling subunits have provided surprising insights into the physiological functions of DAP10 and DAP12, demonstrating that they can either activate or inhibit immune responses. DAP10- and DAP12-associated receptors have been shown to recognize both host-encoded ligands and ligands encoded by microbial pathogens, indicating that they play an important role in innate immune responses.     

Regulation of the mast cell response to the type 1 Fcε receptor

Summary:  The type I Fcε receptor (FcεRI) is one of the better understood members of its class and is central to the immunological activation of mast cells and basophils, the key players in immunoglobulin E (IgE)-dependent immediate hypersensitivity. This review provides background information on several distinct regulatory mechanisms controlling this receptor's stimulus–response coupling network. First, we review the current understanding of this network's operation, and then we focus on the inhibitory regulatory mechanisms. In particular, we discuss the different known cytosolic molecules (e.g. kinases, phosphatases, and adapters) as well as cell membrane proteins involved in negatively regulating the FcεRI-induced secretory responses. Knowledge of this field is developing at a fast rate, as new proteins endowed with regulatory functions are still being discovered. Our understanding of the complex networks by which these proteins exert regulation is limited. Although the scope of this review does not include addressing several important biochemical and biophysical aspects of the regulatory mechanisms, it does provide general insights into a central field in immunology.     

Immunoglobulin variable regions in molecules exhibiting characteristics of innate and adaptive immune receptors

The antigen combining sites of immunoglobulin (Ig) and T cell antigen receptors (TCRs), which are present in all jawed vertebrates, consist of a paired variable (V) domain heterodimer that exhibits varying degrees of germline- and extraordinarily high levels of somatically-derived variation. The near limitless variation in receptor specificity on the surface of individual lymphocytes is the basis for clonal selection in the adaptive immune response. A basic question arises as to whether or not there are other forms of immune-type receptors in vertebrates as well as in invertebrates that derive immune specificity through sequence differences in V domains. Our laboratory has discovered two such families of molecules, the novel immune-type receptors and the variable region-containing chitin-binding proteins. Both families of molecules encode V domains that share some characteristics of adaptive immune receptors but likely mediate innate functions. Presented at the First Robert A Good Society Symposium, St. Petersburg, FL 2006.     

Antimicrobial responses of teleost phagocytes and innate immune evasion strategies of intracellular bacteria

During infection, macrophage lineage cells eliminate infiltrating pathogens through a battery of antimicrobial responses, where the efficacy of these innate immune responses is pivotal to immunological outcomes. Not surprisingly, many intracellular pathogens have evolved mechanisms to overcome macrophage defenses, using these immune cells as residences and dissemination strategies. With pathogenic infections causing increasing detriments to both aquacultural and wild fish populations, it is imperative to garner greater understanding of fish phagocyte antimicrobial responses and the mechanisms by which aquatic pathogens are able to overcome these teleost macrophage barriers. Insights into the regulation of macrophage immunity of bony fish species will lend to the development of more effective aquacultural prophylaxis as well as broadening our understanding of the evolution of these immune processes. Accordingly, this review focuses on recent advances in the understanding of teleost macrophage antimicrobial responses and the strategies by which intracellular fish pathogens are able to avoid being killed by phagocytes, with a focus on Mycobacterium marinum.     

The expanding roles of ITAM adapters FcRγ and DAP12 in myeloid cells

Summary:  The adapter proteins DAP12 and FcRγ associate with a wide spectrum of receptors in a variety of innate immune cells to mediate intracellular signaling pathways when their cognate receptor is engaged. These adapter proteins are coupled to their receptors through charged residues within the transmembrane regions of the adapter and receptor. DAP12 and FcRγ contain specific protein domains (referred to as immunoreceptor tyrosine-based activation motifs) that serve as the substrates and docking sites for kinases, allowing amplification of intracellular signaling reactions. Recent research has broadened the repertoire of receptors that utilize these adapters for signaling to include not only novel immunoglobulin superfamily members but also cytokine receptors, integrins, and other adhesion molecules. There is abundant evidence that these multifunctional signaling adapters also mediate inhibitory activity, downmodulating signaling from Toll-like receptors and other heterologous receptors. In this review, we discuss the newly described receptors that utilize DAP12 and/or FcRγ adapters to modulate innate immune responses.