硬骨鱼新型免疫球蛋白的研究进展

鱼类是最早出现免疫球蛋白的动物,鱼类免疫球蛋白在鱼类的特异性体液免疫应答中发挥重要的作用。一直以来,人们认为在硬骨鱼中仅存在IgM和IgD两种免疫球蛋白,而2005年以来,陆续在斑马鱼、虹鳟鱼及鲤鱼等硬骨鱼中发现了新型免疫球蛋白,分别命名为IgZ、IgT及IgM-IgZ等。这些新型免疫球蛋白不仅在基因结构上很特别,而且呈现出多样性,在不同种的硬骨鱼中的功能也不完全相同,同一种鱼中的IgT也呈现多样性。虽然目前对于鱼类新型免疫球蛋白的研究刚刚起步,对其功能了解较少,但有研究表明IgT在硬骨鱼的粘膜免疫中发挥重要的作用,且认为它与IgA是同源的。该文拟对硬骨鱼中发现的新型免疫球蛋白的结构特点、基因组成和分布模式及功能差异作一简要综述。     

Teleost intestinal immunology

Teleosts clearly have a more diffuse gut associated lymphoid system, which is morphological and functional clearly different from the mammalian GALT. All immune cells necessary for a local immune response are abundantly present in the gut mucosa of the species studied and local immune responses can be monitored after intestinal immunization. Fish do not produce IgA, but a special mucosal IgM isotype seems to be secreted and may (partly) be the recently described IgZ/IgT. Fish produce a pIgR in their mucosal tissues but it is smaller (2 ILD) than the 4–5 ILD pIgR of higher vertebrates. Whether teleost pIgR is transcytosed and cleaved off in the same way needs further investigation, especially because a secretory component (SC) is only reported in one species. Teleosts also have high numbers of IEL, most of them are CD3-?⁺/CD8-α⁺ and have cytotoxic and/or regulatory function. Possibly many of these cells are TCRγδ cells and they may be involved in the oral tolerance induction observed in fish. Innate immune cells can be observed in the teleost gut from first feeding onwards, but B cells appear much later in mucosal compartments compared to systemic sites. Conspicuous is the very early presence of putative T cells or their precursors in the fish gut, which together with the rag-1 expression of intestinal lymphoid cells may be an indication for an extra-thymic development of certain T cells. Teleosts can develop enteritis in their antigen transporting second gut segment and epithelial cells, IEL and eosinophils/basophils seem to play a crucial role in this intestinal inflammation model. Teleost intestine can be exploited for oral vaccination strategies and probiotic immune stimulation. A variety of encapsulation methods, to protect vaccines against degradation in the foregut, are reported with promising results but in most cases they appear not to be cost effective yet. Microbiota in fish are clearly different from terrestrial animals. In the past decade a fast increasing number of papers is dedicated to the oral administration of a variety of probiotics that can have a strong health beneficial effect, but much more attention has to be paid to the immune mechanisms behind these effects. The recent development of gnotobiotic fish models may be very helpful to study the immune effects of microbiota and probiotics in teleosts.     

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.     

Immunological Characterization of the Teleost Adipose Tissue and Its Modulation in Response to Viral Infection and Fat-Content in the Diet

The immune response of the adipose tissue (AT) has been neglected in most animal models until recently, when the observations made in human and mice linking obesity to chronic inflammation and diabetes highlighted an important immune component of this tissue. In the current study, we have immunologically characterized the AT for the first time in teleosts. We have analyzed the capacity of rainbow trout (Oncorhynchus mykiss) AT to produce different immune mediators and we have identified the presence of local populations of B lymphocytes expressing IgM, IgD or IgT, CD8α⁺ cells and cells expressing major histocompatibility complex II (MHC-II). Because trout AT retained antigens from the peritoneal cavity, we analyzed the effects of intraperitoneal infection with viral hemorrhagic septicemia virus (VHSV) on AT functionality. A wide range of secreted immune factors were modulated within the AT in response to VHSV. Furthermore, the viral infection provoked a significant decrease in the number of IgM⁺ cells which, along with an increased secretion of IgM in the tissue, suggested a differentiation of B cells into plasmablasts. The virus also increased the number of CD8α⁺ cells in the AT. Finally, when a fat-enriched diet was fed to the fish, a significant modulation of immune gene expression in the AT was also observed. Thus, we have demonstrated for the first time in teleost that the AT functions as a relevant immune tissue; responsive to peritoneal viral infections and that this immune response can be modulated by the fat-content in the diet.     

The immune response in fish: a review

The literature relating to the immune response of fish has been reviewed. Non-specific immune mechanisms similar to those of other vertebrate classes occur in fish. Similarly specific cell-mediated immunity has been demonstrated at all levels of evolution, from the cyclostomes to the teleosts. A humoral antibody system also occurs in all classes offish but varies considerably in relation to phylogenetic status. In the cyclostomes, only immuno-proteins with properties intermediate between the immunoglobulms of vertebrates and the non-specific agglutinins and lysins of invertebrates have been demonstrated. In the elasmo-branchs and chondrosteans 7 and 19s irnmunoglobulins of IgM type occur. In holosteans the 19s form is predominant whereas in teleosts, 7 and 19s forms occur, with some evidence of specialization in the 7s form. In the phylogenetically most advanced fish, the Dipnoi, two immunoglobulin classes, structurally analogous to IgM and IgG, have been described.
A characteristic feature of both cell-mediated and antibody mediated immune responses in fish is their dependence upon environmental temperature. There is also evidence that, in some species at least, nutritional factors and behaviour patterns may also influence the immune response.
Attempts at artificial immunization of fish against infectious disease have met with varied success. It is probable that better results could be achieved with live vaccine strains, particularly if applied under conditions optimal for the immune responses.     

Systemic and mucosal immunity induced by oral somatic transgene vaccination against glycoprotein B of pseudorabies virus using live attenuated Salmonella typhimurium

Glycoprotein B mediates the absorption and penetration of the pseudorabies virus in the form of an immunodominant Ag, and represents a major target for the development of new vaccines. This study evaluated the efficiency of live attenuated Salmonella typhimurium SL7207 for the oral delivery of DNA vaccine encoding the pseudorabies virus glycoprotein B (pCI-PrVgB) in vivo, leading to the generation of both systemic and mucosal immunity against the pseudorabies virus Ag. An oral transgene vaccination of pCI-PrVgB using a Salmonella carrier produced a broad spectrum of immunity at both the systemic and mucosal sites, whereas the intramuscular administration of a naked DNA vaccine elicited no mucosal immunoglobulin (Ig)A response. Interestingly, the Salmonella-mediated oral transgene vaccination of the pseudorabies virus glycoprotein B biased the immune responses to the Th2-type, as determined by the IgG2a/IgG1 ratio and the cytokine production profile. However, oral vaccination mediated by Salmonella harbouring pCI-PrVgB showed inferior protection to systemic immunization against virulent pseudorabies virus infection. The expression of transgene delivered by Salmonella bacteria in antigen-presenting cells of both the systemic and mucosal-associated lymphoid tissues was further demonstrated. These results highlight the potential use of live attenuated S. typhimurium for an oral transgene pseudorabies virus glycoprotein B vaccination to induce broad immune responses.     

Hidden talents of natural killers: NK cells in innate and adaptive immunity

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells and producing immunoregulatory cytokines. Herein, we discuss recent studies that indicate that NK cells span the conventional boundaries between innate and adaptive immunity. For example, it was recently discovered that NK cells have the capacity for memory‐like responses, a property that was previously thought to be limited to adaptive immunity. NK cells have also been identified in multiple tissues, and a subset of cells that specialize in the production of the T_H17 cytokine IL‐22, NK‐22s, was recently described in mucosal‐associated lymphoid tissue. Finally, we review work that shows that NK cells develop at sites that were traditionally thought to be occupied only by adaptive immune cells, including the thymus and lymph nodes.     

NALT- versus Peyer's-patch-mediated mucosal immunity

Recent studies indicate that the mechanism of nasopharynx-associated lymphoid tissue (NALT) organogenesis is different from that of other lymphoid tissues. NALT has an important role in the induction of mucosal immune responses, including the generation of T helper 1 and T helper 2 cells, and IgA-committed B cells. Moreover, intranasal immunization can lead to the induction of antigen-specific protective immunity in both the mucosal and systemic immune compartments. Therefore, a greater understanding of the differences between NALT and other organized lymphoid tissues, such as Peyer's patches, should facilitate the development of nasal vaccines.     

Modulation of immunity against herpes simplex virus infection via mucosal genetic transfer of plasmid DNA encoding chemokines

In this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1beta (MIP-1beta) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4(+) T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1alpha, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1alpha appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4(+) T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.     

Synthesis and characterization of membrane immunoglobulin, Ia, and H-2 molecules of thy 1+ AKR/J lymphomas.

Three AKR lymphomas displaying B cell and T cell characteristics have been described. Because of the proclivity of normal AKR/J mice to develop T cell lymphomas, and the rarity of lymphomas with dual characteristics, the B cell markers of these tumors were studied more intensively. Fluorescence data with class-specific anti-immunoglobulin reagents demonstrated that the tumor cells stained only with class-specific anti-IgM reagents. Because of the possibility that the surface Ig was passively acquired and of reports that certain anti-mu-chain sera react with "IgT", chemical characterization of the immunoglobulin molecules was performed. Using 3H-leucine internal labeling, we showed that all three tumor lines synthesized the immunoglobulin found on their surface, and that the immunoglobulin had the chemical and immunologic characteristics most typical of monomeric surface IgM, and was composed of mu-chains and light chains. The Ia antigens found on these cells were also examined. These antigens were also synthesized by the cells and were present in the same molecular form and in the same approximate quantity as Ia antigens on normal spleen cells.     

Evaluation of non-specific immune components from the skin mucus of olive flounder (Paralichthys olivaceus)

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish under intensive aquaculture and at prolonged low temperatures from relevant infections due to inadequate adaptive immune responses. In the present study we aimed to understand the mucosal immunity of an economically important mariculture fish, olive flounder (Paralichthys olivaceus) by evaluating the immune components from its skin mucus. The activities of lysozyme (233.33+/-171.82 units mg(-1)), trypsin-like protease (42.84+/-1.249 units mg(-1)), alkaline phosphatase (0.376+/-0.005 units mg(-1)) and esterase (0.170+/-0.006 units mg(-1)) were detected in the skin mucus. Transferrin was identified by MALDI-TOF/MS analysis. ELISA and immunoblot assays using anti-flounder IgM monoclonal antibody showed the presence of a significant level (1.80+/-0.001, n=3) of monomer immunoglobulin M (IgM) with approximate molecular weight of 160 and 25 kDa under non-denaturing and denaturing states, respectively. Skin mucus showed strong antibacterial activity against tested fish pathogenic bacteria. In addition, skin mucus successfully agglutinated (HA titre 2(8)), but completely failed to haemolyse, rabbit erythrocytes. In conclusion, the major immune components of the skin mucus, identified in the present study, are possibly involved in the broad spectrum non-specific immunity of olive flounder.     

Fish immunoglobulins and the genes that encode them

The nature of fish antibodies (concentrating primarily on the most studied species of bony and cartilaginous fishes) is discussed in terms of their immunoglobulin biochemistry and immunobiology. The major serum immunoglobulin (IgM) is described in detail, and structural variants of IgM are discussed in terms of their distribution in different fish species, and different anatomical sites within a fish (e.g. blood, mucus, bile). Structural variation in IgM includes the size of the constituent heavy and light polypeptide chains, and the extent to which they are covalently associated with one another. The intramolecular heterogeneity of binding sites for antigen on IgM is reviewed and possible mechanisms for the phenomenon are presented. The evidence suggests that some, but not all, species of fish possess a detectable J chain in their IgM. The general nature of the fish immune response is that IgM antibody of moderate affinity is produced and prolonged or repeated immunization: (a) fails to produce a switch to production of a non-IgM class of antibody, and (b) fails to induce substantial increases in the affinity of the specific antibodies. Evidence supports a conclusion that fish lack the typical secondary antibody response seen in mammals, and possess antibodies of limited heterogeneity. Our current understanding of the genetic basis for fish antibodies is presented. Fish appear to utilize the same basic genetic elements as mammals to encode and regulate the expression of their immunoglobulins. The teleost heavy chain (IgH) locus resembles that of mammals and amphibia in its organization. The IgH locus of elasmobranchs is arranged in a unique multicluster organization. The light chain loci of elasmobranchs are organized analogously to the heavy chain locus (in multiclusters). The structure of the light chain locus of teleosts is presently unknown. Teleost fish utilize a unique pattern of RNA processing to generate the secreted and membrane receptor forms of the IgM heavy chain. The genes encoding the unique low molecular weight Ig heavy chain found in skates and rays have been cloned and sequenced, and also display the multicluster pattern of organization. Teleost fish appear to have normal numbers of variable regions: it is hypothesized (but as yet unproven) that the failure of their IgM to increase in affinity is due to a deficiency of somatic hypermutational mechanisms in their Ig gene variable regions during B lymphocyte differentiation.     

Plasmacytoid dendritic cells and the regulation of immunoglobulin heavy chain class switching

By substituting the heavy chain constant region of IgM and IgD with that of IgG, IgA or IgE, immunoglobulin class switching endows antibodies with novel effector functions that enhance the ability of the immune system to effectively clear invading pathogens. Plasmacytoid dendritic cells critically link innate immunity with adaptive immunity by producing massive amounts of type 1 IFN in response to viruses. We have recently found that type 1 IFN triggers class switching by inducing myeloid dendritic cells to upregulate the expression of BAFF and APRIL, two powerful B cell-activating molecules. In this paper, we propose that IFN-producing plasmacytoid dendritic cells modulate class switching by activating B cells through both T cell-dependent and T cell-independent pathways. A better understanding of these pathways may facilitate the development of novel antiviral vaccine strategies and aid in identifying new therapies for antibody-mediated autoimmune disorders, such as lupus.     

The early postnatal development of salivary antibody and immunoglobulin response in children orally colonized with a nonpathogenic,probiotic strain of<Emphasis Type="Italic">E. coli</Emphasis>

The development of levels of secretory immunoglobulins (SIgs) in newborns' saliva was examined under physiological conditions and after artificial colonization with nonpathogenic, probiotic bacterial strain E. coli O83. Higher levels of secretory immunoglobulin M (SIgM) and secretory immunoglobulin A (SIgA) were detected in the saliva of breast-fed children when compared with those of bottle-fed infants. SIgM was found earlier than SIgA, the levels of both SIgM and SIgA decreased after weaning. Breastfeeding actively stimulates local immunity on mucosal membranes of newborn infants. Early mucosal colonization with nonpathogenic E. coli bacteria stimulates the mucosal immune system to produce specific antibodies as well as nonspecific secretory immunoglobulins.     

Characterisation of mucosal and systemic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance

Mucosal and systemic antibody production in rainbow trout, Oncorhynchus mykiss (Walbaum), was evaluated following different antigen delivery routes. A BIAcore instrument (Pharmacia) allowed direct detection of antibody-antigen interactions by surface plasmon resonance changes. These interactions were measured in real-time without secondary reagents or extraneous labels. Groups of rainbow trout were immunised with a hapten-carrier antigen consisting of fluorescein isothiocyanate (FITC) conjugated to keyhole limpet haemocyanin (KLH) or phosphate buffered saline (PBS) pH 7.2. Antigens were administered intraperitoneally (i.p.) with or without Freund's complete adjuvant (FCA) or peranally (p.a.) directly to the gastrointestinal (GI) tract. Serum and mucosal anti-FITC responses were significantly (P<0.05) higher in FITC-KLH/FCA groups, clearly showing that adjuvant incorporation enhances mucosal as well as sytemic immunity. Antigen uptake and processing in fish immunised p.a. and i.p. without adjuvant was much less efficient and resulted in relatively low levels of serum and mucosal antibody production. Interestingly, mucosal responses in these groups peaked prior to serum responses suggesting possible early stimulation of mucosal defences. Mucosal antibody production in fish receiving FITC-KLH/FCA correlated more closely with serum responses, indicating possible transfer of serum derived antibody to mucosal sites. Mucosal and serum responses were confirmed as immunoglobulin (Ig) by subsequent reactivity with an anti-trout serum IgM monoclonal antibody (1.14) passed over flow cells containing anti-FITC antibodies. Further analysis showed significantly lower (P<0.05) reactivity of early mucus anti-FITC components (4 weeks post-immunisation) to 1.14. Purified serum and mucus Ig from non-immunised fish showed different protein banding patterns by SDS-PAGE under reducing conditions. Immunoblotting with 1.14 also showed weak reactivity to mucus Ig in control fish while reacting strongly to mucus Ig from immunised fish. These data suggest that early mucosal responses in trout may consist of heterogeneous forms of Ig differing in characteristics to serum Ig. BIAcore analysis in this context and as a means of measuring antibody response proved useful, and has the potential to become a valuable new tool in the study of fish immunology.     

Gene gun-mediated DNA immunization primes development of mucosal immunity against bovine herpesvirus 1 in cattle

Vaccination by a mucosal route is an excellent approach to the control of mucosally acquired infections. Several reports on rodents suggest that DNA vaccines can be used to achieve mucosal immunity when applied to mucosal tissues. However, with the exception of one study with pigs and another with horses, there is no information on mucosal DNA immunization of the natural host. In this study, the potential of inducing mucosal immunity in cattle by immunization with a DNA vaccine was demonstrated. Cattle were immunized with a plasmid encoding bovine herpesvirus 1 (BHV-1) glycoprotein B, which was delivered with a gene gun either intradermally or intravulvomucosally. Intravulvomucosal DNA immunization induced strong cellular immune responses and primed humoral immune responses. This was evident after BHV-1 challenge when high levels of both immunoglobulin G (IgG) and IgA were detected. Intradermal delivery resulted in lower levels of immunity than mucosal immunization. To determine whether the differences between the immune responses induced by intravulvomucosal and intradermal immunizations might be due to the efficacy of antigen presentation, the distributions of antigen and Langerhans cells in the skin and mucosa were compared. After intravulvomucosal delivery, antigen was expressed early and throughout the mucosa, but after intradermal administration, antigen expression occurred later and superficially in the skin. Furthermore, Langerhans cells were widely distributed in the mucosal epithelium but found primarily in the basal layers of the epidermis of the skin. Collectively, these observations may account for the stronger immune response induced by mucosal administration.