Nonspecific Cytotoxic Cells Activity Research Articles

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus)

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Costimulatory Molecules CD80/86 Trigger Non-Specific Cytotoxic Cell of Nile tilapia (Oreochromis niloticus) to Kill CIK Cells

The teleost non-specific cytotoxic cell (NCC), as the evolutionary precursors of NK cells, is an important cytotoxic cell population in the innate immune system of teleost. We have recently realized that costimulatory CD80/86 have conservation in structural and interactional features with its ligand CD28 in Nile tilapia (Oreochromis niloticus). However, the ability of CD80/86 to regulate NCC activity has not been fully investigated. In the present study, we first obtained the recombinant fusion CD80/86 protein from O. niloticus (rOn-CD80/86). Then, NCC incubation with rOn-CD80/86 resulted in a significant production of NCC effector cytokines, including tumor necrosis factor-alpha, cellular apoptosis susceptibility and NK-lysin. Furthermore, NCC treatment with rOn-CD80/86 could significantly improve the ability to kill kidney cells of Grass carp (CIK) and up-regulate the activities of caspase-1 and caspase-3 in CIKs. The yeast, two-hybrid assay showed that On-CD80/86 cannot directly interact with non-specific cytotoxic cell receptor protein-1 of O. niloticus (On-NCCRP-1). The single-cell RNA-Seq data of Nile tilapia head kidney lymphocytes analysis found On-CD28 did not exhibit expression on NCCs subsets. The above results suggest that costimulatory molecules On-CD80/86 is independent of On-NCCRP-1 and On-CD28 receptor in modulating NCC killing activity in vitro of Nile tilapia. The results also provide more insights into the mechanism of NCC activity regulation.

Expression changes of non-specific cytotoxic cell receptor (NCCRP1) and proliferation and migration of NCCs post-Nocardia seriolae infection in Northern Snakehead

Non-specific cytotoxic cells (NCCs) are essential to the cytotoxic cell-mediated immune response in teleost. The fish non-specific cytotoxic cell receptor protein 1 (NCCRP1) plays an important role as a membrane protein in the recognition of target cells and the activation of NCC. However, the roles of fish NCCs during pathogen infection require comprehensive studies. In this study, the coding sequence of northern snakehead (Channa argus) nccrp1 (Canccrp1) was cloned. Canccrp1 contains an open reading frame of 690 bp, encoding a peptide of 229 amino acids with a conserved F-box-associated domain (FBA) and proline-rich motifs (PRMs). Transcriptional expression analysis revealed that the constitutive expression of Canccrp1 was higher in the immune-related organs, such as liver, kidneys, and spleen. Moreover, mRNA and protein expression of Canccrp1 gradually increased in the spleen at 1–6 days post infection (dpi) with Nocardia seriolae, in addition to reaching peak expression in both the kidneys and liver at 2 dpi. A polyclonal antibody prepared against recombinant CaNCCRP1 effectively labeled NCCs in peripheral blood and different tissues. Then, immunofluorescence (IF) staining showed that the number of NCCs was significantly increased and showed a scattered distribution in the early stages of N. seriolae infection (2 and 4 dpi) before the forming of granulomas. At the late stages of N. seriolae infection (6 dpi), more NCCs migrated to preexisting granulomas, showing significant coaccumulation with N. seriolae. All these results clearly indicate the expression changes of CaNCCRP1, and the number and localization changes of NCCs post-N. seriolae infection, implying potential roles for fish NCCs in the antimicrobial infection process in fish.

Fish natural killer enhancing factor-A (NKEF-A) enhance cytotoxicity of nonspecific cytotoxic cells against bacterial infection

Natural killer enhancing factor (NKEF)-A/B is a member of Peroxiredoxin (Prxs) family, which is named for the function of enhancing NK cells activity. NKEF also plays essential roles in multiple physiology/pathology processes including inflammation regulation, cancer development and redox reactions. However, the regulatory effects of fish NKEF on immune cells remain largely unknown. In this study, the full-length cDNA of NKEF-A (Accession No. MK584553, designated as On-NKEF-A) was identified from Nile tilapia (Oreochromis niloticus). On-NKEF-A encoded a 198 amino acid peptide with molecular mass of 22.085 kDa. On-NKEF-A protein contained a typical 2-Cys family domain, two active sites (51aa and 172aa) that were conserved in mammals, birds, amphibians and fish. Phylogenetic analysis showed that On-NKEF-A had the closest relationship with Zebra mbuna (Maylandia zebra) NKEF. The On-NKEF-A transcription was present in all examined tissues or organs. And the relative high expression levels of On-NKEF-A was found in head kidney leucocytes and nonspecific cytotoxic cells (NCC). After Streptococcus agalactiae stimulation, On-NKEF-A was significantly up-regulated in head kidney, spleen, gill and skin. Also, On-NKEF-A was markedly induced post S. agalactiae, LPS and poly I:C stimulation in head kidney-derived NCC. Moreover, On-NKEF-A was mainly distributed in cytoplasm of fathead minnow cells (FHM cells). The further in vitro analysis found that the recombinant protein of On-NKEF-A (rOn-NKEF-A) could induce the expression of various molecular markers of B cells, macrophages and NCC, enhanced the cytotoxicity of NCC via increasing the effectors expression. The present data collectively indicate that On-NKEF-A participates in anti-bacterial immune response via regulating NCC activity, which will provide new ideas to further explore the defense mechanism of fish against bacteria.

Molecular cloning of NCCRP-1 gene from orange-spotted grouper (Epinephelus coioides) and characterization of NCCRP-1+ cells post Cryptocaryon irritans infection

Nonspecific cytotoxic cells (NCCs) are an important cytotoxic cell population in the innate teleost immune system. The receptor designated “NCC receptor protein 1” (NCCRP-1) has been reported to be involved in the recognition and activation of NCCs. In this study, the full-length cDNA of Epinephelus coioides NCCRP-1 (ecnccrp-1) was cloned. The open reading frame (ORF) of ecnccrp-1 is 699bp, encoding a 232 amino acid protein that includes proline-rich motifs at the N-terminus and is related to the F-box associated family. Although a bioinformatics analysis showed that EcNCCRP-1 had no signal peptide or transmembrane helices, a polyclonal antibody directed against recombinant EcNCCRP-1 efficiently labeled a membrane protein in the head kidney, detected with Western blot analysis, which indicated that the protein localized to the cell surface. RT-PCR showed that the constitutive expression of ecnccrp-1 was higher in the lymphoid organs, such as the trunk kidney, spleen, head kidney, and thymus, and lower in brain, heart, fat, liver, muscle, and skin. After infection with Cryptocaryon irritans, the transcription of ecnccrp-1 was analyzed at the infected sites (skin and gills) and in the systemic immune organs (head kidney and spleen). At the infected sites, especially the skin, ecnccrp-1 expression was upregulated at 6h post infection, reaching peak expression on day 3 post the primary infection. However, the expression patterns differed in the systemic immune organs. In the spleen, ecnccrp-1 was gradually increased in the early infection period and decreased sharply on day 3 post the primary infection, whereas in the head kidney, the transcription of ecnccrp-1 was depressed during almost the whole course of infection. An immunohistochemical analysis showed that EcNCCRP-1+ cells accumulated at the sites of infection with C. irritans. These results suggested that NCCs were involved in the process of C. irritans infection in E. coioides.

Effect of low concentration of endosulfan in vivo on cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus splenocytes

Effect of low concentration of endosulfan in vivo on cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus splenocytes

Evaluation of the immune response against immature viral particles of infectious pancreatic necrosis virus (IPNV): A new model to develop an attenuated vaccine

Infectious pancreatic necrosis virus (IPNV) is a worldwide problem affecting both freshwater and seawater fish.Vaccines developed against IPNV are not as efficient in the field as they are in tests. Moreover, research in the development of vaccines against IPNV has often shown that vaccines can stimulate the immune response of fish antibodies but do not protect efficiently against IPNV. In fact, sometimes dead infected fish show high antibody titers against IPNV. This suggests that the magnitude of total antibodies stimulated by the vaccine is not necessarily related to the level of protection against IPN, suggesting that a new method is needed to evaluate vaccine stimulation of the immune system. We propose in vitro evaluation of the non-specific cytotoxic cells (NCC) of the innate immune response, in addition to humoral specific response.Moreover, it is necessary to develop innovative methods to improve fish vaccines. In this work, IPNV replicative intermediaries (provirus) were used to inject rainbow trout fry, which is the most vulnerable state to IPNV. To evaluate the immune response triggered by this vaccine, NCC and total and neutralizing antibodies against IPNV and the provirus were determined. Results indicated that NCC activity in rainbow trout fry is triggered by IPNV infection. Both IPNV and the provirus stimulate humoral and NCC immune response in rainbow trout fry. Although the total antibodies triggered by the provirus were half of that triggered by IPNV infection, the number of neutralizing antibodies was similar in the two treatments. This suggests that the ratio of neutralizing antibodies is higher among the antibodies stimulated by provirons than among those stimulated by IPNV infection. Thus, immature provirus is sufficient to activate immune response and is a good candidate as an attenuated vaccine in rainbow trout fry. In addition, neutralizing antibodies, together with non-specific cytotoxic activity, are a more suitable strategy to evaluate new vaccines than humoral immune response alone.

The gene structure and expression of the non-specific cytotoxic cell receptor protein (NCCRP-1) in Atlantic cod ( Gadus morhua L.)

The non-specific cell receptor protein (NCCRP-1) serves an important function in target cell recognition and activation of non-specific cytotoxic cells in teleosts. Atlantic cod NCCRP-1 was identified in a suppression-subtractive cDNA library and NCCRP-1 from Atlantic salmon, rainbow trout, Japanese medaka and fathead minnow was found deposited in the GenBank as EST sequences. The predicted amino acid sequences of these receptors contain the characteristic functional domains representing NCCRP-1, and phylogenetic analyses support the identification of five NCCRP-1 orthologues. Cod NCCRP-1 is shorter and has a different intron/exon organization from the common carp and channel catfish counterparts, but shows high extent of conservation in NCCRP-1 signature motives. Quantitative real-time PCR analyses showed that the gene expression of cod NCCRP-1 was higher in the lymphoid organs, head kidney (90-fold) and spleen (30-fold), compared to the organ with lowest expression. NCCRP-1 gene expression was not induced by in vitro treatment of head kidney cells with polyinosinic polycytidylic acid (poly I:C) or lipopolysaccharide (LPS), or by in vivo injections with poly I:C or formalin killed Vibrio anguillarum. These results show that the cod NCCRP-1 gene is differentially expressed in organs, and that gene expression is not induced by the tested treatments.

Molecular characterization of the nonspecific cytotoxic cell receptor (NCCRP-1) demonstrates gilthead seabream NCC heterogeneity

Teleost fish NCCs (nonspecific cytotoxic cells) are thought to be the evolutionary precursors of the mammalian NK cells. A novel mechanism mediating the NCC-mediated cytotoxicity has been described in teleosts. Now, this NCC receptor protein-1 (NCCRP-1) was characterized in gilthead seabream. The NCCRP-1 full-length sequence contains 1036 bp with an open reading frame of 702 bp. A comparison of the predicted 233-amino acid protein with several fish orthologues indicates a highly conserved sequence containing the F-box associated (FBA) domain and proline-rich motifs (PRM) characteristics of this family. The phylogenetical tree shows that seabream NCCRP-1 belongs to the NCCRP subfamily within the FBA family of proteins. This is a single copy gene with a constitutive and ubiquitous expression as determined by RT-PCR and flow cytometry. The results show that lymphocytes, monocyte/macrophages and acidophilic granulocytes from lymphoid tissues express the receptor, both at gene and protein level. Immunofluorescence microscopic observations confirm the previous results. The implications of this receptor on seabream NCC activity are discussed.

The gene and promoter structure of non-specific cytotoxic cell receptor protein-1 (NCCRP-1) in channel catfish ( Ictalurus punctatus)

Non-specific cytotoxic cells (NCCs) are essential to the cytotoxic cell-mediated immune response in teleost. The fish non-specific cytotoxic cell receptor protein 1 (NCCRP1) plays an important role as a membrane protein in the recognition of target cells and the activation of NCC. However, the roles of fish NCCs during pathogen infection require comprehensive studies. In this study, the coding sequence of northern snakehead (Channa argus) nccrp1 (Canccrp1) was cloned. Canccrp1 contains an open reading frame of 690 bp, encoding a peptide of 229 amino acids with a conserved F-box-associated domain (FBA) and proline-rich motifs (PRMs). Transcriptional expression analysis revealed that the constitutive expression of Canccrp1 was higher in the immune-related organs, such as liver, kidneys, and spleen. Moreover, mRNA and protein expression of Canccrp1 gradually increased in the spleen at 1–6 days post infection (dpi) with Nocardia seriolae, in addition to reaching peak expression in both the kidneys and liver at 2 dpi. A polyclonal antibody prepared against recombinant CaNCCRP1 effectively labeled NCCs in peripheral blood and different tissues. Then, immunofluorescence (IF) staining showed that the number of NCCs was significantly increased and showed a scattered distribution in the early stages of N. seriolae infection (2 and 4 dpi) before the forming of granulomas. At the late stages of N. seriolae infection (6 dpi), more NCCs migrated to preexisting granulomas, showing significant coaccumulation with N. seriolae. All these results clearly indicate the expression changes of CaNCCRP1, and the number and localization changes of NCCs post-N. seriolae infection, implying potential roles for fish NCCs in the antimicrobial infection process in fish.

Natural cytotoxic cells of gilthead seabream: maximum percentage of lysis

The maximum percentage of lysis of head-kidney non-specific cytotoxic cells (NCC) against mammalian tumour cells (L1210 and K562) in the marine teleost gilthead seabream (Sparus aurata L.) was studied. The present data indicate the short period of time necessary for gilthead seabream NCC to form conjugates and deliver a lethal hit. The maximum percentage of lysis observed demonstrates that seabream NCC activity against L1210 tumour cells is faster than against K562 tumour cells. This kinetic parameter suggests that fish NCC show a less efficient cytotoxic activity than their mammalian counterparts.The possibility of applying theoretical treatments to systems consisting of lower vertebrate non-specific cytotoxic cells and tumour targets, similar to those applied to mammals, is considered, and the phylogenetic implications of our findings are discussed.

Activation of nonspecific cytotoxic cells (NCC) with synthetic oligodeoxynucleotides and bacterial genomic DNA: Binding, specificity and identification of unique immunostimulatory motifs

We have analyzed the effects of synthetic oligodeoxynucleotides (sODNs) and bacterial DNA (bDNA) on the in vitro activation of NCC. Teleost NCC recognition of DNA appeared to differ from that which occurs in higher vertebrates. NCC contain at least two different receptor specificities for DNA. Both oligodeoxyguanosine 20-mers (dG20) and 5′-TGCTGCTTGTGCTTGTGCTT-3′ (4GC-2T) bound specifically to NCC. The existence of different receptor specificities was indicated by reciprocal cold target inhibition experiments. dG20 competed with 4GC-2T binding but sODNs composed of GpC or CpG nests did not compete with recognition by NCC of the dG20. ODN binding by NCC primarily depended on the presence of GpC or CpG nests with a preference for -G- serving as the anchor nucleotide. Secondarily, and similar to models of ODN activation in mammals, palindrome sequences of pu–pu-CpG-py–py activated NCC cytotoxicity. Additional analysis of the requirements for ODN activation indicated that guanosine could not substitute for adenosine as a purine spacer and that CpG motifs containing flanking thymidine (i.e.-GTCpGTT-) augmented the activity of the sODN containing this flanking base. Other evidence for the participation of both G and C in the recognition of specific nucleotides by NCC was that poly-dC20, dA20 or dT20 had no activating properties. Methylation of all cytosine nucleotides within an ODN abrogated activation. A canonical ODN motif of 5′-C/AT/AGCTT-3′ can now be suggested for teleosts. Additional studies were done to examine the effects of in vitro treatment of NCC with bDNA. bDNA from three different disease isolates of Streptococcus iniae activated NCC cytotoxicity. Treatment of the bDNA with DNase abrogated the enhancement of cytotoxicity. Also, treatment of NCC with eukaryotic DNA had no effects on cytotoxicity. These studies suggested that NCC recognize bacterial nonmethylated DNA. The consequences of these interactions may be increased innate and acquired anti-bacterial immunity.

The non-specific cytotoxic cell receptor (NCCRP-1): molecular organization and signaling properties

The evolutionary precursor to mammalian natural killer cells in teleost fish is called non-specific cytotoxic cells (NCC). NCC collaborate with other non-specific effector mechanisms to provide innate resistance during acute stress responses. The NCC receptor protein (NCCRP-1) contains 238 amino acid residues and is believed to be a type III membrane protein with three distinct functional domains. The antigen-binding domain has been mapped to amino acids nos. 104–119. The intracellular C-terminus contains a high concentration of potential phosphorylation sites (Y, S, T). Indeed, we have shown that activation of NCC by crosslinking of NCCRP-1 leads to receptor tyrosine and serine phosphorylation. The N-terminus of the molecule is also inside the cells and has as well signature amino acids, proline-rich motifs (PRM), that are indicative of functional relevance. The cytokine/hormone receptor-like PRMs are known docking sites for JAK kinases. We have evidence that following activation, NCCRP-1 comes in contact with JAK kinase and as a result of this interaction, STAT 6 is translocated into the nucleus. These results suggest that NCCRP-1 may play a dual role in the activation of NCC: first, as an antigen recognition molecule necessary for target cell lysis, and second, as an initiator of cytokine release from NCC. Both of these processes are required for a competent innate immune response.

Phosphorylation-induced activation of tilapia nonspecific cytotoxic cells by serum cytokines.

Cytokines as soluble mediators of immunity are important in understanding immunological mechanisms against infectious organisms and during stress conditions. In the present study, the role of protein tyrosine phosphorylation is assessed in the activation of nonspecific cytotoxic cells (NCC) from tilapia Oreochromis niloticus by cytokine-like serum factors. NCC are the teleost equivalent of mammalian natural killer (NK) cells. In teleost fish, NCC are important mediators of innate immunity against bacterial and parasite insult and tumor growth. We have previously shown that exposure of tilapia (a tropical fish) to cold water temperatures (3 to 5 min at 5 to 10 degrees C) produces physiological stress responses characterized by immediate phenotypic and immunological changes. The serum obtained from stressed tilapia contains a 'stress activating serum factor' (SASF) which passively increases in vitro naive NCC cytotoxicity 2- to 4-fold over control levels. In an effort to identify the mechanisms of activation of cytotoxicity by SASF, the phosphorylation status of tyrosine residues in proteins from treated NCC was determined. NCC were incubated with heat-inactivated or untreated stress serum and Western blots of the cell lysates were probed with anti-phosphotyrosine monoclonal antibodies (mabs). The levels of tyrosine phosphorylation in several proteins of the SASF-activated NCC were higher than in control cells. Increased tyrosine phosphorylation was also induced by incubation of NCC in the presence of the tyrosine phosphatase inhibitor Na orthovanadate (vanadate). In every case, an increase in phosphorylation status shown by Western blotting was correlated with increases in cytotoxic activity of NCC against HL-60 target cells. The enzyme inhibitor Herbimycin A (HA) has been previously used to inhibit the activity of the src-family of tyrosine kinases. In the present study, a 4 h pretreatment of NCC with HA (2 microM), followed by treatment with SASF blocked the activation of cytotoxicity produced by SASF. These results suggested that activation of NCC by cytokine-like factors is mediated through activation of the src family of protein tyrosine kinases. Activation was associated with increased phosphorylation and higher cytotoxic effector functions.

In vivoactivation of tilapia nonspecific cytotoxic cells byStreptococcus iniaeand amplification with apoptosis regulatory factor(s)

An important component of immediate innate responses of tilapia to stress is the release within minutes of soluble cytokine-like substances into the peripheral circulation. These cytokine-like stress factors bind nonspecific cytotoxic cells (NCC) and produce 3–4-fold increased cytotoxicity. In the present study, the in vivo responses of tilapia NCC following injection with different isolates of intact killed Streptococcus iniae was investigated. Activated cytotoxicity of NCC in the peripheral blood (PB) was produced by increased specific activity of resident cells rather than increased numbers. Tilapia injected intravenously (i.v.) with killed S. iniae produced different cytotoxicity responses compared to fish injected intraperitoneally (i.p.). In the spleen (S) and anterior kidney (AK), there was no correlation between S. iniae isolate and cytotoxicity response at 4, 8 or 24h following i.p. injection. The NCC response following i.v. injection of killed bacteria was different. Within minutes following i.v. injection, NCC cytotoxicity from the PB increased 100% compared to naı̈ve controls. The existence of subsets of differentiated NCC in the PB was suggested because i.v. injection had no amplification effects on NCC from the AK or S. Likewise, NCC from the PB only appeared to exhibit a degree of antigen specificity.S. iniae strain #173 produced activation of cytotoxicity compared to isolates #164 and ATCC. Evidence for soluble factor (cytokine?) involvement in increased cytotoxicity was obtained by passive activation of NCC with serum from #173 (i.v.) injected fish. Incubation of this serum with control (naı̈ve) NCC produced large increases in the cytotoxicity of labelled HL-60 target cells. Similarly obtained serum from fish injected with ATCC and #164 isolates had no amplification activity. Studies were also performed to study the mechanism(s) of passive activation. Flow cytometric analysis revealed that NCC from the S, AK and PB constitutively expressed cytosolic (not membrane) FasL. Stress serum treated NCC obtained from the peripheral blood produced an increase in the expression of FasL, CAS and FADD by Western blot examination. These data indicated that cytokine like factors in the serum of stressed tilapia activate increased NCC cytotoxicity(possibly) by stimulating the expression of proteins involved in activation of programmed cell death.

Activation-Induced Programmed Cell Death of Nonspecific Cytotoxic Cells and Inhibition by Apoptosis Regulatory Factors

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian lymphokine-activated natural killer cells. The cytotoxic activities of NCC are enhanced by stress-activated serum factors (SASF) present in tilapia acute-phase serum. In the present study purified NCC and xenogeneic target HL-60 tumor cells and nuclei were distinguishable in mixtures determined by flow cytometry. NCC activated by target HL-60 cells undergo activation-induced programmed cell death (AIPCD) during 12- to 16-h killing assays as shown by Annexin-V binding and nuclear DNA fragmentation results. Annexin-V binding studies also demonstrated that NCC kill HL-60 cells by an apoptotic mechanism. NCC are protected from AIPCD by 4-h preincubation in 50% SASF. Pretreatment also produced more than a fourfold increase in NCC cytotoxicity (effector/target (E:T) ratio = 100:1). In the absence of SASF preincubation, the percentage of apoptotic NCC increased from 8 to 91% at E:T ratios of 1:0 and 1:1, respectively. Kinetic studies (E:T = 10:1) demonstrated that the percentage of NCC exhibiting HL-60-dependent AIPCD increased between 0.1 and 12 h and then decreased inversely with total cell necrosis over the next 60 h. Preincubation of NCC with SASF protected NCC from AIPCD for over 72 h. Crosslinkage of the NCCRP-1 receptor with monoclonal antibody (mab) 5C6 produced AIPCD between 1 and 100 μg/mL mab concentrations. Preincubation with SASF completely protected NCC from mab 5C6-dependent AIPCD. SASF-mediated protection of NCC from AIPCD was dependent upon divalent cations, as demonstrated by increases in DNA hypoploidy of 38, 67, and 88% following preincubation in the presence of 10, 100, and 1000 μM EDTA, respectively. SASF also protected NCC from glucocorticoid- (i.e., dexamethasone) induced apoptosis. Combined, these results demonstrated that NCC activity is down-regulated by AIPCD. Release of SASF into the peripheral circulation may prevent negative regulation of NCC by AIPCD by increasing recycling capacity. Results are discussed in the context of the effects of acute stressors on innate immunity.

Regulation of innate immunity in tilapia: activation of nonspecific cytotoxic cells by cytokine-like factors

Exposure of tilapia ( Oreochromis niloticus) to water temperatures of 10–15°C for 3–5 min produces physiological stress responses characterized by immediate phenotypic and immunological changes. In the present study, this general stress response was utilized as a model system to study innate immunity mediated by soluble factors and cytotoxic cells. Acute innate cytotoxic responses of nonspecific cytotoxic cells (NCC) in the peripheral blood (PBL), anterior kidney (AK) and spleen (SPL) were measured. Following temperature stress, the levels of NCC activity depended on the presence of soluble factors and on the cell compartments from which the NCC were obtained. NCC from PBL of stressed tilapia had 30× or greater cytotoxic activity compared to nonstressed PBLs from controls. NCC activity from the AK and SPL of stressed tilapia was lower than controls. Flow cytometric analysis of NCC in each tissue showed that increased cytotoxicity was not produced by increased numbers of NCC. To determine the mechanism of amplification of cytotoxicity, NCC from nonstressed tilapia were passively treated with serum from temperature stressed tilapia. Serum containing the “stress activated serum factor” (SASF) passively increased naive NCC cytotoxicity (from PBL) 3–4 fold. The cytotoxic cell response was inhibited by addition of anti-NCC monoclonal antibody 5C6. These data indicated that NCC are (at least one of) the target cells for SASF. SASF required only 15 min pre-incubation with naive NCC to activate cytotoxicity. Activation was nonreversible and concentration dependent. Pretreatment of NCC with SASF reduced the assay time required to amplify target cell cytotoxicity from 12–24 h to 6 h. SASF amplification of NCC cytotoxicity was not restricted by different histological types of target cells. Determination of select physical/chemical properties of SASF revealed: complete heat inactivation of cytotoxicity amplification following 55°C and 65°C pretreatment; SASF was thermostable at room temperature to 45°C for 15 min; and freeze-thaw treatment reduced but did not completely remove amplification activity. The molecular weight range of SASF activity was identified in a 50–100 kDa fraction obtained by differential dialysis. SASF appears to be a protein sensitive to trypsin digestion.

Natural cytotoxic activity of gilthead seabream ( Sparus aurata L.) leucocytes : Assessment by flow cytometry and microscopy

This paper describes an easy and sensitive flow cytometric assay for assessing the non-specific cytotoxic activity of gilthead seabream ( Sparus aurata L.) head-kidney leucocytes against tumor target cells. Concomitantly, the cytotoxic process and the cell types involved were microscopically studied. The assay was based on the consecutive use of two fluorochromes. The targets were preincubated with 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) and then mixed with effectors. At the end of incubation time propidium iodide (PI) was added. While live effectors were non-fluorescent, live and dead targets retained the DiO (green) fluorescence and non-viable targets and effectors showed PI (red) fluorescence staining. The kinetics of the cytotoxic activity was studied from 10 to 240 min. Lymphocytes, monocyte-macrophages and granulocytes showed non-specific cytotoxic activity, as demonstrated by light and electron microscopy. In conclusion, the technique presented validates the effectiveness of a dual-color flow cytometric assay for assessing the activity of non-specific cytotoxic cells in fish.

In vivo modulation of innate resistance to Edwardsiella ictaluri with a phosphatase inhibitor.

Catfish were treated with the protein phosphatase inhibitor sodium orthovanadate (vanadate) and challenged with the pathogen Edwardsiella ictaluri to investigate the relationship between the in vivo immunoregulatory effects of tyrosine and serine phosphatases on nonspecific modulation of resistance to bacterial infections. Two different infection protocols were used: fish were pretreated by immersion in vanadate and subsequently infected (by immersion) with 1 LD100 E. ictaluri, or fish were injected (intraperitoneally, i.p.) with bacteria and simultaneously treated (by immersion) with 25 microM vanadate. In the absence of vanadate, both infection models produced fulminant infection by 10 or 6 d, respectively. Zero to 48 h treatment with vanadate (by immersion) prior to infection produced 17 to 100% survival of infected fish. In addition to augmentation of inmate immunity, vanadate enhanced acquired immunity to this pathogen. Fish which had vanadate-induced resistance to primary infection were 'immune' to secondary challenge with a LD100 of E. ictaluri. Experiments were done to determine the mechanism(s) of the altered innate resistance. Catfish were injected (i.p.) with E. ictaluri and simultaneously treated (by immersion) with 25 microM vanadate. Assays were done to measure nonspecific cytotoxic cell (NCC) activity at 0, 24, 48, 72 and 96 h post-infection/vanadate treatment. Increased NCC activity at 48 to 96 h post-infection appeared to correlate with resistance to bacterial related mortality. These data indicated that in vivo vanadate treatment of catfish significantly increased resistance to otherwise fulminant E. ictaluri infections. This effect coincided with the initiation of resistance to secondary infections without additional vanadate treatments. Vanadate-modulated resistance in catfish may be associated with augmented NCC activity.

Suppression of natural cytotoxic cell and macrophage phagocytic function in oyster toadfish exposed to 7,12-dimethylbenz[a]anthracene

Studies were undertaken to determine the effects of the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) on the function of oyster toadfish peritoneal non-specific cytotoxic cells (NCC) and macrophages. The functioning of these two cell populations was assessed in terms of their ability to lyse cultured tumour target cells and to phagocytose yeast cells, respectively. Following intraperitoneal (i.p.) injection of 0, 1, 10, 50 and 100 mg DMBA/kg body weight, macrophage activity was suppressed in essentially a linear fashion, whereas NCC activity was virtually obliterated at all doses of DMBA administered. Thus, non-specific cytotoxicity in oyster toadfish has been shown to be substantially more sensitive to DMBA than non-specific phagocytosis.