Cell Activation In Mice Research Articles

Inhibition of class II trans-activator function by HIV-1 tat in mouse cells is independent of competition for binding to cyclin T1

The Tat trans-activator protein from HIV-1 inhibits the function of the class II trans-activator protein (CIITA), resulting in reduced MHC class II gene transcription in human cells. Tat does so by competing with CIITA for binding to cyclin T1, a component of the transcriptional elongation complex PTEFb. Since Tat does not functionally interact with mouse cyclin T1, we decided to examine the ability of Tat to inhibit CIITA in mouse cells. We found that Tat inhibited CIITA activity in mouse cells though this inhibition was independent of cyclin T1. The inhibition required the transcriptional activation domain of CIITA, but did not involve alterations in MHC class II promoter occupancy. Although Tat blocked the interaction between CIITA protein and human cyclin T1, it had no effect on the binding between CIITA and mouse cyclin T1. Therefore, Tat can inhibit the ability of CIITA to activate transcription of MHC class II genes in mouse cells by a mechanism that appears to be distinct from that proposed for human cells.

Natural Killer Cell Activity in Mice after Intraperitoneal Administration of Beryllium Chloride

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the blood, spleen, lymph nodes and abdominal cavity of humans and animals. There are asialo GM1 antigens as well as many other antigens on the surfaces of their membranes, particularly in mice. As NK cells are supposed to play an important role in protecting the living body against the development of tumors and viral infections, they are regarded as one of the immunological parameters for the defense mechanism of the body. Beryllium (Be) is reported to be carcinogenic to humans and animals. The carcinogenic mechanisms of Be, however, have not yet been studied in relation to NK cell activities. The aim of this study is to examine the influence of Be on NK cells to seek a clue to the carcinogenic mechanisms of Be.

Effects of Rotational Stress of Different Duration on NK Cell Activity, Proinflammatory Cytokines, and POMC-Derived Peptides in Mice

Previous studies have shown that the same stressor, depending on intensity, controllability, or duration, can have different effects on the immune system. The purpose of this study was to determine the effect of 10- and 20-min rotation on natural killer (NK) cell activity and also to establish if changes in body temperature, proinflammatory cytokine (IL-1β, IL-6, and TNF-α) levels, and proopiomelanocortin (POMC)-derived peptide (ACTH and β-endorphin) levels parallel the changes in NK cell activity in mice. We found that 10-min rotation significantly increased NK cell activity as compared to both the control (home cage) group and the 20-min-rotation group, while NK cell activity in the 20-min group was not significantly changed compared to the control group. Both 10 and 20 min of rotational stress decreased body temperature and induced significant changes in the proinflammatory cytokine and POMC-derived peptide levels as compared to the control group. The pattern of proinflammatory cytokine expression was quite different between the 10- and 20-min rotation groups. All three proinflammatory cytokines were expressed sequentially (at 0 h after rotation TNF-α, at 6 h IL-1β and IL-6, and at 24 h IL-6) in the 10-min rotation group, while the 20-min rotation group had a small increase in IL-1β (6.7 ± 1.8 pg/ml) at 0 h and increased levels of IL-6 at 6 and 24 h. There was a dissociation of ACTH and β-endorphin expression in both groups resulting in significantly more β-endorphin (p < 0.05) in the 10-min group at 6 h and significantly more ACTH (p < 0.04) in the 20-min group at 6 h. IL-1β and β-endorphin have both been shown to have a direct stimulatory effect on NK cell activity. Therefore, we suspect that the significant increase in both IL-1β and β-endorphin at 6 h in the 10-min-rotation group may be involved in the increased NK cell activity observed at 24 h in the 10-min-rotation group.

Antigen-Specific T Cell Tolerance Down-Regulates Mast Cell Responses in Vivo

Fel d I is the major cat allergen that induces asthma and allergic rhinitis in humans. To investigate the mechanism of allergic responses to this allergen, a mouse model was developed. Mice sensitized to chain 1 of Fel d I exhibited T cell responses, B cell responses, and mast cell responses when challenged with the protein. Subcutaneous injections of peptides containing the dominant T cell epitopes of the allergen induced T cell tolerance in presensitized mice. When challenged with the allergen intratracheally, these tolerized mice produced a decreased amount of histamine in vivo. The decrease in histamine release was not solely dependent on the reduction of allergen-specific IgE. These data show that mast cell activity in mice with an ongoing sensitivity to allergen can be regulated through peptide-induced T cell tolerance.

The long terminal repeat of Jaagsiekte sheep retrovirus is preferentially active in differentiated epithelial cells of the lungs.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a contagious bronchioloalveolar carcinoma of sheep known as sheep pulmonary adenomatosis (SPA; ovine pulmonary carcinoma). JSRV is unique among retroviruses because it transforms the alveolar type II cells and the nonciliated bronchiolar cells (Clara cells) of the lungs; these cells are where JSRV is specifically expressed in both naturally and experimentally SPA-affected sheep. In this study, we investigated the cell specificity of JSRV expression. By transient-transfection assays of 23 different cell lines with a reporter plasmid driven by the JSRV long terminal repeat (LTR), pJS21-luc, we found that the JSRV LTR is preferentially active in cell lines derived from type II pneumocytes and Clara cells (MLE-15 and mtCC1-2 mouse cell lines). Reporter assays using progressive 5' deletions of pJS21-luc allowed us to establish that the JSRV enhancers are able to activate the JSRV proximal promoter in MLE-15 and mtCC1-2 cells, but they have very low activity in mouse cells of other lineages (e.g., NIH 3T3). The JSRV enhancers are able to activate heterologous promoters in both MLE-15 and 3T3 cells, although optimal activity is achieved in MLE-15 cells only with the homologous JSRV promoter. Thus, JSRV cell-specific LTR activity appears to result from an interaction between the enhancer elements and the JSRV proximal promoter elements. By mutation analysis, we established that an upstream NF-kappaB-like element appears to be responsible for approximately 50% of the JSRV LTR transcriptional activity in MLE-15 cells. Electrophoretic mobility shift assays showed evidence of a factor(s) that binds to this sequence. Antibody supershift experiments indicated that the factor(s) is not related to NF-kappaB component p50 or p52. This factor also appeared to be present in cells that do not support a high level of JSRV expression. Finally the JSRV(21) LTR contains putative enhancer binding motifs for transcription factors such as hepatocyte nuclear factor 3 (HNF-3) that are involved in lung-specific gene expression. Cotransfection experiments demonstrated that exogenous HNF-3 is able to enhance the expression of pJS21-luc in NIH 3T3 cells, which normally show minimal enhancer activity for the JSRV LTR.

Modulation of natural killer cell function after exposure to 60 Hz magnetic fields: confirmation of the effect in mature B6C3F1 mice.

In a previous study, we demonstrated that subchronic exposure to pure, linearly polarized 60 Hz magnetic fields (MFs) at flux densities ranging from 0.002 to 1.0 mT induced a modest but statistically significant and reproducible suppression of NK cell activity in young adult B6C3F(1) mice. NK cell activity in mice declines with age and is known to be suboptimal in older animals. The present study was designed to determine if the same MF exposure regimens will suppress NK cell activity in mature (i.e. more than 1 year old) animals. Extending our previous findings, a modest suppressive effect of MFs on NK cell activity in B6C3F(1) mice was found when subchronic exposure was initiated in animals held in quarantine for 1 year prior to exposure. These data demonstrate that MF exposure suppresses NK cell activity in both young and mature adult B6C3F(1) mice. However, because chronic exposure to the same MF parameters used in the NK function studies does not increase the incidence of neoplasia in B6C3F(1) mice, this statistically significant inhibition of NK cell function appears to be of limited biological significance.

Coreceptor-Independent T Cell Activation in Mice Expressing MHC Class II Molecules Mutated in the CD4 Binding Domain

We have previously reported that efficient selection of the mature CD4+ T cell repertoire requires a functional interaction between the CD4 coreceptor on the developing thymocyte and the MHC class II molecule on the thymic epithelium. Mice expressing a class II protein carrying the EA137/VA142 double mutation in the CD4 binding domain develop fewer than one-third the number of CD4+ T cells found in wild-type mice. In this report we describe the functional characteristics of this population of CD4+ T cells. CD4+ T cells that develop under these conditions are predicted to be a CD4-independent subset of T cells, bearing TCRs of sufficient affinity for the class II ligand to undergo selection despite the absence of accessory class II-CD4 interactions. We show that CD4+ T cells from the class II mutant mice are indeed CD4 independent in their peripheral activation requirements. Surprisingly, we find that CD4+ T cells from the class II mutant mice, having been selected in the absence of a productive class II-CD4 interaction, fail to functionally engage CD4 even when subsequently provided with a wild-type class II ligand. Nevertheless, CD4+ T cells from EA137/VA142 class II mutant mice can respond to T-dependent Ags and support Ig isotype switching.

Augmentation of natural killer cell activity in mice by oral administration of transforming growth factor-beta.

The latent form of transforming growth factor-beta (TGF-beta) in human milk and platelets was converted to the active form when conscious, pylorus-ligated mice were given human milk and platelets by intragastric intubation. Oral administration of TGF-beta exerted enhancing effects on the natural killer (NK)-cell activities in spleen and liver. Augmentation of NK-cell activities in spleen was observed for 7 days after oral administration of TGF-beta. TGF-beta at concentrations of 5 and 20 ng produced the greatest augmentation of NK-cell activities in spleen. However, NK-cell activities in spleen were unaffected when TGF-beta was given intravenously. Interleukin (IL)-12 production in spleen was enhanced by oral administration of TGF-beta, but not by intravenous administration of TGF-beta. These findings suggest that large amounts of TGF-beta in human milk are involved in early antiviral protection through the augmentation of NK-cell activities.

Early Preferential Stimulation of γδ T Cells by TNF-α

Abstract Although recent findings indicate that γδ T cells influence both early innate and Ag-specific adaptive host responses, it has remained unclear what triggers γδ T cell reactivity. Investigating very early T cell activation in mouse and human models of bacterial infection, we measured CD69 expression as an indicator of early cellular activation. Both murine αβ and γδ T cells responded polyclonally to systemic bacterial infections, and to LPS. However, γδ T cells responded more strongly to the bacteria and to LPS. In vitro LPS-stimulated human T cells showed a similar differential response pattern. We identified TNF-α as mediator of the early differential T cell activation, and of differential proliferative responses. The stronger response of γδ T cells to TNF-α was correlated with higher inducible expression levels of TNF-Rp75. Among unstimulated splenocytes, more γδ T cells than αβ T cells expressed CD44 at high levels. The data suggest that TNF-Rp75 determines the differential T cell reactivity, and that most γδ T cells in the normal spleen are present in a presensitized state. As TNF-α stimulates activated T cells, it may early preferentially connect γδ T cell functions with those of cells that produce this cytokine, including activated innate effector cells and Ag-stimulated T lymphocytes.

A new internal ribosomal entry site 5' boundary is required for poliovirus translation initiation in a mouse system.

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.

Murine Cytomegalovirus Infection-Induced Polyclonal B Cell Activation Is Independent of CD4+T Cells and CD40

The results of this study demonstrate that murine cytomegalovirus (MCMV) induces polyclonal B cell activation in mice during the acute phase of primary infection. First, flow cytometric analysis revealed that surface expression of CD45R, IgM, and Igκ by splenocytes from MCMV-infected mice was significantly reduced with a concomitant increase in the frequency of surface IgG-expressing cells. Second, ELIspot assays demonstrated that the changes revealed by flow cytometry were paralleled by increases in the numbers of IgG-producing cells, especially those secreting IgG2a. Third, the IgG antibodies from MCMV-infected animals reacted against a variety of self and foreign antigens. MCMV-induced B cell activation was independent of CD4+T-cell-mediated help and CD40, since activation was observed in two models of mice deficient for this T cell subset and in mice deficient for CD40. Reverse transcription–polymerase chain reaction analysis showed that mRNA transcripts for the cytokines IL-6, IL-10, and IFN-γ were rapidly induced following infection with MCMV, but only IL-6 and IFN-γ proteins were detectable by ELISA. In addition, the numbers of cells producing IL-6 and IFN-γ were significantly increased in the spleen. The magnitude of the polyclonal B cell activation response was diminished by 50% in IL-6-deficient mice but not in mice lacking IFN-γ. In the absence of IFN-γ, surface expression and serum levels of IgG2awere reduced while IgG1expression was increased.

Involvement of Catecholamines and Glucocorticoids in Ethanol‐Induced Suppression of Splenic Natural Killer Cell Activity in a Mouse Model for Binge Drinking

Ethanol (EtOH) suppresses splenic natural killer (NK) cell activity in a mouse binge drinking model. Direct effects of EtOH and its metabolites are not the major cause of this suppression. Also, catecholamines do not completely explain this suppression. This implicates the involvement of other neuroendocrine mediators in this suppression. Previous studies in this laboratory have shown that RU 486 at a dosage of 100 mg/kg did not affect EtOH-induced suppression of NK cell activity. However, in the present study, RU 486 at a dosage of 200 mg/kg partially blocked the suppression of NK cell activity induced by EtOH. Moreover, corticosterone at levels expected in the free (unbound) form in EtOH-treated mice decreased NK cell activity in vitro. Nadolol in combination with RU 486 blocked the suppression of NK cell activity in EtOH-treated mice. Although there were reasons to suspect that EtOH-induced changes in the levels of growth hormone or prolactin might also contribute to the suppression of NK cell activity; evidence obtained herein did not indicate such involvement. Thus, glucocorticoids and catecholamines seem to be involved in EtOH-induced suppression of NK cell activity. Together, with the direct effects of EtOH, these neuroendocrine mediators seem to be sufficient to explain all of the suppression of NK cell activity caused by EtOH.

Reovirus Type 3 Chemoimmunotherapy of Murine Lymphoma is Abrogated by Cyclosporine

We have previously shown that the combined modalities of reovirus type 3 and the chemotherapeutic agent 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) synergize to cause the rejection of various murine tumors. Resistance of surviving animals to challenge with homologous but not heterologous tumor suggested that tumor was eliminated through an immune-mediated mechanism. In this study, we showed that mice undergoing therapy-mediated rejection of tumor were able to reject subsequent weekly challenges with EL-4 but not L1210 tumor cells. The mechanism underlying this therapy was investigated using cyclosporine (CS) to suppress immune responsiveness. A dose-related inhibition of therapy was observed with total inhibition occurring at 30 mg/kg/day. Delayed administration of CS at day 14 or later after tumor administration resulted in little or no inhibitory effect. The resistance of cured mice to EL-4 tumor challenge was not affected by CS, which is consistent with the reduced ability of CS to affect secondary immune responses. In addition, CS did not alter natural killer cell activity in mice receiving the BCNU/reovirus therapy. These results suggest that there is an obligatory immune response produced by the BCNU/reovirus therapy which arises early after the administration of the therapy.

Resistance to pseudorabies virus with enhanced interferon production and natural killer cell activity in mice treated with serum thymic factor.

Susceptibility to pseudorabies virus (PRV) infection in mice, which were continuously depleted of natural killer (NK) cell activity by injection of anti-asialo GM1, was examined. Effects of serum thymic factor (FTS) on susceptibility of mice to PRV were also investigated. In mice with depleted NK cell activity, the mortality of PRV-infected mice was markedly high, whereas that of FTS-pretreated mice was significantly lower than the controls. Reduced susceptibility to PRV was demonstrated in mice treated with anti-asialo GM1 antisera before the PRV infection. Such a reduced susceptibility was not observed in mice inoculated with the antisera on day 1 post-infection (PI). To analyze the FTS-induced resistance to PRV infection, NK cell activity, macrophage activity, and interferon (IFN) productions were studied. Interferon production and NK cell activity were enhanced in the FTS-pretreated mice, suggesting that interferon may play an important role in this FTS-induced resistance to PRV infection.

Isolation of multiple activities from mouse FM3A cells which promote homologous pairing of DNA molecules

We describe the detection and partial purification of three homologous pairing activities from extracts of mouse mammary carcinoma FM3A cells. These activities, designated MHP1a, 1b, and 1c, form joint molecules between circular single-stranded DNA and homologous linear duplex DNA and are distinguished from one another by their chromatographic behaviors or isoelectric properties. The reactions promoted by these activities require homologous substrates but not ATP. All the reactions also show Mg 2+ dependence in the absence of exogenous ATP. Analysis of the reaction products has revealed that strand exchange proceeds for lengths of up to at least 271 bp during the homologous pairing reaction. The finding of multiple types of homologous pairing and strand exchange activity in mouse cells may facilitate the elucidation of the mechanism of homologous recombination in somatic mammalian cells.

Effect of abrogation of natural killer cell activity on the course of candidiasis induced by intraperitoneal administration and gastrointestinal candidiasis in mice with severe combined immunodeficiency

Candida albicans CFU per gram of tissue recovered from livers, spleens, and kidneys of 12 severe combined immunodeficiency (scid) and 12 BALB/c mice 5 days after intraperitoneal (i.p.) administration of 10(7) C. albicans cells were not significantly different. Nine scid mice given normal rabbit serum (NRS) as a control and eight scid mice given anti-asialo-GM1 (alpha-ASGM1) had C. albicans CFU per gram recovered from livers and spleens 1 week after i.p. administration of C. albicans that were not significantly different, despite virtual elimination of natural killer (NK) cell activity in mice treated with alpha-ASGM1. At 2 weeks after i.p. administration, despite significantly increased NK cell activity in eight infected NRS-treated scid mice and virtual elimination of NK cell activity by alpha-ASGM1 treatment of eight scid mice, C. albicans CFU per gram recovered from livers and kidneys were not significantly different. At 2 weeks after intragastric administration of 2 x 10(6) C. albicans cells, eight NRS- and eight alpha-ASGM1-treated scid mice had identical proportions colonized with C. albicans and similar C. albicans CFU per gram recovered from feces. There was no evidence of hematogenous dissemination in either group. Similar results were seen 1 week after intragastric administration of 10(7) C. albicans cells. We conclude that NK cell activity is increased by i.p. administration of C. albicans in scid mice, but nontheless, abrogation of NK cell activity is not associated with enhanced susceptibility to candidiasis induced by i.p. administration and also is not associated with enhanced susceptibility to gastrointestinal colonization or hematogenous dissemination after intragastric administration of C. albicans.

Inhibition of interferon stimulation of natural killer cell activity in mice anesthetized with halothane or isoflurane.

Basal cytotoxic activity of NK cells, a subtype of lymphocytes involved in the nonspecific immune response to viruses, tumors, and some bacteria, is altered in the postoperative period. The current study examines the effects of halothane and isoflurane on interferon-induced stimulation of NK cell cytotoxicity in vivo and in vitro. Mice were exposed to either anesthetic on days 10, 5, 1, or -1 relative to interferon treatment on day 0. NK cytotoxicity was assessed 24 h later. Similarly, splenic mononuclear cells containing NK cells were treated with interferon, before or after in vitro exposure with either halothane or isoflurane, and cytotoxicity was determined. In vivo, isoflurane or halothane inhibited subsequent interferon-induced NK cell stimulation (> 90% and 67%, respectively). No inhibition occurred if interferon was given before anesthetic exposure. Significant inhibition of interferon-induced NK cell stimulation could be observed 11 days after anesthesia. In vitro, both anesthetics inhibited the subsequent stimulation of NK cytotoxicity by interferon, however, cytotoxicity of NK cells treated with interferon before anesthetic exposure was comparable to untreated interferon-stimulated NK cells. Halothane and isoflurane inhibit interferon stimulation of NK cytotoxicity in naive (unstimulated) NK cells of the splenic mononuclear cell pool without affecting the cytotoxicity of previously stimulated (interferon) NK cells. This could occur directly by preventing the NK cell from responding or indirectly by altering other cells in the splenic mononuclear cell pool (T cells, macrophages), which then inhibit NK cell induction.

Induction of Human Immunodeficiency Virus-Specific T Cell Responses in Rhesus Monkeys by Synthetic Peptides from gp160

We have previously identified several synthetic peptides from conserved regions of the human immunodeficiency virus (HIV) envelope protein gp160 that have the capacity to induce broadly reactive T cell responses against gp160 in mice of several major histocompatibility complex (MHC) haplotypes. In the present investigation three rhesus monkeys were immunized with a mixture of eight synthetic peptides that are capable of inducing T cell activity in mice. Peripheral blood mononuclear cells (PBMCs) from these monkeys were monitored every 2 weeks for a period of 34 weeks for proliferative responses against individual peptides and recombinant gp160. Peripheral blood mononuclear cells from all three rhesus monkeys showed good proliferative responses with peptides 104 (aa 45-55), 111 (aa 118-130), and 63 (aa 519-543), whereas weak responses were observed with peptides 113 (aa 204-216) and 116 (aa 240-252). Two of the three rhesus monkey-derived PBMC preparations also showed good proliferative responses with peptide 61 (aa 586-598). Significant responses were not observed with peptides 105 (aa 48-61) and R15K (aa 315-329) in any of the monkeys immunized. However, PBMCs from all three monkeys showed significantly high proliferative responses with recombinant gp160, the HIV-1 envelope protein precursor. These results demonstrate that mixtures of synthetic peptides from HIV env gene product can prime gp160-specific T cell responses in rhesus monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of the Arg179 in the active site of human IL-6.

Three internal-amino acid deletions of amino acids 171-179 of human interleukin 6 (IL-6) were introduced at the cDNA level. While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation-specific anti-(IL-6) monoclonal antibodies showed that only mutant delta 177-179 does not present major alterations in folding. This finding, together with the observation that delta 177-179 is not able to compete with IL-6 for binding to the soluble human IL-6 receptor, suggested that some or all of these three residues participate to the composition of the receptor-binding site of human IL-6. A large number of single-amino-acid-substitution mutants were generated in residues 177, 178 and 179. Their detailed analysis revealed that Arg179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding. The only substitution which preserved some residual activity was the conservative Arg to Lys change. This demonstrates the absolute requirement for a positive charge in position 179 for the interaction of human IL-6 with its receptor.

Natural killer cell activity in mice infected with free-living amoeba with reference to their pathogenicity

The purpose of this observation was to investigate the natural killer cell activities in mice infected with pathogenic free-living amoeba, Naegleria fowleri and Acanthamoeba culbertsoni according to the infection doses, and infected with non-pathogenic free-living amoeba, Naegleria gruberi. The natural killer cell activity was examined by means of target binding capacity, active NK cell and maximum recycling capacity of the mice after inoculating free-living amoebae with low and high doses. The mice infected with 1,103, 1,105 A. culbertsoni trophozoites showed mortality rates of 6.9% and 65.5%, respectively. The mice infected with 1,104, 1,105 N. fowleri trophozoites showed mortality rates of 5.9% and 72.2%, respectively. The NK cell activities in all experimental groups increased significantly on day 1 after infection as compared with control group, and then remarkably declined thereafter, there was no difference of the cytotoxic activity of the NK cells in mice among inoculation doses of pathogenic free-living amoebae. The target binding capacities of NK cells and percentages of activated NK cells in mice infected with pathogenic free-living amoebae were significantly increased a day after infection, as compared with control group. There was no difference of the maximal recycling capacities of NK cells in all experimental groups as compared with control group. There was significant difference in the cytotoxic activity and single cell cytotoxicity of NK cells between the experimental groups infected with pathogenic free-living amoebae and that infected with non-pathogenic free-living amoebae.