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Abstract: An influx of neutrophils followed a short time later by an influx of macrophages to the infected site plays a key role in innate immunity against Escherichia coli infection. We found in this study that Vδ1−/− mice exhibited impaired accumulation of peritoneal macrophages but not neutrophils and delayed bacterial clearance after intraperitoneal inoculation with E. coli. Peritoneal γδ T cells from E. coli-infected wild-type mice produced CCL3/MIP-1α and CCL5/RANTES in response to γδ T CR triggering in vitro, while such production was not evident in γδ T cells from E. coli-infected Vδ1−/− mice. Neutralization of CCL3/MIP-1α by a specific monoclonal antibody in vivo significantly inhibited the accumulation of macrophages in the peritoneal cavity after E. coli infection, resulting in exacerbated bacterial growth in the peritoneal cavity. These results suggest that Vδ1+ γδ T cells bridge a gap between neutrophis and macrophages in innate immunity during E. coli infection mediated by production of CC chemokines, enhancing macrophage trafficking to the site of infection. [Copyright &y& Elsevier]

Recombinant human granulocyte colony-stimulating factor (rh G-CSF) enhanced resistance of mice against Listeria monocytogenes (LM) as determined by survival and bacterial growth. Mice pretreated with rh G-CSF twice daily for 5 days survived better than untreated animals to the challenge with LM. Number of bacteria in peritoneal cavity (PC) and spleen was lower in treated mice than that in the control group. RhG-CSF increased mainly polymorphonuclear cells (PMN) in blood and spleen. After LM inoculation, a larger number of PMN and monocyte-macrophages accumulated in PC and spleen of tested mice. In addition, PMN primed in vivo with rh G-CSF released more superoxide anions when stimulated with phorbol myristate acetate. The inhibition of bacterial growth in PC and spleen could be ascribed to the accumulation of phagocytic cells at the infection sites and the increased oxidative metabolism. The results provided further evidence of the important contribution of G-CSF and neutrophils, as target cells, to the host defence against the intracellular bacteria. [ABSTRACT FROM AUTHOR]

In flow microfluorometry analysis of thymus and lymph node cells of C57BL/6(I-E-,Mlsb) mice rendered neonatally tolerant to (C57BL/6 × AKR/J)F1 (I-E+,Mlsb/a) lymphoid cells, both CD4+ and CD8+ cells showed a striking reduction in the number of Vβ6+ cells capable of recognizing Mlsa in the context of major histocompatibility complex (MHC) class II molecules, indicating that clonal deletion of Vβ6+ cells by Mlsa antigen occurs just at a stage of immature Vβ6lowCD4+CD8+thymocytes. On the other hand, the number of Vβ11+ cells capable of recognizing I-E was markedly reduced in CD4+ cells, but CD8+ cells showed only partial (20%) reduction of such a population. The clonal deletion of Vβ11+ cells by I-E may begin at the transitional stage from Vβ11lowCD4+CD8+ to Vβ11highCD4+CD8- single-position cells, and Vβ11lowCD4+CD8+ cells differentiating to Vβ11highCD4-CD8+ cells seem to be resistant to clonal deletion. Vβ11+ T cells are also stimulated by staphylococcal enterotoxin A (SEA) irrespective of expression of CD4 or CD8. Nearly all of both Vβ11+CD4+ and Vβ11+CD8+ lymph node T cells were deleted by the injection of SEA every other day from birth. In their thymi, both Vβ11+CD4+CD8- and Vβ11+CD4 CD8+ single-positive thymocytes were deleted, and the proportion of Vβ11low thymocytes was lower than that of normal mice. The clonal deletion of Vβ11+ T cells by SEA injection occurs at a stage of immature Vβ11lowCD4+CD8+ double-positive thymocytes, resulting in deletion of both Vβ11+CD4+ and Vβ11+CD8+ T cells. [ABSTRACT FROM AUTHOR]

We have previously reported that T-cell receptor (TcR) γδ-bearing T cells precede TcR αβ-bearing T cells in appearance in the thymus after whole-body irradiation. In the present study, the kinetics of appearance of intestinal intra-epithelial lymphocytes (IEL) was examined in mice after whole-body irradiation with a lethal dose of 9.5 Gy or with a sublethal dose of 6 Gy. The number of CD3+ IEL decreased to the lowest value 4 days after irradiation with 9.5 Gy, and thereafter increased to half as many as the normal level by day 7. Thy-1+TcRαβ- IEL and Thy-TcRβ IEL recovered considerably by day 7 after the irradiation, whereas Thy-1+TcRαβ+ IEL and Thy-1+TcRαβ+ IEL hardly recovered at this stage. All mice died within 12 days after irradiation with a lethal dose of 9.5 Gy. On the other hand, when irradiation dose was decreased to 6 Gy, all mice survived beyond 40 days after irradiation. The number of CD3+ IEL recovered to the normal level by 10 days after irradiation with 6 Gy. Consistently with the results in mice irradiated with a lethal dose, the first cells to increase in IEL of mice irradiated with a sublethal dose were TcRγδ+ IEL expressing Thy-1 antigen. The number of Thy-1+TcRγδ+ IEL increased to approximately two-fold as many as that in normal mice by day 10, while TcRαβ+ IEL began to increase in number from day 20 after irradiation and recovered to the normal level by day 40 after irradiation. Thus, sequential appearance of TcRγδ+ and TcRαβ+ IEL was evident after irradiation, similar to that seen in the thymus after irradiation. The IEL on day 10 after sublethal irradiation, which is composed mainly of Thy-1+TcRγδ+ IEL, exhibited a strong cytolytic activity against P815 in the presence of anti-CD3 mAb, suggesting that the early appearing Thy-1+TcRγδ+ IEL may play important roles in epithelial immunity at an early stage after irradiation. [ABSTRACT FROM AUTHOR]

Murine thymocytes are divided into four major populations on the basis of expression of CD4 and CD8 antigens. The bulk of evidence favours the view that CD4-CD8- cells can develop into CD4-CD8+ and CD4+CD8- cells via the CD4+CD8+ stage in the thymus. However, CD4-CD8+ and CD4+CD8- thymocyte subsets contain not only CD3+ mature cells but also CD3- immature cells, which seem to be intermediate cells between CD4-CD8- and CD4+CD8+ cells. Here we demonstrate mouse strain differences in the proportion of immature single-positive thymocyte subsets in thymus at the steady or developing state. In C3H mice, immature CD4+ CD8- is dominant in proportion over CD4-CD8+ in foetal thymus and in donor-derived thymocytes at an early stage of bone marrow transplantation. On the other hand, immature CD4 CD8+ is dominant over CD4+CD8- during T-cell development in the case of B10.BR mice. An intermediate pattern was shown in the case of Fi mice. Both of these immature single-positive subsets gave rise to doublepositive cells after 24 hr culture. These results suggest that there exist two distinct differential pathways; one is from CD4- CD8- cells to CD4+ CD8+ cells via CD4-CD8+ cells, and another is via CD4+CD8- cells, and that an application of the 'CD8 pathway' or 'CD4 pathway' seems to be genetically destined by BM-derived cells but not by thymic stromal cells. [ABSTRACT FROM AUTHOR]

In order to search for a possible role of abnormally proliferating T cells in developing autoimmune disease in lpr mice, and to define the difference of the T ceils among various lpr-congeneic mice with different clinicopathological findings, the T-cell receptor (TcR) Vβ gene expression in the enlarged lymph nodes (LN) of C3H/HeJ-lpr/lpr (C3H-lpr), C57BL/6-1pr/lpr (B6-lpr) and MRL/Mp-lpr/lpr (MRL-lpr) mice was analysed. A RNA blot analysis using several Vβ-specific probes showed that the Vβ3 gene, whose products are important for recognizing Mlsb/a, was used in B6-lpr and MRL-lpr with the Mlsb/b but not in C3H-lpr with the Mlsb/a. The Vβ5 gene, which is selectively related to I-E molecules, was predominantly used in B6-lpr(I-E-) but not in C3H-lpr(I-E+) nor MRL-lpr(I-E+). Similarly, the V12 gene was also expressed in B6-lpr but not in C3H-lpr. To compare in detail the Vβ repertoire among lpr mice with different major histocompatibility complex (MHC) backgrounds, the Vβ gene sequences in the cDNA libraries from LN cells of C3H-lpr were analysed, following the recent investigation of B6-lpr mice (Ohga et al., 1989). Eleven β-chain cDNA out of 32 β cDNA in B6-lpr and 24 β-chain cDNA out of 55 β cDNA in C3H-lpr were found to contain sequences with open reading frames that potentially encode functional TcR β-chain. The fequencies of the messages in the cDNA libraries from these mice were consistent with the RNA blot analysis using Vβ3- and Vβ5-specific probes. It was notable that 36% of the functional β-chain mRNA in B6-lpr and 50% of the β mRNA in C3H-lpr expressed the Vβ8 gene family. When the TcR Vβ gene expression was compared between the LN cells in C3H-lpr, B6-lpr and MRL-lpr, as reported by Singer et al. (1986), the usage of Vβ genes other than the Vβ8 gene family in B6-lpr (H-2b) LN cells differed significantly from those in C3H-lpr (H-2k) and MRL-lpr(H-2k). The results presented here indicate that the usage of Vβ genes is heavily influenced by the genetic background of lpr mice, similar to normal mice, but with preferential usage of the Vβ8 gene family as a common structural feature in lpr gene-induced cell populations. [ABSTRACT FROM AUTHOR]

It has been reported previously that environmental antigens such as intestinal microflora play an important role in an age-associated increase in number of T-cell receptor γ/δ-bearing T cells. To extend the scope of this finding, this study examined the influences of local inflammation on the accumulation and/or proliferation of γ/δ T cells in athymic nude mice injected subcutaneously with complete Freund's adjuvants (CFA). The inguinal lymph nodes (ILN) in nude mice injected with CFA in their hind footpads 7 days previously contained an increased number of CD3+CD4-CD8 cells. Increased levels of proliferative responses against syngeneic stimulator cells were noted in the LN cells of CFA-injected nude mice in association with increases in expression of γ-and δ-chain gene messages. Furthermore, the LN cells showed an early proliferative response to purified protein derivative (PPD) from Mycobacterium boris. These results suggest that local inflammation by CFA may induce functional γ/δ T cells in nude mice, which may proliferate rapidly at the inflamed sites and represent a first line of defence in such animals against the invasion of various pathogens. [ABSTRACT FROM AUTHOR]

Clonal deletion oft cells capable of recognizing both host-type M1s and donor-type M1s occurred in the peripheral mature T-cell pool in radiation bone marrow chimeras of two H-2-compatible M1s disparate strain combinations of AKR/J(H-2k,Thy-1.1,Mls-1a) and C3H/He(H-2k,Thy-1.2,M1s-1b). In order to determine further the stage at which the clonal deletion occurs in thymus, we examined the kinetics of thymocytes bearing Vβ6 capable of recognizing M1s-1a in both C3H/He→AKR/J and AKR/J→C3H/He chimeras. An almost complete replacement from host-derived cells to donorderived cells occurred by Day 21 after reconstitution in both chimeras. At this stage, CD4+CD8+ double-positive thymocytes contained an appreciable number of cells that expressed Vβ6 on their surface, albeit at Iow intensity, whereas CD4 or CD8 single-positive thymocytes which expressed a high density of Vβ6 were virtually abolished in both C3H→AKR and AKR→C3H chimeras on Day 21. These results suggest that clonal deletion of self-M1s-reactive T cells begins at an early stage when the thymocytes interact with the early appearing donor-derived haematopoietic cells and relatively radio-resistant host-derived cells in thymus of radiation bone marrow chimeras. [ABSTRACT FROM AUTHOR]

To search the abnormality in prethymic T-cell precursors in lpr/lpr(lpr) mice, rearrangement and expression of T-cell receptor (TcR) genes were investigated in long-term cultured bone marrow (LTBM) cells of lpr mice, in which the developmental steps of T-cell purcursors may be better synchronized than those in bone marrow (SM) cells. Neither rearrangment nor expression of TCR γ and δ genes were detected in the LTBM cells from +/+ control mice, whereas some γ gene rearrangements were detected in those derived from lpr mice, irrespective of the genetic background. When BM cells or LTSM cells from lpr mice were transplanted into supralethally irradiated +/+ mice, the lpr-derived BM cells appeared earlier in the thymus of the recipient mice than +/+-derived BM cells and the recipients suffered from lethal wasting syndrome. In addition, the lpr-derived SM cells showed higher activity in colony-forming unit spleen (CFUs) than the +/+-derived SM cells. These results suggest that the T-cell progenitors in the SM of lpr mice may be different not only in quantity but also in quality from those of +/+ mice. [ABSTRACT FROM AUTHOR]

We have previously shown that extrathymic rearrangements of T-cell receptor (TcR) γ and δ chain genes occur in the peripheral lymphoid tissues of athymic nude mice. To further determine where the TcR gene rearrangements occur in nude mice, we investigated the rearrangement and expression of the TcR genes in the long-term cultured bone marrow (LTBM) cells which were homogenous in developments without mature T cells as assessed by FACS analysis. The LTBM derived from euthymic mice contained IcR γ and δ chain genes in germline configuration, while gene rearrangements of both locus were detected in the LTBM cells from nude mice. These results suggested that γ and δ gene rearrangements do occur in the bone marrow cells of nude mice and that the T-call precursors in bone marrow may be increased in frequency in such animals. [ABSTRACT FROM AUTHOR]

The role of mononuclear phagocyte system (MPS) in the induction of suppressor T cells which depress the delayed footpad reaction to sheep erythrocytes (SR BC) was studied in mice in which MPS was blocked or stimulated. Colloidal carbon and diethyl- stilbestrol were used for blockade and stimulation respectively. Adoptive transfer of suppressor T cells was achieved by spleen cells of mice immunized intra- peritoneally with varying doses of SRBC. In non-treated mice, 1 × 109 SRBC were required to induce suppressor T cells, while 6 × 108 could not induce the suppressor T cells. In MPS-blocked mice, however, even 6 × 108 SRBC could induce the suppressor T cells. On the other hand, 3 × 108 SRBC were required for the induction of suppressor T cells in MPS-stimulated mice. These results suggest that the activity of macrophages as scavenger cells modulates the subsequent induction of suppressor T cells after immunization with high doses of SRBC. [ABSTRACT FROM AUTHOR]

Delayed footpad reactions and acquired cellular resistance to Listeria monocytogenes were studied in mice whose mononuclear phagocyte system (MPS) had been blocked or stimulated. Colloidal carbon was used for the blockade of MPS and Corynebacterium parrum used for the stimulation. Strong delayed footpad reactions. On the other hand, the i.v. rejection of 3 × 101; listeria induced an appreciable level mice, while in MPS-stimulated mice, i.v. injection with even 4 × 103; listeria could not induce such strong delayed footpad reaction. On the other hand, the i.v. injection of 3 × 10¹ listeria induced an appreciable level of delayed footpad reaction only in MPS-blocked mice. Acquired cellular resistance was depressed by MPS stimulation, whereas it was augmented by MPS blockade. These results suggested that non-specific activity of MPS modulates subsequent immune responses after inoculation of listeria. [ABSTRACT FROM AUTHOR]

The delayed footpad reaction to sheep erythrocytes (SRBC) was studied in mice whose mononuclear phagocyte system (MPS) was blocked or stimulated. Colloidal carbon or carrageenan was used for the blockade of MPS and Corynebacterium parvurn or diethylstilbestrol used for the stimulation. The optimal dose of SRBC for the induction of the delayed footpad reaction was lower in MPS-blocked mice than in non-treated mice, whereas a high dose of SRBC was required for the induction of the strongest delayed footpad reaction in MPS-stimulated mice. These results suggest that non-specific phagocytic function of MPS modulates subsequent immune responses after administration of antigens. [ABSTRACT FROM AUTHOR]

An autosomal recessive gene, lpr, is responsible for lymphoproliferation and autoimmunity of lpr mice, in which background genes are also known to influence the development of autoimmune disease. To define the differences in abnormally proliferating T cells between C57BL/6-lpr/lpr and MRL/Mp-lpr/lpr mice, and to try and understand the influence of background in the differing expression of autoimmune disease in both strains, we analysed the sequences of T cell antigen receptor Vβ genes expressed in the cells from the enlarged lymph nodes of C57BL/6-lpr/lpr mice. Eleven β cDNAs out of the 38 Cβ-specific cDNAs contained sequences with open reading frames from the beginning of the variable region to the expected termination codons at the end of the constant regions. Notably, 36% of the functional β-chain mRNAs expressed Vβ8.3 genes, whereas Vβ8.3 and Vβ8.2 genes were not found. These results are consistent with a relatively lower frequency of the Vβ8.1 or Vβ8.2 expressing cells in the hypertrophic lymph nodes of C57BL/6-lpr/lpr mice, detected by KJI6-133 monoclonal antibody. Interestingly, other Vβ genes expressed in these mice were completely distinct from those in MRL/Mp-lpr/lpr mice as described by Singer et al (1986), The different distribution of Vβ genes expressed in C57BL/6-lpr/lpr from that in MRL/Mp-lpr/lpr mice might be related to the differences in the genetic background and the expression of lpr gene-associated autoimmunity. [ABSTRACT FROM AUTHOR]

The purpose of this paper was to study the γδ T-cell receptor repertoire and target specificity following stimulation of peripheral T cells of BALB/c mice with autologous or bacterial ligands. The expression of Vγ and Vδ chain families in T cells that had been expanded by stimulation in syngeneic mixed lymphocyte culture or with purified protein derivative (PPD) was determined by the semiquantitative DNA polymerase chain reaction (PCR) method. Responder T cells to either of these stimuli strongly expressed both Vδ5 and Vδ6 genes. However, addition of the ML 30 anti-murine heat shock protein (hsp) 60 monoclonal antibody (mAb) to the cell culture selectively inhibited only the expansion of Vδ5 T cells. A Vδ5 T-cell hybridoma (KMT-5), which recognized syngeneic splenic and fibrosarcoma Meth A cells but not allogeneic cells, was produced by cell fusion from autoreactive blast cells. Incubation of the KMT-5 hybridoma in the presence of ML 30 antibody blocked the stimulation of interleukin-2 (IL-2) secretion by syngeneic target cells. It was also found that the DNA of KMT-5 hybridoma and of the autoreactive γδ T cells contained the BALB/c invariant delta (BID) chain sequence. It is concluded from these results that BALB/c peripheral Vδ5 T cells recognize an autologous hsp 60 target specificity in a Vδgene restricted manner. We also propose that T cells of this V gene family may be involved in the immune surveillance of certain tumours and intracellular infections. [ABSTRACT FROM AUTHOR]

CD30 ligand (CD30L, CD153), a member of the tumor necrosis factor (TNF) superfamily, and its receptor CD30 are important for differentiation and activation of CD4+ T helper type 17 (Th17) cells. In this report, we demonstrate that the interleukin 17A (IL-17A)-producing γδ T cells normally developed in the fetal thymus, whereas Vγ1−Vγ4− γδ T cells expressed Vγ6/Vδ1 gene transcript selectively decreased in mucosa-associated tissues in naive CD30KO or CD30LKO mice. Moreover, CD30 and CD30L were expressed preferentially by Vγ1−Vγ4− γδ T cells in naive mice. The bacteria clearance was attenuated by the impaired response of the IL-17A-producing γδ T cells and decreased infiltration of neutrophils in CD30KO or CD30LKO mice. In vivo administration of agonistic anti-CD30 monoclonal antibody restored the ability of protection against Listeria monocytogenes by enhancing Vγ1−Vγ4− γδ T cells producing IL-17A not only in wild-type but also CD30LKO mice. Taken together, it appears that CD30L/CD30 signaling plays an important role in the maintenance and activation of IL-17A-producing γδ T cells presumably bearing Vγ6 in the mucosa-associated tissues of mice. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC)-based immunotherapy is a potent anticancer modality. In DC-based immunotherapy, allogeneic DC may be an alternative source, but the usefulness of allogeneic DC in DC-based immunotherapy is still controversial. When used for immunotherapy, three factors may affect the efficiency of an allogeneic DC-driven antitumour response: (1) survival time, which is affected by T-cell alloresponses; (2) major histocompatibility complex incompatibility with the host cells in the context of antigen presentation; and (3) the role of host-derived professional antigen-presenting cells (pAPC). In addition, it is unclear which injection route is preferable when using allogeneic DC. In this study, we demonstrate that semi-allogeneic DC, which share half of the genes of the recipient, are more effective when used via the intratumoural (i.t.) injection route, rather than the subcutaneous (s.c.) injection route, for the induction of efficient antitumour effects and the generation of a significant tumour-specific CD8 T-cell response. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ. Allogeneic bone marrow transplantation (BMT) models, which permit us to separately assess the three factors described previously, show that while all three factors are important for efficient antitumour effects, the control of the alloresponse to injected DC is the most crucial for host-derived pAPC to function well when DC are administered intratumourally. This information may be useful for DC-based cancer immunotherapy under circumstances that do not allow for the use of autologous DC. [ABSTRACT FROM AUTHOR]

We reported that invariant NKT-cell knockout (iNKT KO) mice are resistant to the induction of intrathymic chimerism and clonal deletion in the cyclophosphamide (CP)-induced tolerance system (CPS). However, another report shows that clonal deletion with chimerism may be intact in iNKT KO recipients in a bone marrow transplantation model. We also reported that pretreatment with anti-Thy1.2 mAb, which reduces the number of T cells and iNKT cells, promotes allograft tolerance across H-2 barriers in the CPS. In this study, we evaluated the efficacy of T-cell depletion in the CPS, and the relationship between the role played by iNKT cells in central tolerance and mixed chimerism. BALB/c (H-2d) wild-type, or iNKT KO (Jα18−/−) mice were pretreated with 20–100 μg of anti-Thy1.2 mAb and given 108 donor DBA/2 (H-2d) spleen cells on Day 0, and 200 mg/kg CP on Day 2. Pretreatment with T-cell depletion resulted in higher levels of mixed chimerism, increased intrathymic clonal deletion of donor-reactive cells, and the induction of skin graft tolerance in iNKT KO recipients in CPS. This suggests that the high levels of mixed chimerism overcame the resistance to CP-induced tolerance in iNKT KO mice. Consistently, the enhancement of mixed chimerism by injection of tolerant donor spleen cells (SC) rendered iNKT KO recipients susceptible to CP-induced tolerance. These results suggest that iNKT-cell-mediated immunoregulation of central tolerance is evident at low levels of peripheral mixed chimerism in the CPS. [ABSTRACT FROM AUTHOR]

Cyclophosphamide (CP)-induced tolerance is a mixed chimerism-based tolerance and is one of the strategies used to induce transplant tolerance. Toll-like receptor (TLR) agonists are reportedly able to abrogate the induction of tolerance by activating alloreactive T cells, or by inhibiting Treg cells. However, little is known about the effect of the immune response mediated by TLR on mixed chimerism-based tolerance protocols. In this study, we evaluated the influence of lipopolysaccharide (LPS), which is best known as an TLR4 agonist, on CP-induced tolerance. BALB/c (H-2d) mice received a conditioning regimen consisting of 108 donor DBA/2 (H-2d) spleen cells (SC) on day 0 and 200 mg/kg CP on day 2. A single dose of 20 μg LPS was injected on day −2, 0, 7, or 35. Our results showed that LPS infusion at any time point resulted in chronic rejection of donor skin grafts and the abrogation of mixed chimerism in 33–60% of recipients. We found a correlation between skin graft acceptance and higher levels of mixed chimerism. Flow cytometric analysis revealed that donor-reactive T cells were permanently eliminated, regardless of LPS infusion. In conclusion, LPS-infusion had little influence on the immune response of donor-reactive T cells, but had a significant effect on the induction and maintenance of mixed chimerism in CP-induced tolerance. [ABSTRACT FROM AUTHOR]

Protection against Mycobacterium tuberculosis not only depends on CD4+ T helper type 1 (Th1) cells but, also, on CD8+ T cells. Interleukin (IL)-15 has an important function in the maintenance of memory CD8+ T cells. In the present study, we examined the efficacy of recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) secreting fusion protein antigen (Ag) 85B murine IL-15 (rBCG-Ag85B-IL15) in providing protection against M. tuberculosis infection. The levels of major histocompatibility (MHC) class Ib (H2-M3)-binding TB2- or MHC class Ia (H-2Db)-binding MPT64-specific CD8+ T cells producing interferon (IFN)-gamma were significantly higher after immunization with rBCG-Ag85B-IL15 than after immunization with rBCG secreting Ag85B (rBCG-Ag85B). The levels of purified protein derivative- or Ag85B-specific CD4+ T cells producing IFN-gamma were also higher in mice immunized with rBCG-Ag85B-IL15 than in mice immunized with rBCG-Ag85B. Mice immunized with rBCG-Ag85B-IL15 exhibited CD8+ and CD4+ T cells responses that were stronger than those in mice immunized with rBCG-Ag85B, as well as robust protection in the lung against intratracheal challenge of M. tuberculosis. Thus, rBCG-Ag85B-IL15 vaccination capable of inducing efficient cell-mediated immunity might be used as an effective vaccine for tuberculosis. Copyright © 2008 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Background Bacterial infection occasionally exacerbates asthma, although the cellular and molecular mechanisms have not been well defined. An involvement of mast cells has been suggested, as lipopolysaccharides (LPS)-induced cytokine production from mast cells in vitro. Objective This study was undertaken to examine the effects of LPS inhalation on mast cell functions and allergen-specific immune responses in a murine model of asthma. Methods Female BALB/c mice or mast cell-deficient W/Wv mice were immunized intraperitoneally with ovalbumin (OVA). Mice were challenged with aerosolized OVA or OVA with LPS daily from day 21 to day 24. Twenty-four hours after the last challenge, airway inflammation and OVA-specific immune responses were examined. Allergen-specific T cell responses were further analysed by adoptively transferring OVA-specific CD4+ T cells. Expression of chemokines in the lung was also examined. Results LPS inhalation with OVA resulted in exacerbated airway infiltration, which was not evident in mast cell-deficient mice. IL-5 production by mast cells in the lung was enhanced by LPS inhalation. OVA-specific IgE production as well as proliferation, cytokine production and local infiltration of OVA specific T-helper lymphocytes type 2 (Th2) were also enhanced. Up-regulated expression of Th2- and/or eosinophil-attracting chemokines was observed in the lung of mice inhalated with LPS. Conclusions LPS inhalation exacerbates airway inflammation, which is accompanied by mast cell activation and enhanced Th2 responses. These observations provide clues towards understanding the mechanisms of bacterial infection-induced exacerbation of the clinical features of asthma. [ABSTRACT FROM AUTHOR]

Background and aims: Interleukin (IL)-15 is a member of the IL-2 family, stimulating dendritic cells, natural killer (NK) cells, NK T cells and memory CD8+ T cells. IL-15 levels were elevated in the intestinal mucosa of inflammatory bowel diseases. Here we investigated the involvement of IL-15 in the pathogenesis of acute and chronic dextran sulphate sodium (DSS) induced colitis. Methods: IL-15 knockout (KO) mice and control C57BL/6 mice were used to induce colitis with DSS in their drinking water. Survival rate, clinical activity of diseases, extent of tissue damage, leucocyte population, and cytokine production of lamina propria (LP) cells of the large intestines were assessed. Results: IL-15 KO mice exhibited resistance to DSS induced acute colitis, as reflected by lower lethality, weight loss, clinical scores, and histological scores compared with those in control mice (p

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced human monocyte-derived macrophage (GM-Mφ) or macrophage CSF (M-CSF)-induced human monocyte-derived Mφ (M-Mφ) are distinct in terms of the resistance to Mycobacterium tuberculosis. To elucidate the role of molecules involved in the functional differences between these Mφs, we investigated the gene expression profiles using microarray. After culture of CD14+ monocytes with CSFs, Mφs were cultured with or without bacillus Calmette–Guérin (BCG) (GM-Mφ-BCG and M-Mφ-BCG). The gene expression profiles from these cells were compared. Chemokines highly expressed in M-Mφs were selected and evaluated for anti-mycobacterial activity and superoxide production. FN1 and FCGR2B were the most up-regulated genes in GM-Mφ and M-Mφ, respectively. After stimulation with BCG, three chemokine genes ( Osteopontin ( SPP1), CXC chemokine ligand 7 ( CXCL7) and CC chemokine ligand 11 ( CCL11)) were highly expressed in M-Mφ-BCG when compared to those in GM-Mφ-BCG. A significantly increased resistance to M. tuberculosis H37Ra was observed after the stimulation of GM-Mφ with SPP1 or CXCL7. Superoxide production levels of SPP1- or CXCL7-stimulated GM-Mφs were higher than those of GM-Mφs without stimulation. These results indicate that both SPP1 and CXCL7 might have a role in the resistance against mycobacteria, at least in part, through augmenting reactive oxygen intermediate production in Mφs. [ABSTRACT FROM AUTHOR]

Background Products from the fermentation process of soybeans by Bacillus subtilis ( natto) have been shown to possess anti-tumour and immunomodulatory activities. However, the formulations previously examined were not chemically pure, and this is a major limitation for elucidation of the molecular mechanisms for their activities. Objective In order to determine which components in soybean mucilage exert immunostimulatory activities, we examined the activities of their purified forms in vitro and in vivo in mice. Methods B. subtilis ( natto) and fractions including levan and poly-γ-glutamic acid (γ-PGA) from fermented soybean mucilage were prepared. Levels of cytokine production by mouse macrophage cells after treatment with the fractions were measured by means of ELISA. In vivo effect of levan delivered intragastrically on ovalbumin (OVA)-specific T-helper type 2 (Th2) response with IgE production was examined in BALB/c mice that had been immunized intraperitoneally with OVA. Results Levan but neither γ-PGA nor killed B. subtilis ( natto) was found to exert strong activity to induce production of IL-12 p40 and TNF-α by macrophage cell lines in vitro. Results of experiments using Toll-like receptor (TLR) 4-deficient mice and TLR4-transfected human cell line indicated that TLR4 is involved in pattern recognition of levan. Oral administration of levan in vivo significantly reduced the serum levels of OVA-specific IgE and Th2 response to OVA in mice immunized with OVA. Conclusion Levan is an immunostimulatory moiety in products from the fermentation process of B. subtilis ( natto) and may be useful for prevention of allergic disorders with IgE production. [ABSTRACT FROM AUTHOR]

Background and aims: Surgical management of extrahepatic cholestasis is frequently complicated by bacterial translocation and severe liver injury. The aim of this study was to clarify the involvement of Toll-like receptors (TLRs) in the pathogenesis of bacterial translocation and liver injury in obstructive cholestasis. Methods: TLR2 deficient (TLR2-/-), MyD88-/-, Jα281-/-, gld/gld, and lpr/lpr mice, all of which have a C57BL/6 background, and C3H/HeN and TLR4 mutated C3H/HeJ mice were subjected to bile duct ligation (BDL). Faecal IgA and serum alanine aminotransferase levels were determined after BDL. Apoptosis was examined by histological and flow cytometric analyses of cells from Peyer's patches and the liver. Results: The size and number of B cells in Peyer's patches markedly decreased on day 3 after BDL. Increased apoptosis in Peyer's patch B cells was evident on day 1 after BDL in control mice but not in lpr/lpr, MyD88-/-, or C3H/HeJ mice. On the other hand, TLR2 and Fas ligand expression on intrahepatic NK1.1+T cells increased on day 1 after BDL in C57BL/6 mice. Liver injury and apoptosis were evident on day 1 after BDL in control and C3H/HeJ mice but were significantly reduced in TLR2-/-, Jα281-/-, gld/gld, and lpr/lpr mice. Conclusions: TLR4 and TLR2 may play important roles in Fas dependent apoptosis in Peyer's patch B cells and hepatocytes, respectively, at an early stage after BDL in mice. [ABSTRACT FROM AUTHOR]

Summary Major histocompatibility complex (MHC) class I-specific inhibitory receptors are expressed not only on natural killer (NK) cells but also on some subsets of T cells. We here show Ly49 expression on γ/δ T cells in the thymus and liver of β2-microglobulin-deficient (β2m-/-) and C57BL/6 (β2m+/+) mice. Ly49C/I or Ly49A receptor was expressed on NK1.1+γ/δ T cells but not on NK1.1-γ/δ T cells. The numbers of NK1.1+γ/δ T cells were significantly smaller in β2m+/+ mice than in β2m-/- mice with the same H-2b genetic background. Among NK1.1+γ/δ T cells, the proportions of Ly49C/I+ cells but not of Ly49A+ cells, were decreased in β2m+/+ mice, suggesting that cognate interaction between Ly49C/I and H-2Kb is involved in the reduction of the number of Ly49C/I+ γ/δ T cells in β2m+/+ mice. The frequency of Ly49C/I+ cells in NK1.1+γ/δ T cells was lower in both lethally irradiated β2m+/+ mice transplanted with bone marrow (BM) from β2m-/- mice and lethally irradiated β2m-/- mice transplanted with BM from β2m+/+ mice than those in adult thymectomized BM-transplanted chimera mice. These results suggest that reduction of Ly49C/I+ NK1.1+γ/δ T cells in β2m+/+ mice is at least partly due to the down-modulation by MHC class I molecules on BM-derived haematopoietic cells or radioresistant cells in the thymus. [ABSTRACT FROM AUTHOR]

Summary Cytomegalovirus (CMV) causes severe opportunistic infection in immunocompromised hosts. The importance of conventional αβ T cells in protection against CMV infection has been well documented. However, the role of the second T‐cell population (which express the γδ T‐cell receptor) in CMV infection is not known. In the present study, we analysed the function and protective role of γδ T cells in a murine cytomegalovirus (MCMV) infection model. After intraperitoneal infection with MCMV, the number of γδ T cells increased in the liver and peritoneal cavity from day 3, and reached a peak on day 5. The γδ T cells showed an activated T‐cell phenotype and predominantly expressed Vγ1, which is known to be expressed by heat‐shock protein 65 (hsp 65)‐specific γδ T cells. Analysis of cytokine expression demonstrated that the MCMV‐induced γδ T cells expressed interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) but not interleukin‐4 (IL‐4), implying their participation in the cell‐mediated immune response against MCMV. Depletion of γδ T cells by anti‐T‐cell receptor (TCR) γδ monoclonal antibody (mAb) treatment resulted in significant increase of virus titre and decrease of IFN‐γ in the liver on day 3 after MCMV infection, which further supports the importance of γδ T cells in early protection against infection. Finally, the MCMV‐induced γδ T cells produced IFN‐γ in vitro in response to hsp 65. Our results suggest that γδ T cells participate in early protection against MCMV infection through recognition of hsp 65 and production of IFN‐γ. [ABSTRACT FROM AUTHOR]

We examined the host defence mechanism against infection with Listeria monocytogenes, a facultative intracellular bacterium, in mice with murine acquired immunodeficiency syndrome (MAIDS) caused by LP-BM5 murine leukaemia virus (MuLv) infection. Although LP-BM5 MuLV infection in C57BL/6 mice leads to a stage of immunodeficiency characterized by severe compromise of cell-mediated immunity, the mice with established MAIDS infected with LP-BM5 8 weeks previously, showed resistance to an intraperttoneal infection with Listeria monocytogenes. These MAIDS mice also showed resistance to a lethal dose of secondary listerial challenge, while the delayed-type hypersensitivity response to heat-killed Listeria (HKL) was severely impaired in MAIDS mice. The resistance of MAIDS mice to listerial infection was mediated by CD4+ αβ T cells but neither by γδ T cells nor natural killer (NK) cells. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) were produced by CD4+ T ceils from Listeria-infected MAIDS mice in response tO the in vitro stimulation with HKL, whereas IFN-γ but not IL-10 were produced by those from Listeria-infected control mice. These results suggest that T-helper 0 (Th0)-like immune responses of CD4+ T cells occur and participate in host defence mechanisms against listerial infection in MAIDS mice. [ABSTRACT FROM AUTHOR]

We have previously reported that heat-shock protein (hsp) 60-reactive T-cell receptor (TCR)γδ+ T cells appear in the peritoneal cavity during the early stage of infection with Listeria monocytogenes in mice. In this study, we examined the kinetics of TCRγδ+ T cells during listeriosis in F344 ruts by flow cytometry using a V65 monoclonal antibody (mAb) directed to a constant determinant of rat TCRγδ chains. TCRγδ+ T cells significantly increased in the peritoneal cavity on day 6 and then decreased by day 10 after infection, in parallel with the kinetics of hsp 60 expression in the peritoneal macrophages during listeriosis in F344 rats. Most of the early appearing TCRγδ+ T cells were of the CD4- CD8αβ+ CD5+ lymphocyte function-associated antigen (LFA)-1αhigh CD45RC- interleukin-2 receptor (IL-2R) α- phenotype, although a significant fraction of the TCRγδ+ T cells expressed CD8σ only. The increase in TCRγδ+ T cells during listeriosis was prominent in F1 (F344 × Lewis) rats but only marginal in Lewis rats, which was correlated with the expression level of hsp 60 in the peritoneal macrophages. The peritoneal TCRγδ+ T cells in naive F344 rats appeared to proliferate significantly in response to recombinant hsp 60 (rhsp 60) derived from Mycobacterium bovis bacillus Calmette-Guérin (BCG). These results imply that the early appearance of hsp 60-reactive TCRγδ+ T cells during listerial infection can be generalized across species. [ABSTRACT FROM AUTHOR]

Conversion of Salmonella typhimurium to L-forms, both in vitro and in vivo, resulted in the expression of proteins cross-reacting to the mycobacterial 65000 MW heat-shock protein (hsp). Immunization of C3H/HeJ mice with a protective dose of stable L-form S. typhimurium induced γδ T cells in the liver, in accordance with the multiplication of L-form Salmonella in Kupffer cells. The number of γδ T cells decreased after the intracellular growth of L-form Salmonella plateaued. Persistance of the L-forms in Kupffer cells, however, allowed hepatic γδ T cells to increase within 48 hr of infection with virulent S. typhimurium. Thus, the intrahepatic colonization of L-form Salmonella seems to keep γδ T cells on standby, but the emergence of these T cells does not correlate with the expression of L-form hsp. In addition, Kupffer cells colonized by L-forms constitutively synthesized mRNA for interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). These results suggest that conversion of S. typhimurium to L-forms in phagocytic cells builds up and maintains acquired resistance, conferred by live-cell vaccines of S. typhimurium, against murine typhoid. [ABSTRACT FROM AUTHOR]

We investigated the host defense mechanism in primary infection with Listeria monocytogenes in non-obese diabetic (NOD) mice at pre-diabetic stage showing an impaired responsiveness of the αβ T cells to T-cell receptor (TCR) triggering. The NOD mice showed a deteriorated resistance at the late stage after an intraperitoneal infection with L. monocytogenes compared with BALB/c and C57BL/6 mice as assessed by bacterial growth in organs. Consistent with our previous findings, a prominent increase in number of γδ T cells was evident at the early stage after infection, while generation of Listeria-specific αβ T cells was impaired in these mice. In vivo administration of anti-TCR γδ monoclonal antibody (mAb) allowed L. monocytogenes to grow exaggeratedly in the NOD mice. These results imply that γδ T cells may be mainly involved in protection against primary infection with L. monocytogenes in NOD mice. [ABSTRACT FROM AUTHOR]

Interleukin-10 (IL-10) is a cytokine that regulates various macrophage functions. To elucidate the involvement of endogenous IL-10 in the early stage of murine salmonellosis, we examined the effect of anti-IL-10 monoclonal antibody (mAb) administration on the host defence mechanism against Salmonella choleraesuis infection. The in vivo administration of anti-IL-10 mAb significantly enhanced host resistance at the early stage of Salmonella infection, as assessed by bacterial growth in the peritoneal cavity and the liver. Enhanced levels of monokine mRNA, including IL-1α, tumour necrosis factor-α (TNF-α) and IL-12, were observed from day I after infection in the peritoneal macrophages in anti-IL-10 mAb-treated mice compared with those in control mAb-treated mice. Mice treated with anti-IL-10 mAb exhibited significantly higher levels of interferon-γ (IFN-γ) in the peritoneal exudate's and major histocompatibility complex (MHC) class II expression on the peritoneal macrophages on days 3 and 5 after infection. Notably, in vivo anti-IL-10 mAb brought about an increment of γδ T cells in the peritoneal cavity at the early phase of infection, which was correlated with the expression of endogenous heat-shock protein 60 (HSP60), which is implicated as a potential ligand for γδ T cells, in the infected macrophages. Our results suggest that the neutralization of endogenous IL-10 accelerates some macrophage functions and, consequently, the activation of immunocompetent cells, including & grave;δ cells, at the early stage of infection, resulting in an enhanced host defence against Salmonella infection.Interleukin-10 (IL-10) is a cytokine that regulates various macrophage functions. To elucidate the involvement of endogenous IL-10 in the early stage of murine salmonellosis, we examined the effect of anti-IL-10 monoclonal antibody (mAb) administration on the host defence mechanism against Salmonella choleraesuis infection. The in vivo administration of anti-IL-10 mAb significantly enhanced host resistance at the early stage of Salmonella infection, as assessed by bacterial growth in the peritoneal cavity and the liver. Enhanced levels of monokine mRNA, including IL-1α, tumour necrosis factor-α (TNF-α) and IL-12, were observed from day I after infection in the peritoneal macrophages in anti-IL-10 mAb-treated mice compared with those in control mAb-treated mice. Mice treated with anti-IL-10 mAb exhibited significantly higher levels of interferon-γ (IFN-γ) in the peritoneal exudate's and major histocompatibility complex (MHC) class II expression on the peritoneal macrophages on days 3 and 5 after infection. Notably, in vivo anti-IL-10 mAb brought about an increment of γδ T cells in the peritoneal cavity at the early phase of infection, which was correlated with the expression of endogenous heat-shock protein 60 (HSP60), which is implicated as a potential ligand for γδ T cells, in the infected macrophages. Our results suggest that the neutralization of endogenous IL-10 accelerates some macrophage functions and, consequently, the activation of immunocompetent cells, including & grave;δ cells, at the early stage of infection, resulting in an enhanced host defence against Salmonella infection. [ABSTRACT FROM AUTHOR]

Neonatally thymectomized (NTx) mice, whose ability to mount antigen-specific cell-mediated immunity is reported to be generally defective, were found to be capable of mounting a normal level of acquired cellular resistance (ACR) and delayed footpad reaction (DFR) to Listeria monocytogenes. The present study was done in order to determine the functional differences of T cells contributing to the protection against L. monocytogenes between NTx and sham-operated mice. In mice immunized with viable L. monocytogenes, the absolute number of splenic T cells was significantly lower in NTx mice compared with sham-operated mice. When the ability of immune T cells to transfer ACR and DFR was examined by passive transfer, lymphocytes from immune NTx mice conferred a higher level of ACR and DFR on naive recipient mice, despite the marked difference in total number of T cells compared with immune Sham mice. Antigen-specific proliferation and interleukin-2 (IL-2) production by splenic T cells from immune NTx mice were significantly lower than in those from immune Sham mice. The proliferative response of T cells to exogenous IL-2 was also lower in NTx group. These results suggest that the requirement for the IL-2-driven T-cell proliferation system is basically low in the generation of effector T cells specific for L. monocytogenes. [ABSTRACT FROM AUTHOR]

We previously reported that the number of TNF-α-producing cells was increased in the liver of patients with type C chronic liver disease. To understand further the pathophysiology of this change, we examined serum levels of two soluble TNF receptors, TNF-αRI (p55)and -αRII (p75), and IL-10, all of which act as TNF-α buffer, and IL-15, a novel cytokine sharing many immunological activities with IL- 2, using ELISA methods. We studied control individuals and patients with type C chronic liver disease, including asymptomatic hepatitis C virus (HCV) carriers with persistently normal serum ALT values, and those with chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Both types of sTNF-αR closely correlated with disease progression. Patients with LC and HCC had significantly elevated levels for sTNF-αRII compared with the other patient groups and controls. Serum IL-10 levels were significantly greater in all chronic liver disease groups than in controls. With respect to IL-15, the values were high in CH, LC and HCC compared with those of controls. Notably, HCC patients showed highest values for both IL-10 and IL-15, with significant differences from the other patient groups. Serial determinations revealed that interferon (IFN) treatment for CH patients resulted in the suppression of circulating lL-10 and IL-15 levels along with decrease in serum aminotransferase values. Both cytokines remained at decreased levels after cessation of therapy in patients who went into clinical and virological remission. On the other hand, treatment did not affect serum levels of sTNF-αRs, These findings indicate that serum levels of these molecules correlated with disease progress in chronic HCV infection, and that IL-10 and IL-15 may reflect the degree of inflammation in the liver. It is also suggested that both cytokines may be related to the development of HCC. [ABSTRACT FROM AUTHOR]