The nonopsonic allogeneic cell phagocytosis of macrophages detected by flow cytometry and two photon fluorescence microscope

Phagocytosis, one of the apparent functions for macrophages, represents an early and crucial event in triggering host defenses against invading pathogens as well as allo- or xenogeneic rejection. Now, some methods have been used in detecting the opsonic phagocytosis of macrophages in xenogeneic settings. Efficient nonopsonic phagocytosis analysis method has not been established yet. In the present studies, allogeneic lymphocytes pre-labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) or derived from green fluorescent protein transgeneic B6 mice (GFP-B6 mice) were co-incubated with primary murine peritoneal macrophages (PEMs) for 1–2 h or were injected into murine peritoneal cavity for 30 to 240 min. Assays by flow cytometry (FCM) and two photon laser scanning microscope (TPM) showed an efficient uptake of both allogeneic lymphocytes and xenogeneic chicken red blood cells. The continuing process of nonopsonic phagocytosis of allogeneic lymphocytes by PEMs was recorded by TPM. Furthermore, the phenotype differences of PEMs with or without phagocytosis of allogeneic cells were determined by three-color FCMs. Significantly upregulated expressions of CD11b, CD44, TLR2 and TLR4 on PEMs were observed as early as 6 h after phagocytosis of allogeneic cells. Our present data indicated that the FCM and TPM combined method is a practical approach to detect macrophage nonopsonic phagocytosis of allogeneic lymphocytes and to identify the phenotype alteration of macrophages after phagocytosis.     

Comparison of activation requirements and activation phenotype of allogeneic and xenogeneic rodent responses in vivo and in vitro

BACKGROUND: Both discordant and concordant xenogeneic responses are dominated by humoral immunity. Recent advances in molecular engineering approaches may largely prevent rejection by means of this pathway, leaving the cellular arm of the immune response as the principal remaining barrier to successful engraftment. METHODS: To characterize further the cellular response to xenogeneic tissues, we used the intracellular fluorescent marker CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) to track the mitotic record of T cells (and T cell subsets) after either xenogeneic or allogeneic activation in vitro or in vivo. Activation marker expression was monitored by simultaneous labeling with antibodies for either CD25 or CD134. RESULTS: The in vitro and in vivo responses of Lewis lymphocytes were generally similar in magnitude and timing comparing activation with allogeneic or xenogeneic stimulators. However, the xenogeneic T cell precursor frequency was found to be markedly higher than that previously reported and were comparable to that seen in allogeneic responses. Xenogeneic responses were unique in the continued expression of activation markers in later division cycles. In addition, CD4 and CD8 T-cell proliferation was highly dependent on stimulator class II expression, highlighting the importance of CD4 T cells and the indirect pathway in the xenogeneic response. CONCLUSIONS: An unexpectedly high precursor frequency was detected for xenogeneic cellular responses in the rat anti-mouse combination and was comparable to that seen in allogeneic responses. Differences in xenogeneic versus allogeneic activation profiles exist that may result from the cellular pathways used for activation.     

Characteristics of direct and indirect activation of human T cells against allogeneic and porcine xenogeneic cells/peptides

Abstract: The immunological consequences of direct versus indirect activation of T lymphocytes is of considerable interest both in allogeneic and in xenogeneic transplantation. In this paper, the activation of human T cells by allogeneic and xenogeneic porcine antigen-presenting cells (APC) (direct activation), or by donor-derived peptides presented on autologous APC (indirect activation) was studied in vitro. The direct activation of purified human T lymphocytes by allogeneic and xenogeneic porcine stimulator cells led to marked proliferative responses, IL-2 and IFN-γ production. The ratio of CD4/CD8 of responsive cells upon direct activation was the same for allogeneic and xenogeneic stimulation. Also, the CD25 expression on total and on CD8+ lymphoblasts was similar in allogeneic and xenogeneic responses. The frequency of alloreactive and xenoreactive CTLp following direct activation was 7.7 ± 1.4 / 10⁴ and 3.7 ± 1.3 / 10⁴, respectively. Direct activation of proliferation and IL-2 and IFN-γ production in both experimental situations was completely blocked by Cyclosporin A (CsA). 15-deoxyspergualin (DSG), which is thought to prevent antigen processing, had little effect. Thus, when it comes to direct activation of T lymphocytes xeno- and allospecific responses were qualitatively and quantitatively comparable. However, some differences between allogeneic and xenogeneic responses were observed when indirect activation was studied using stimulator cells from peripheral blood depleted of both adherent cells and of SLA class II positive cells or when human allogeneic or xenogeneic fibroblasts were used as stimulators. Indirect activation following recognition of alio- or xenogeneic peptides in a responder HLA-restricted fashion resulted in proliferation, IL-2 and IFN-y production, albeit lower than in direct combinations. Indirect activation also induced a lower proportion of CD25+ lymphoblasts than direct activation. Furthermore, indirect xenoactivation resulted preferentially in proliferation and CD25 expression in the CD4+ cell population, and in contrast to direct activation, not only CsA but also DSG inhibited activation. Thus, our findings indicate that direct and indirect activation of human responder T cells stimulated by allogeneic and/or xenogeneic porcine cells/peptides results in immune responses with similar characteristics. Only the proliferation of CD8+ cells and their expression of CD25 and of functional IL-2 receptors was weaker following xeno- than alloactivation, reasons for which are discussed. We conclude that indirect activation of T cell reactivity against xenogeneic peptides leads to an efficient immune response, likely to be of clinical importance for rejection of xenogeneic transplants devoid of professional antigen-presenting cells.     

IFN-gamma but not IL-4 is important for mouse CD4+ T cell-mediated macrophage activation following their exposure to pig cells in vitro

In order to investigate the mechanism by which CD4+ T cells and macrophages interact in the xenogeneic immune response, murine CD4+ T cells and macrophages were used as responder cells in culture with irradiated fetal pig spleen cells (FPSC) as pig xenogeneic stimulators. In this in vitro model, murine CD4+ T cells and macrophages were cultured individually, or together with FPSC. In addition, mouse CD4+ T cells were also cultured with autologous macrophages which were previously stimulated by FPSC. The cultured murine cells were analyzed for expression of CD4+ T cell and macrophage activation markers (cell surface markers and cytokines) as well as cytokine production. CD4+ T cells and macrophages cultured alone or together without FPSC showed unchanged low levels of expression of activation markers. Coculture of macrophages with FPSC and in the absence of CD4+ T cells induced increased expression levels of all the activation markers examined except B7.2 and ICAM-1. Addition of CD4+ T cells to the coculture further enhanced this up-regulation. Coculture of CD4+ T cells with FPSC-stimulated macrophages, but not naive macrophages, or FPSC alone, resulted in significantly increased numbers of CD4+ T cells coexpressing their activation markers, especially IFN-gamma and CD40L, and this expression was enhanced further by including FPSC in the coculture. The activation of both CD4+ T cells and macrophages in their coculture with FPSC was suppressed by neutralizing IFN-gamma but not IL-4. Our results demonstrated that interaction of CD4+ T cells and autologous macrophages was required for their optimal activation in response to pig xenogeneic stimulation. The mechanisms involved included cell-cell and/or cytokine interactions, and in particular IFN-gamma mediated communication was involved. Macrophages activated by pig cells in the absence of CD4+ T cells were able to activate naive CD4+ T cells, thus providing an important communication pathway between innate immune activation and a T cell mediated response in xenograft rejection.     

Antisense deoxyoligonucleotides or antibodies to murine MD-1 inhibit rejection of allogeneic and xenogeneic skin grafts in C3H mice

BACKGROUND: Altered expression of murine MD-1, a molecule controlling expression of members of the interleukin (IL)-1 receptor family of signaling proteins, regulates antigen-presenting cell-induced alloreactions. We investigated the effect of treatment with antisense deoxyoligonucleotides or antibodies to MD-1 in vivo on allogeneic and xenogeneic skin graft survival and the immune responses in transplanted mice. METHODS: C3H mice received C57BL/6 or Lewis rat skin grafts, followed by i.v. injections of anti-MD-1 antibody or antisense oligonucleotides or control reagents at 48-hr intervals. Survival was monitored. In separate studies, mice were sacrificed at 5-day intervals. Serum was analyzed for circulating MD-1 antigen, and peritoneal cells for surface expression of MD-1. The proliferative and cytolytic response of lymphocytes harvested from treated animals and restimulated in vitro with allo- or xenogeneic cells, and the cytokines produced, was measured. Graft histology was assessed at 11 days after transplantation. RESULTS: Treatment with anti-MD-1 oligonucleotides or antibodies suppressed rejection of both xeno- and allogeneic grafts, decreased induction of graft-specific cytotoxic T cells, increased production of type-2 cytokines (IL-4 and IL-10), and decreased production of type-1 cytokines (IL-2 and interferon-gamma). Serum levels of MD-1 were suppressed, as was expression of MD-1 on the surface of antigen-presenting cells. Grafts from MD-1-treated mice showed little lymphocyte infiltration, and no signs of graft necrosis. CONCLUSION: Our data suggest a critical in vivo role for MD-1 expression in regulating graft rejection, as well as in the concomitant sensitization of T cells and their cytokine production profile, which parallels the rejection response.     

Effect of DSG on xenogeneic immune reactivity with special emphasis on human anti-pig cellular reactions in vitro

Abstract: The effect of the immunosuppressive drug 15-deoxyspergualin (DSG) on xenogeneic human anti-porcine cellular reactivity in vitro, including MLR induced proliferation, interleukin-2 (II-2) production, generation of cytotoxic cells, and the effect on antibody-dependent cellular cytotoxicity (ADCC), were compared with the effects of cyclosporin A (CsA) and/or FK506. The cytotoxic response was evaluated for both direct and indirect pathways for antigen presentation. In addition, the effects of DSG and CsA on antibody production to pig peripheral blood lymphocytes (PBL) in mice was studied. The degree of immunosuppression of xenogeneic and allogeneic cellular responses was compared. CsA and FK506 effectively inhibited proliferation and II-2 production induced by allogeneic human PBL or xenogeneic porcine PBL, whereas DSG did not have any effect on these responses. However, DSG suppressed both the allogeneic and xenogeneic in vitro induced cytotoxic responses, to the same level whether induced via the direct or indirect pathways of immune activation. In contrast, CsA inhibited cytotoxicity induced by xenogeneic cells via the direct but not via the indirect pathway. No effect of FK506 and DSG on ADCC was demonstrated.
A 5-day treatment with DSG or CsA of mice immunized with pig PBL partly suppressed antibody production. In DSG treated mice anti-pig PBL antibodies were produced, but titers were lower than in nontreated or CsA treated mice. The results indicate that DSG may be more effective than CsA/FK506 in inhibiting cytotoxic responses and antibody production induced by xenogeneic pig cells. A possible explanation could be that cytotoxicity induced via the indirect activation pathway of xenoreactivity is mediated to a high degree by CD3^- CD16⁺ (natural killer) NK-like cells, and that stimulation of these cells may be more sensitive to DSG than to CsA/FK506.     

Human CD8 responder cells can be directly activated to proliferate and to produce IL-2 following stimulation by allogeneic, but not by xenogeneic, porcine blood mononuclear cells

Abstract: In order to determine the precise nature of human T lymphocytes reactivity against porcine stimulator cells, purified CD4₊ and CD8+ human peripheral T lymphocytes have been tested for their responsiveness against porcine stimulator cells. In a xenogeneic mixed lymphocyte reaction (MLR), CD4+ T cells were capable of proliferating as a result of the recognition of porcine peripheral blood lymphocytes (PBL), whereas CD8+ T cells were unresponsive. A proliferative response of CD8+ T cells could be restored by treatment with human IL-2, but not by IL-lα, IL-lβ, or IL-6. Production of IL-2 was not detected in the xenostimulated CD8+ responder cells, nor could IL-2 production be restored by the addition of IL-lα, IL-1β, or IL-6. The presence of human CD4₊ responder cells was crucial both for a xenoproliferative response and for IL-2 synthesis. However, when the expression of the IL-2 receptor (CD25) on the CD8₊ T cells was analyzed, no difference was detected between xenostimulated and allostimulated CD8₊ T cells. When the development of cytotoxic T cells in xenogeneic and allogeneic MLRs was compared, the cytotoxic activity exhibited by purified CD8+ T cells in xenogeneic MLR was significantly lower than that in the allogeneic combination. In the xenogeneic combination, exogenous IL-2 reconstituted the cytotoxicity by purified CD8₊ T cells; however, IL-lα, IL-lβ, or IL-6 did not.
Our results show that purified human CD4₊ T cells respond directly against pig PBMCs, whereas purified CD8₊ T cells do not. Furthermore, responsiveness in CD8₊ T cells is completely restored by the addition of human IL-2.     

Toll-like receptor 4 plays a role in macrophage phagocytosis during peritoneal sepsis

Background

Peritoneal sepsis is a significant cause of mortality in infants with necrotizing enterocolitis, caused in part by impaired bacterial clearance. Recent studies have identified toll-like receptor-4 (TLR4) as a receptor for endotoxin (lipopolysaccharide [LPS]). We hypothesized that TLR4 regulates bacterial clearance from the peritoneal cavity and sought to investigate whether macrophage phagocytosis was involved.

Methods

Peritoneal sepsis was induced in mice expressing either functional TLR4 (TLR4-wild-type [WT]) or mutant TLR4 by intraperitoneal injection of either live Escherichia coli or LPS. Phagocytosis was assessed by measuring the uptake of opsonized red cells. To assess bacterial clearance, we irrigated peritoneal cavities of injected animals with saline and plated it on gram-negative selective media.

Results

LPS significantly increased the rate of phagocytosis by peritoneal macrophages from TLR4-WT mice, but not in those from TLR4-mutant mice, suggesting a role for TLR4 in phagocytosis. LPS also increased the rates of phagocytosis in cultured macrophages expressing TLR4, confirming these findings. The yield of gram-negative bacteria obtained from the peritoneal cavities of septic TLR4-WT mice was greater than that from TLR4 mutants, consistent with TLR4-dependent alterations in their septic course.

Conclusions

We conclude that TLR4 plays a critical role in the response to intraperitoneal E. coli through effects on phagocytosis by macrophages, suggesting the possibility of using TLR4 as a therapeutic target in diseases of peritoneal sepsis.     

Xenogeneic human anti-pig cytotoxicity mediated by activated natural killer cells

Abstract: Even if hyperacute rejection, which is mediated by human natural antibodies (nAb) and complement, could be prevented, xenoreactive human anti-pig cellular responses may lead to delayed and/or chronic xenograft rejection. Among the cell populations participating in such rejection, NK cells have been proposed as an important component. In this study we report the in vitro cytotoxic activity of natural killer (NK) cells obtained from healthy human donors against porcine target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) and purified NK cells (CD16+/CD56+, CD3-, CD20-, CD33-) exhibited little or no cytotoxic activity when tested on porcine phytohemagglutinin (PHA)-stimulated lymphoblasts or bone marrow- or aortic-derived endothelial cell lines in the presence of serum-free medium. Killing was considerably higher in the presence of human decomplemented plasma, containing xenoreactive nAb, or purified Gal(α1,3)Gal-reactive antibodies, suggesting that antibody dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells is an important mechanism involved in xenogeneic cytotoxicity. After incubation of human PBMC for 6 days in the presence of irradiated xenogeneic porcine or allogeneic stimulator cells, or in the presence of exogenous interleukin 2 (IL-2), the cytotoxic activity of the bulk cultures as well as that of isolated NK cells (separated from stimulated bulk cultures) against xenogeneic targets increased considerably, and corresponded to an increased NK-specific lysis of K562 target cells. Cell surface staining and flow cytometry showed that CD16+/CD56+, CD3- NK cells composed ca. 25% of short-term (6 days) xenogeneic, allogeneic, or IL-2 stimulated bulk cultures. In summary, these data suggest that, in contrast to allogeneic cell- mediated killing, xenogeneic human anti-porcine cytotoxicity includes an important contribution from NK cells.     

HSP70 enhances macrophage phagocytosis by interaction with lipid raft-associated TLR-7 and upregulating p38 MAPK and PI3K pathways

BACKGROUND: Exogenous Heat Shock Protein-70 (HSP70), a product of necrotic cell death, binds the lipid raft microdomains of macrophages and, within minutes, stimulates the phagocytosis and presentation of internalized antigens. The aim of this study was to identify (a) the receptor on the lipid raft microdomain that interacts with HSP70 and (b) the subsequent signaling pathways that mediate HSP70-enhanced phagocytosis. METHODS: Cells including RAW264.7, bone-marrow-derived macrophages from TLR7-/- mice or controls and cells subjected to genetic methods reducing the mRNA expression of TLR7 were used to examine the interaction of HSP70 with TLR7. The effect of HSP70-TLR7 interaction on phagocytosis was assessed using phagocytosis assays described earlier. RESULTS: HSP70 binds Toll-like receptor-7 (TLR7) on the lipid raft microdomain of macrophage plasma membrane. Subsequent signaling is mediated through phosphoinositide 3-kinase (PI3K) and the up-regulation of the p38 MAP kinase pathways, both known activators of the phagocytic mechanisms. Reduced expression of TLR7 either via short interfering RNA for TLR7 (siRNA-TLR7) or using bone-marrow derived macrophages from TLR7-/- mice show that, as macrophages lose expression of TLR7, their ability to mediate HSP70-induced phagocytosis undergoes a corresponding diminution. Similarly, disruption of lipid rafts or blocking HSP70-TLR7-interaction or treatment with wortmannin and SB203580, inhibitors of PI3K or p38 MAPK, respectively, abrogates HSP70-induced macrophage phagocytosis. CONCLUSIONS: The interaction of HSP70 and LR-associated TLR7, two phylogenetically conserved molecules, activates a rapid, membrane-bound signaling pathway that enhances phagocytosis, a vital innate defense mechanism. This study elucidates critical mechanistic elements that mediate HSP70-enhanced phagocytosis by macrophages.     

Requirement of MyD88 for macrophage-mediated islet xenograft rejection after adoptive transfer

BACKGROUND: Porcine antigen primed and CD4+ T-cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. METHODS: In this study macrophages which lack the universal toll-like receptor (TLR) adaptor MyD88 were used to investigate the role of TLR in the recognition and activation of macrophages in islet xenograft rejection. Macrophages were isolated from rejecting MyD88(-/-) and wild-type C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Both wild-type C57BL/6 and MyD88(-/-) mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation, and the grafts were heavily infiltrated with CD4+ T cells and macrophages. However, graft infiltrating macrophages from rejecting MyD88(-/-) recipients demonstrated impaired up-regulation of TLR expression and impaired activation phenotype, when compared to those from rejecting C57BL/6 recipients. Transfer of NOD-SCID recipients with macrophages from rejecting C57BL/6 mice resulted in NPCC xenograft rejection along with massively infiltrated macrophages 8 days after transfer, whereas NPCC xenografts in NOD-SCID mice transferred with macrophages from rejecting MyD88(-/-) mice remained intact until the end of this study, 90 days after transfer, with insulin-positive islets and no infiltration by macrophages. CONCLUSION: This study demonstrates that deletion of MyD88 causes impaired macrophage activation after pig islet xenotransplantation. However, graft survival is not prolonged and xenografts are rejected rapidly by alternate mechanisms.     

Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis

Purpose

Various strategies, such as the generation of alpha-1,3-galactosyltransferase knocked-out pigs and CD55 transgenic pigs, have been investigated to inhibit pig to human xenogeneic rejection. Our aim is to develop strategies to overcome the hurdle of not only hyper acute rejection, but also that of cellular xenogeneic rejection (CXR). Although macrophages have been well known to play a critical role in CXR, monocyte/macrophage-mediated xenogeneic rejection has not been well studied. In this study, we evaluated the effect of CD200 in xenogeneic rejection by macrophages.

Methods

Naïve swine endothelial cells (SEC) and SEC/CD200 were co-cultured with M0 macrophages and the cytotoxicity was measured by a WST-8 assay. The phagocytosis of SEC and SEC/CD200 by macrophages was analyzed by flow cytometry.

Results

While CD200 failed to suppress a significant amount of cytotoxicity against SEC by monocytes, M0 macrophage-mediated cytotoxicity was significantly suppressed by human CD200. The phagocytosis by M0 macrophages was also tested. The phagocytosis assay revealed that human CD200 suppresses M0 macrophage-mediated phagocytosis.

Conclusions

Our findings indicate that human CD200 suppresses the xenogeneic rejection by CD200R⁺ macrophages and that the generation of hCD200 transgenic pigs for use in xenografts is very attractive for preventing the macrophage-mediated rejection.

     

Simultaneous inhibition of B7 and LFA-1 signaling prevents rejection of discordant neural xenografts in mice lacking CD40L

Abstract: Transplantation of embryonic human neural tissue can restore dopamine neurotransmission and improve neurological function in patients with Parkinson's disease. Logistical and ethical factors limit the availability of human embryonic allogeneic tissue. Embryonic xenogeneic neural tissue from porcine donors is an alternative form of donor tissue, but effective immunomodulatory techniques are warranted for neural xenotransplantation to become clinically feasible. We transplanted embryonic porcine ventral mesencephalic tissue into the brains of adult untreated C57BL/6 mice, untreated CD40L-/–mice and CD40L-/–mice that received injections of anti-LFA-1, CTLA4Ig or both compounds. Double-treated CD40L-/–mice had large grafts with high numbers of dopaminergic neurons 4 wk after transplantation. The grafts were completely devoid of lymphocytes, macrophages and activated microglia. Untreated C57BL/6 mice had rejected their grafts. Untreated CD40L-/–mice and CD40L-/–mice treated with monotherapy of anti-LFA-1 or CTLA4Ig had smaller grafts and more microglial and lymphocytic infiltration than double-treated CD40L-/–mice. We conclude that immunomodulation with concomitant inhibition of LFA-1 and B7 signaling in the perioperative period in CD40L-/–mice prevented the rejection of discordant neural xenografts. The treatment most likely reduced antigen presenting capacity and interfered with the costimulatory signaling needed for T cell activation to occur.     

CD4+ T cells, without CD8+ or B lymphocytes, can reject xenogeneic skin grafts

Abstract: Previous experiments have shown that rejection of xenogeneic skin grafts by mice is particularly dependent on CD4⁺ T cells. There are two possible explantations for this finding: either 1) "help" provided by CD4⁺ T cells is essential for CD8⁺ T cell-, B cell-, or NK cell-mediated effector mechanisms of rejection, or 2) CD4⁺ cells are themselves responsible for rejection, perhaps by some nonspecific effector mechanism. To examine these two hypotheses, we transplanted pig skin onto SCID mice and then reconstituted the mice with selected subpopulations of lymphocytes. Mice that did not received CD4⁺ T cells were unable to reject their xenografts, whereas those receiving CD4⁺ cells could do so in the absence of CD8⁺ cells or B cells and even when additionally depleted of NK cells by treatment with anti-Asialo GM1 antibody. Additional experiments were performed both in vivo and vitro to confirm the absence in test mice of CD4⁺ or CD8⁺ and B lymphocytes, respectively. These results suggest that CD4⁺ T cells are not only necessary for rejection of xenogeneic skin grafts by mice, but that they can do so without CD8⁺ cells or B cells, and probably without NK cells. Since CD4⁺ cells in mice have been shown to recognize xenogeneic antigens indirectly, this suggests that a nonspecific effector mechanism may be involved in the rejection of xenografts. In these experiments allogeneic skin grafts behave quite differently as they could not be rejected by this mechanism.     

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.     

Flow cytometric analysis of mesothelial cells in peritoneal dialysis effluent

According to recent reports, deformity and size of mesothelial cells reflect mesothelial condition. We applied flow cytometry (FCM) to the analyzation of mesothelial cells in peritoneal dialysis effluent (PDE) and the relationship between the period of peritoneal dialysis (PD) and peritoneal function. Eighteen patients treated for two to 89 months by PD were selected. Their dialysate: plasma creatinine ratio (D/P creatinine)was 0.67 +/- 0.086 (0.53 to 0.87). Overnight PDE was drained and centrifuged. The cell population of peritoneal cells identified by anti-cytokeratin, CD14 and 45 antibodies was studied by FCM. Cytokeratin positive cells were identified as mesothelial cells, distinct from macrophages, granulocytes or lymphocytes. The forward scatter (FSC) of cytokeratin positive cells, fluorescence intensity of cytokeratin and percentage of cytokeratin-positive cells in PDE were 395.6 +/- 55.5 (298.31 to 527.72), 333.9 +/- 272.9 (67.55 to 1,071.95), and 6.75 +/- 6.1% (0.44 to 21.14), respectively. There was a positive correlation between D/P creatinine and FSC, and a negative correlation between D/P creatinine and cytokeratin fluorescence intensity or the percentage of cytokeratin positive cells. However, there was no correlation between the period of PD and FSC, cytokeratin fluorescence intensity or the percentage of cytokeratin-positive cells. It was suggested that the alteration of mesothelial cells is not necessarily influenced by the period of PD, but influences peritoneal function. It was found that the analysis of cell population by FCM reflects the morphological and functional changes in the peritoneum of patients on PD.     

Association of CD8+ natural killer T cells in the liver with neonatal tolerance phenomenon

BACKGROUND: Neonatal tolerance is a very interesting phenomenon, because even allogeneic skin grafts are not rejected in these mice at the adult stage. However, the underlying mechanism remains unclear. METHODS: In this study we prepared such tolerant C57BL/6 (B6) mice (H-2b) by the injection of allogeneic lymphocytes of BALB/c origin (H-2d) at the neonatal stage. RESULTS: The total number of liver lymphocytes in these tolerant mice was found to increase when it was examined at the adult stage. Nevertheless, the retention of allogeneic lymphocytes that were injected at the neonatal stage was highest in the spleen. It is speculated that these allogeneic lymphocytes stimulate the hepatic immune system via the portal vein and that such stimulation maintains the tolerance phenomenon. Indeed, these tolerant mice showed elevated levels of IL-2R beta+ CD3int cells (i.e., extrathymic T cells) and NK1.1+ CD3int cells (i.e., NKT cells) in the liver. Even more interestingly, the number and proportion of CD8+ NKT cells, which are usually a minor population in normal mice, increased among NKT cells in the liver of tolerant mice. This became much more prominent when tolerant mice were grafted with allogeneic (H-2d) skin. CONCLUSION: In conjunction with additional data from a cell-transfer experiment and a splenectomy experiment, our results suggest that CD8+ NKT cells in the liver of tolerant mice might be intimately associated with the neonatal tolerance phenomenon.     

Comparison of in vivo lymphocyte proliferation between allogeneic and xenogeneic heart transplantation in mice

There are controversial in vitro data comparing the strength of the cellular immune response between allogeneic and xenogeneic stimulator/responder combinations. The present study therefore compares in vivo lymphocyte proliferation using heart transplantation (HTx) models in mice. Heterotopic HTx into BALB/c mice was performed using donor organs from mice (BALB/c and C57BL/6) or Lewis rats. Intraperitoneally given bromodeoxyuridine (BrdU) was incorporated into the DNA and was subsequently analyzed by flow cytometry. On postoperative days 3 and 5, proliferation of splenocytes, CD4(+) T-lymphocytes, and CD19(+) B-lymphocytes was significantly higher after xenogeneic than after allogeneic and isogeneic HTx. No significant difference was observed when proliferation of CD8(+) lymphocytes was determined. The increased in vivo proliferation after xenotransplantation may reflect an earlier and probably stronger cellular immune response compared to allogeneic transplantation. The higher CD4(+) lymphocyte proliferation underscores the importance of this cell population in xenograft rejection.     

Transplantation of allogeneic or xenogeneic bone marrow within the donor stromal microenvironment

Successful engraftment of hematopoietic stem cells requires a supportive hematopoietic stromal microenvironment (HSM). Defects in the HSM associated with aplastic anemia, myelofibrosis, or caused by intensive ionizing radiation and chemotherapy generally result in failure of bone marrow (BM) engraftment. Transplantation of donor BM within donor HSM may therefore provide optimal conditions for allogeneic BM transplantation. We have transplanted donor hematopoietic cells together with their own HSM to improve acceptance of allogeneic or xenogeneic BM. The non-myeloablative treatment used induced tolerance to murine allografts and provided conditions for the life-long acceptance of allogeneic HSM. Allogeneic BM transplanted within it's own HSM under the kidney capsule caused less graft-versus-host disease than BM transplanted i.v. Tolerance in mice to xenogeneic (rat) HSM was less complete. Ectopic ossicles were small and contained fewer hematopoietic cells. However, simultaneous transplantation of rat BM and HSM to preconditioned mice improved engraftment of rat BM compared with transplantation of BM alone. Donor hematopoietic cells survived longer on their own HSM than on HSM of recipients.