Injection Of Parental Spleen Cells Research Articles

Fas-mediated cytotoxicity by intestinal intraepithelial lymphocytes during acute graft-versus-host disease in mice

BACKGROUND & AIMS: Host-derived intestinal intraepithelial lymphocytes (IELs) increase in number during acute graft-versus-host disease (GVHD) in mice. In the present study, we examined Fas-mediated cytotoxicity by host-derived IELs against Fas-expressing target cells to see whether Fas/Fas ligand (Fas-L) interaction is involved in the pathogenesis of enteropathy during acute GVHD. METHODS: Acute GVHD was induced by injection of parental spleen cells into nonirradiated F1 mice. The expression of Fas antigen on the intestinal epithelial cells (IECs) was examined by flow cytometry, and the expression of messenger RNA (mRNA) for Fas-L, interleukin 2, and interferon gamma in host-derived IELs was assessed by reverse-transcription polymerase chain reaction. Fas- mediated cytotoxicity by host-derived IELs was assessed using Fas- transfected cells, IECs, and Fas immunoglobulin Fc fusion protein (Fas Fc). RESULTS: Fas antigen was constitutively expressed on the cell surface of IECs before and after GVHD induction. Although Fas-L mRNA was not detected or detected scarcely in either alphabeta or gammadelta IELs before GVHD induction, both IELs expressed high levels of mRNA for Fas-L and interferon gamma after GVHD induction. Host-derived IELs during acute GVHD showed cytotoxicity against Fas-transfected target cells and IECs, which was partly blocked by addition of Fas Fc. CONCLUSIONS: Fas/Fas-L-mediated cytotoxicity by host-derived IELs may be partly responsible for the enteropathy during acute GVHD. (Gastroenterology 1997 Jul;113(1):168-74)

Natural Killer Cells Inhibit the Development of Autoantibody Production in (C57BL/6 × DBA/2) F1 Hybrid Mice Injected with DBA/2 Spleen Cells

We investigated the role of natural killer (NK) cells in the development of autoantibody production in which (C57BL/6 × DBA/2) F1 (BDF1) hybrid mice were injected intravenously with spleen cells (SC) from parental DBA/2 mice (treated BDF1 mice). Treated BDF1 mice began to show an increase in serum anti-dsDNA antibody 2 weeks after injection, while the NK activity of their SC transiently increased initially in the first 1 to 2 weeks after injection, but subsequently decreased dramatically. Flow cytometric analysis suggested that this sequential change in NK activity correlated with the absolute number of host-derived NK1.11 cells in SC from treated BDF1 mice. We demonstrated that the level of anti-dsDNA in serum is directly influenced by the level of NK activity in treated BDF1 mice. Depletion of NK cells by administration of anti-NK1.1 mAb accelerated the development of autoantibody production, whereas augmentation of NK activity by administration of poly-(I:C) inhibited the development of autoantibody production. This inhibitory effect of poly(I:C) was abolished by prior depletion of NK cells. Interestingly, suppression of autoantibody production was seen only when poly(I:C) was administrated within 1 week after injection of parental SC. Last, we demonstrate that adoptive transfer of interleukin-2 (IL-2)-activated NK cells had a protective effect against the development of autoantibody production. These findings imply that NK cells may have a protective role in lupus-like disease especially in its early stage. In addition, it opens up the possibility that adoptive immunotherapy with IL-2-activated NK cells can delay or even prevent the development of autoimmune disease.

Synergistic effect of murine cytomegalovirus on the induction of acute graft-vs-host disease involving MHC class I differences only. Analysis of in vitro T cell function.

The development of acute graft-vs-host disease (GVHD) is a common outcome after the injection of fully MHC disparate parental T cells into unirradiated F1 mice. Murine cytomegalovirus (MCMV) infection has been previously shown to augment the development of acute GVHD in the parent-into-F1 (P----F1) model, such that 10-fold fewer parental cells are required. In the present study, we have investigated the effect of MCMV infection on the induction of non-lethal GVHD that occurs in P----F1 combinations involving MHC class I only or class II only differences. Using P----F1 combinations involving either an H-2K only difference or an H-2D only difference, MCMV infection of F1 mice 3 days before the injection of parental spleen cells led to a profound T cell immunodeficiency that strongly resembled that observed in acute GVHD. Further studies examining the H-2K disparate P----F1 combination, C57Bl/6---- (C57Bl/6xB6.C-H-2bm1) F1 and combined MCMV infection showed that the immunodeficiency is characterized by a profound loss of in vitro Th cell production of IL-2 and an intrinsic defect in T effector function as shown by an inability of rIL-2 to restore defective CTL responses. Additional experiments in these mice revealed the presence of suppressor cells as well as significant parent-anti-F1 CTL activity possibly accounting for the suppressor effect. This pattern of immunodeficiency was not seen after the administration of either MCMV or MHC class I disparate parental cells alone. MCMV infection did not detectably alter the immunodeficiency observed in a P----F1 combination involving a MHC class II difference only. These results indicate that MCMV infection can alter the pattern of GVHD in the setting of an MHC class I disparity, but not in the setting of class II disparity, such that it resembles acute GVHD. These results may have relevance to the human transplant setting where intercurrent CMV infection has been associated with an adverse clinical outcome.

Immunomodulation by low-dose methotrexate. I. Methotrexate selectively inhibits Lyt-2+ cells in murine acute graft-versus-host reactions.

We have studied the effect of methotrexate in murine acute graft vs host (GvH) disease at concentrations analogous to those used in human rheumatoid arthritis. The GvH reaction was induced by i.v. injection of parental spleen cells into a normal F1 recipient. The acute suppression of T cell function in GvH mice was prevented by methotrexate given orally for 10 days at 1.0 or 0.5 mg/kg but not at 0.25 mg/kg. T cell mitogen response and IL-2 secretion that were inhibited in GvH mice were restored by methotrexate. Protection from immunosuppression in drug-treated GvH mice lasted at least 3 wk after drug dosing was stopped. The mechanism of the protective effect appears to be a preferential inhibition of donor and host Lyt-2+ Ts cell proliferation. In mixing experiments we found that methotrexate inhibited Ts function in GvH mice. By dual fluorescence labeling we showed that the engraftment of donor Lyt-2+ cells was prevented by drug treatment. This was not true of donor L3T4+ cells which were clearly present in the spleens of GvH mice after methotrexate treatment. These donor L3T4 cells were functional in that they induced the production of anti-DNA autoantibodies in the methotrexate-treated GvH mice.

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells.

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.

Abrogation of hybrid resistance to bone marrow engraftment by graft-vs-host-induced immune deficiency.

Lethally irradiated F1 mice, heterozygous at the hematopoietic histocompatibility locus Hh-1, which is linked with H-2Db, reject bone marrow grafts from H-2b parents. This hybrid resistance (HR) is reduced by prior injection of H-2b parental spleen cells. Because injection of parental spleen cells produces a profound suppression of F1 immune functions, we investigated whether parental-induced abrogation of HR was due to graft-vs-host-induced immune deficiency (GVHID). HR was assessed by quantifying engraftment of H-2b bone marrow in F1 mice with the use of splenic [125I]IUdR uptake; GVHID, by the ability of F1 spleen cells to generate cytotoxic T lymphocytes (CTL) in vitro. We observed a correlation in the time course and spleen cell dose dependence between loss of HR and GVHID. Both GVHID and loss of HR were dependent on injection of parental T cells; nude or T-depleted spleen cells were ineffective. The injection of B10 recombinant congenic spleens into (B10 X B10.A)F1 mice, before grafting with B10 marrow, demonstrated that only those disparities in major histocompatibility antigens that generated GVH would result in loss of HR. Thus, spleens from (B10 X B10.A(2R]F1 mice (Class I disparity only) did not induce GVHID or affect HR, whereas (B10 X B10.A(5R))F1 spleens (Class I and II disparity) abrogated CTL generation and HR completely. GVHID produced by a class II only disparity, as in (B10 X B10.A(5R))F1 spleens injected into (B6bm12 X B10.A(5R))F1 mice, was also sufficient to markedly reduce HR to B10 bone marrow. This evidence that GVHID can modulate hematopoietic graft rejection may be relevant to the mechanisms of natural resistance to marrow grafts in man.

Selective effects of graft-versus-host reaction on disaccharidase expression by mouse jejunal enterocytes.

Graft-versus-host reaction (GvHR) was induced in neonatal mice to produce crypt hyperplasia with and without stunted villi. Lactase activity was measured along individual villi of control and GvHR mice using quantitative cytochemistry. Lactase activity increased in control mice as enterocytes migrated over the lower part of the villus. This increase was followed by a period when lactase activity remained approximately constant. Effects produced by GvHR on this normal profile of development included an extension of the distance on the villus over which enterocytes could continue to increase lactase activity, a reduction in the time needed for an enterocyte to express lactase activity at maximal rate, and an overall decrease in the maximal lactase activity expressed by mature enterocytes. These effects have been quantified by fitting logistic curves to the experimental data. Parallel biochemical analyses of intestinal homogenates showed sucrase, isomaltase, trehalase and maltase activities to increase markedly 7-8 days after the injection of parental spleen cells. Attention is drawn to similarities between these results and steroid induced precocious development of intestinal function in neonatal mice.

Parental spleen cells accelerate the development of intestinal brush border structure and function in neonatal mice

1. 1. The influence of parental spleen cells on the postnatal development of brush border microvillus membrane structure and the ability to transport lysine and alanine has been studied in the mouse jejunum during the second week of postnatal life. 2. 2. Control tissue taken from 7–11 day old mice has an unchanging crypt-villus structure and a low enterocyte migration rate of about 1 μm hr −1. 3. 3. Microvillus elongation in crypt enterocytes takes 6 days to complete under these conditions. 4. 4. Lysine and alanine transport begin 2 days after structural differentiation has ceased. 5. 5. Parental spleen cells injected into 1–2-day-old F 1 mice cause crypt cell hyperplasia, villus shortening and a 3–6-fold increase in enterocyte migration rate after a period of 8 days. 6. 6. These effects are associated with large reductions in the time needed to complete microvillus membrane development and first express absorptive function. 7. 7. Lysine and alanine transport begin approximately 6 hr after structural differentiation has ceased under these conditions. 8. 8. Adaptive changes in the development of enterocyte structure and function, induced by injection of parental spleen cells, bear some resemblance to other changes found to occur normally at weaning and in adult animals subjected to controlled changes in diet and environmental temperature. 9. 9. The possibility that common principles govern enterocyte adaptation and that some of these still apply in an intestine undergoing an immune reaction is discussed.

Postinflammatory increase of pathogenicity of lymphocytic inocula in the peritoneal cavity.

Passive transfer of experimental allergic encephalomyelitis in rats by intraperitoneal injection of cells from draining lymph nodes was usually unsuccessful unless very large numbers of cells or conditioning procedures were employed. If the ip injection was done during the healing phase of a chemical peritonitis, then its effectiveness was greatly increased, even exceeding that of the iv route. The same effect of a healing chemical peritonitis was observed in adrenalectomized rats. A regional graft-versus-host disease produced by ip injection of parental spleen cells into hybrid recipients was greatly augmented in the healing phase of a chemical peritonitis. A host-versus-graft reaction against allogeneic spleen cells was histologically detectable only when the cells were injected after a chemical peritonitis. All these results are explicable as the consequence of a postinflammatory increase of absorption of lymphocytic inocula into the draining lymph nodes. The production of a chemical peritonitis is likely to be useful wherever immunologic and pathologic experimentation requires ip inoculations.

Suppressive influence of surgical stress on the graft-versus-host reaction in mice.

The influence of surgical stress on the local graft-versus-host reaction (GVHR) in F1 mice was studied. Skin incision 1 day prior to injection of parental spleen cells produced impairment of popliteal lymph node enlargement; however, this effect was not observed when GVHR was induced 3 and 5 days after operation. Strong GVHR suppressive activity of spleen cells was observed three hours after leg amputation before a decrease in thymus weight became evident. The GVHR suppressive activity declined by six hours later, but a second peak of 60% inhibition was observed after 24 h. This suppressive activity completely disappeared by treatment with anti-Thy 1.2 and complement. This shows that the GVHR is suppressed by surgical stress, and that this suppression is due to suppressor T lymphocytes.

Graft-Versus-Host Reactions in F 1 Hybrid Mice: MHC-Restriction-Independent Generalized Depression of Virus-Specific Cytotoxic T Cell Response

A graft-versus-host reaction (GVHR) was induced in F 1 hybrid mice by intravenous injection of parental spleen cells. A possible effect generated by recipient F 1 mice of a GVHR on the restriction specificities of anti-viral cytotoxic T cells was investigated. (A/J × C57BL/6) F 1 recipients were injected with 1 × 10 8 or 2 × 10 7 spleen cells from either parent. Zero to two weeks later during an acute GVHR or 12 to 26 weeks later during a chronic GVHR, spleen cells from these F1 recipients were assayed for their capacity to generate vaccinia (or lymphocytic choriomeningitis) virus-specific cytotoxic T cells. No effect was seen when parental cells were injected with virus on the same day. During the acute phase of the GVHR, recipients of spleen cells of both doses or from either parent injected 7 or 14 days previously generated markedly fewer cytotoxic T cells with respect to both parental restriction specificities. In the more chronic situation only, recipients of 1 × 10 8 parental spleen cells showed depressed virus-specific cytotoxic responses, again both restriction specificities were affected comparably. Therefore, a GVHR depresses generation of virus-specific cytotoxic T cells in a dose- andtime-dependent way, but does not measurably disturb the balance of parental 1 versus parental 2 restriction specificity in an F 1 recipient.

Analysis of murine T lymphocyte markers during the early phases of GvH-associated suppression of cytotoxic T lymphocyte responses.

Graft-vs-host- (GvH) associated immune-suppression was generated by injecting nonirradiated (B6 X C3H)F1 hybrid mice with 40 to 60 X 10(6) B6 or C3H parental spleen cells. Susceptibility to suppression as well as the potential of parental donor lymphocytes to generate suppressor activity was investigated in allogeneic cytotoxic T cell responses by mixing spleen cells from the inoculated F1 recipients with syngeneic responder cells from untreated mice. The susceptibility to suppression in F1 recipients, as well as the potential of parental lymphocytes to induce suppression in F1 mice, varied with the age of the mice. Cellular analysis revealed that GvH-suppression was mediated by 500 R radiosensitive parental T cells during the period tested (from day 5 until day 8 after injection of B6 parental spleen cells). In contrast, selected F1 lymphocytes from recipients injected with parental spleen cells failed to exhibit suppressor cell activity. The sensitivity of parental GvH suppressor cells to anti-Lyt reagents was time dependent. Thus, suppressor cells were sensitive to monoclonal anti-Lyt-1 or anti-Lyt-2 and C (usually until day 7 after injection of parental spleen cells); subsequently, suppression became insensitive to anti-Lyt-1, but remained sensitive to anti-Lyt-2 and C treatment.

Further studies on the protection by an F1 tumour against GvHR induced in F1 mice by injection of parental spleen cells.

Further studies on the protection by an F 1 tumour against GvHR induced in F 1 mice by injection of parental spleen cells

Protection against graft vs. host-associated immunosuppression in F1 mice. I. Activation of F1 regulatory cells by host-specific anti-major histocompatibility complex antibodies.

Injection of parental spleen cells into unirradiated F1 hybrid mice results in suppression of the potential to generate cytotoxic T lymphocyte (CTL) responses in vitro. In an attempt to protect the F1 mice from immunosuppression, the recipients were injected with antibodies specific for major histocompatibility complex (MHC)-encoded antigens of the F1 mice 24 h before inoculation of the parental spleen cells. 8-14 d later, the generation of CTL responses in vitro against H-2 alloantigens was tested. Alloantiserum directed against either parental haplotype of the F1 strain markedly diminished the suppression of CTL activity. Furthermore, monoclonal antibodies recognizing H-2 or Ia antigens protected the F2 mice from parental spleen cell-induced suppression. Although this study has been limited to reagents that recognize host H-2 determinants, these findings do not necessarily imply that protection against graft vs. host (GvH) can be achieved only with anti-MHC antibodies. However, protection was observed only by antibodies reactive with F1 antigens, and small amounts of the alloantibodies were sufficient to diminish CTL suppression. Adoptive transfer of spleen cells from syngeneic F1 mice treated with anti-h-2a alloantiserum 24 h previously provided protection equal to that of injection of the recipients with alloantibodies. The cells necessary for this effect were shown to be T cells and to be radiosensitive to 2000 rad. This cell population is induced by antisera against F1 cell surface antigens and effectively counteracts GvH-associated immuno-suppression.

The presence of a tumour in F1 mice partially inhibits the GvH reaction following injection of parental spleen cells.

(A x CBA(T6)F1 mice bearing F1 mammary carcinomas for 9 days were injected i.v. with A strain spleen cells. The A spleen cells were from either non-immune donors or mice which had received an i.p. injection of F1 tumours cells 9 days previously, and were thus immune to the CBA component of the tumour. Fourteen days after receiving parental spleen cells, the F1-tumour-bearing mice were killed and their spleen ratios and indices were determined as an index of the severity of the graft-versus-host reaction (GvHR) induced. The spleen indices were compared with those in non-tumour-bearing F1 mice, receiving aliquots of the same parental cell suspensions. At the higher doses of A spleen cells, the presence of an F1 tumour reduced the GvHR. At the same time, in 3/5 experiments, the weight of the F1 tumour in mice injected with immune A spleen cells was less than that in F1 mice receiving the same number of tumour cells but no spleen cells. A reduction in GvHR and a decrease in tumour weight was not seen when F1 mice carrying an A strain tumour were injected with A strain spleen cells immune to an F1 tumour. Adding 23-day F1 tumour-bearing F1 spleen cells to A spleen cells did not reduce the GvHR induced in further non-tumour-bearing F1 recipients by the parental cells. This was evidence against the presence of suppressor cells in the tumour-bearing F1 spleen. When 51Cr-labelled A strain spleen cells were injected into F1 mice, some of which had a tumour and therefore an enlarged spleen, there was an inverse relation between the size of the spleen and the number of parental cells therein, per g spleen 4 h after injection. It is thus suggested that the reduction in GvHR in F1-tumour-bearing F1 mice, after injection of parental spleen cells, is due first to a reduction in the concentration of donor cells in the recipient spleen (i.e. the same number of donor cells in a larger spleen) and second to pre-occupation of the donor cells in reacting to the tumour.

Effect of allogeneic cell interaction and graft-versus-host reaction on the antibody response to Escherichia coli 0127 antigen

The influence of allogeneic cell interaction and GVH reaction on the immune response to Escherichia coli antigen was investigated. Addition of CBA/J spleen cells to cultures of nu/nu spleen cells stimulated a significant increase in the nude PFC response to SRBC but had no significant effect on the immune response to E. coli antigen. Similarly, the induction of a GVH reaction in F 1 mice by the injection of parental spleen cells also had no significant effect on the immune response to the bacterial antigen. These results suggest that the immune response to E. coli is not affected by products of thymus-derived cells.

Immunosuppressive effect of graft-versus-host reaction

GVHR was elicited in adult F 1 hybrids by iv injection of parental spleen cells. The F 1 hybrids were then immunized with θ-AKR antigen. Plaque-forming cells (PFC) producing antibodies lytic for AKR thymocytes were enumerated in spleens of experimental animals using the method of cytolysis in agar-gel. The number of PFC found in spleens of animals grafted with parental cells was significantly lower than the number in spleens of animals grafted with syngeneic cells. The degree of suppression of immune response to θ-AKR depended on: (1) the duration of GVHR at the time of immunization, (2) the parental strain and the number of parental cells grafted to evoke GVHR, and (3) the source of cells grafted. Thymus and bone marrow cells when grafted together showed a synergistic effect in suppression of the host's immune response. Immunosuppression of the response to θ-AKR seems to be a more sensitive indicator of GVHR in adult F 1 hybrids than splenomegaly, which is commonly used as a means of assessment of GVHR. The possible role of immunosuppression in the pathogenesis of GVHR is briefly discussed.

Radiosensitive nature of paravascular infiltrate-producing potential of parental spleen cells

The injection of spleen cells from donors of parental strain into F 1 hybrid mice results in paravascular infiltration (PVI) in the portal tracts of the recipients' livers. The infiltrates can be enumerated. Whole-body irradiation (802 r) of the recipients before the injection of parental spleen cells does not reduce the number of PVI formed. Irradiation (1500 r) of the donors eradicates the capacity of their spleen cells to produce PVI. In vivo radiation inactivation experiments indicate that the LD 37 for donor PVI-producing cells is approximately 47 r and the D 0 is 102 r. These data support the contention that the formation of PVI is a function of donor cells and depends upon cell division.

Changes in the activity of the reticulo-endothelial system (RES) following the injection of parental spleen cells into F1 hybrid mice

Changes in the activity of the reticulo-endothelial system (RES) following the injection of parental spleen cells into F1 hybrid mice

Changes in the activity of the reticuloendothelial system (RES) following the injection of parental spleen cells into F1 hybrid mice

Changes in the activity of the reticuloendothelial system (RES) following the injection of parental spleen cells into F1 hybrid mice