Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop.

We have revealed that about one and a half thousand tiny clusters, filled with one thousand closely packed lymphocytes, can be found throughout the murine small and large intestinal mucosa. They are located in crypt lamina propria (cryptopatches; CP) and can be first detected at 14-17 d after birth. A large fraction of lymphocytes in CP expresses c-kit, IL-7R, Thy1 and a lymphocyte function-associated antigen, LFA-1, whereas most of them remain CD3-, TCR alpha beta-, TCR gamma delta-, sIgM-, and B220-. The population size of IL-2R alpha+, HSA+ and Pgp-1+ subsets is variable (20-50%) and the composition of CD8+, Ly-1+, and CD4+ subsets is smaller but also variable (3-20%). In the small intestine, CP do not contain cells undergoing apoptosis nor cells bearing RAG-1 molecules, but do contain dendritic stromal cells bearing CD11c/CD18 molecules. The frequency of DNA replicating cells in CP is higher than that in Peyer's patches (PP), is lower than that in the thymic cortex and is almost comparable with that in the thymic medulla. The numbers of CP remain the same in aged mice (> 114 wk) but double after estrogen treatment even though the thymi are attenuated sharply in both conditions. Thus, with respect to histogenesis, lymphocyte composition and tissue level of cellular behavior, neither PP, isolated lymphoid follicles, peripheral LNs, nor thymus are identical with CP. Finally, CP are virtually absent in lamina propria of IL-7R-deficient mice that display a profound reduction in thymic and peripheral lymphoid cellularity. By contrast, CP are present in germ-free mice and in athymic (nu/nu), SCID, TCR beta x delta-/-, RAG-2-/-, PP-deficient (aly/aly), stem cell factor (Sl/Sld) and c-kit (W/Wv) mutant mice. Taking all of these results together, CP are the first identification of gut-associated murine lymphoid tissues where the generation of IL-7-dependent lympho-hematopoietic progenitors for T and/or B cell descendants may start to take place at the age of commencement of weaning.

Tumor necrosis factor alpha/cachectin and interleukin 1 beta initiate meningeal inflammation.

Although previous studies using human cytokines in rabbits and rats have provided evidence of the participation of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) in the meningeal inflammatory cascade, the results obtained by several groups of investigators have been discordant or, at times, contradictory. In the present study, homologous cytokines were applied to the rabbit meningitis model. Intracisternal administration of 10(2)-10(5) IU of purified rabbit TNF-alpha (RaTNF-alpha) produced significant cerebrospinal fluid (CSF) inflammation. A similar response was observed after intracisternal inoculation of 5-200 ng of rabbit recombinant IL-1 beta (rrIL-1 beta). Preincubation of these two mediators with their specific antibodies resulted in an almost complete suppression of the CSF inflammatory response. In animals with Haemophilus influenzae type b lipooligosaccharide-induced meningitis, intracisternal administration of anti-rrIL-1 beta, anti-RaTNF-alpha, or both resulted in a significant modulation of meningeal inflammation. Simultaneous administration of 10(3) IU of RaTNF-alpha and 5 ng of rrIL-1 beta resulted in a synergistic inflammatory response manifested by a more rapid and significantly increased influx of white blood cells into the CSF compared with results after each cytokine given alone. These data provide evidence for a seminal role of TNF-alpha and IL-1 beta in the initial events of meningeal inflammation.

Generation of intestinal T cells from progenitors residing in gut cryptopatches.

Cryptopatches (CPs) are part of the murine intestinal immune compartment. Cells isolated from CPs of the small intestine that were c-kit positive (c-kit+) but lineage markers negative (Lin-) gave rise to T cell receptor (TCR) alphabeta and TCR gammadelta intestinal intraepithelial T cells after in vivo transfer or tissue engraftment into severe combined immunodeficient mice. In contrast, cells from Peyer's patches and mesenteric lymph nodes, which belong in the same intestinal immune compartment but lack c-kit+Lin- cells, failed to do so. These findings and results of electron microscopic analysis provide evidence of a local intestinal T cell precursor that develops in the CPs.

Tumor necrosis factor inhibiting angiogenesis in vitro.

The effect of recombinant human tumor necrosis factor (TNF) on the capillary growth was evaluated using an in vitro angiogenesis model recently developed. Sprague-Dawley rat microvessel fragments, from epididymal fat pads, seeded onto the confluent culture of myofibroblastic cells from the same tissue origin, gave rise to microvascular networks on and in the multilayered myofibroblastic cells. In contrast, the capillary growth from the vessel fragments was markedly inhibited in the presence of 10-1,000 U TNF/ml. Monoclonal antibody against TNF completely neutralized the capillary growth inhibitory activity of TNF. The mode of angiogenesis inhibitory action of TNF was also examined by use of bovine capillary endothelial (BCE) cells and rat myofibroblastic cells. TNF exerted growth inhibitory and cytotoxic actions against BCE cells cultivated on the various basement membrane components, such as extracellular matrix secreted by BCE cells, fibronectin, laminin, and type IV collagen. An irreversible damage to most of the BCE cells was observed ultrastructurally after 60 hours' exposure to TNF. TNF, however, did not injure the myofibroblastic cells but rather stimulated their growth. These findings indicate that TNF inhibits in vitro capillary growth by its direct cytostatic and cytotoxic actions to microvascular endothelial cells.

Inhibition of tumor-induced migration of bovine capillary endothelial cells by mouse and rabbit tumor necrosis factor.

The effect of tumor necrosis factor (TNF) on the tumor-induced endothelial migration was evaluated with the use of a phagokinetic track assay. TNF from both ddy mice and Japanese albino rabbits (sp act, 3-5 X 10(5) U/mg, respectively) was found to inhibit the migration of bovine capillary endothelial (BCE) cells stimulated by factors from tumor cells, such as the medium conditioned with mouse sarcoma 180 cells or human meningioma extract. These TNF preparations, however, did not affect the spontaneous migration of the BCE cells. When mouse TNF was further fractionated by polyacrylamide gel electrophoresis, only TNF-positive fractions showed an inhibitory activity on the tumor-induced endothelial motility. Moreover, monoclonal antibody against rabbit TNF completely neutralized its migration-inhibitory activity. These findings indicate that the observed inhibitory effect of TNF preparations on the endothelial motility evoked by tumor is exclusively ascribed to the function of TNF. This activity presumably is involved in the suppression of tumor angiogenesis in vivo.

Actions of tumor necrosis factor on cultured vascular endothelial cells: morphologic modulation, growth inhibition, and cytotoxicity.

Exposure to highly purified preparations of murine tumor necrosis factor (TNF) resulted in distinct morphologic changes and proliferation inhibition of cultured endothelial cells from the bovine aorta and capillary and of those from the human umbilical vein. The TNF preparation also inhibited the proliferation of bovine aortic smooth muscle cells but failed to inhibit the proliferation of bovine and human fibroblasts. The cytotoxic activity of the TNF preparation was prominent only in bovine capillary endothelial cells. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and upon PAGE only, of the TNF preparations, the fractions representing growth inhibitory and cytotoxic activities against bovine capillary endothelial cells exactly corresponded to those containing migrated TNF. Treatments of the TNF preparations with heat and polymyxin B revealed that the effects on endothelial cells were not due to possibly contaminated endotoxin. Moreover, growth inhibitory and cytotoxic actions of rabbit TNF on bovine capillary endothelial cells were completely neutralized by the addition of an anti-rabbit TNF monoclonal antibody. These results suggest that TNF causes morphologic changes in, growth inhibition of, and cytotoxicity against, the vascular endothelial cells.

Abnormality in the early signal transduction pathway is responsible for the impaired proliferative response and low K+ current in a T-cell clone by stimulation with anti-CD3 antibody.

Two T-cell clones were established from young and old C57BL/6 mice, respectively. The proliferative response to anti-CD3 stimulation was significantly greater in the young (YT5) than in the old (OT13) T cell clone. However, a similar high response was observed in both T-cell clones upon stimulation with phorbol myristate acetate (PMA) and ionomycin (INM). The calcium-dependent K+ current (IK(Ca)) was recorded with the patch-clamp method in these T-cell clones. With anti-CD3 stimulation, the amplitude of the outward K+ current was significantly lower in OT13 than in YT5 cells. With stimulation with PMA and INM, however, no significant difference in IK(Ca) was obtained between the two types of cells. The level of proliferative response of T-cells to mitogens was well reflected by the amplitude of IK(Ca). Some abnormality in the early pathway of signal transduction, which led to the intracellular Ca2+ influx, appears to be responsible for the impaired proliferation of the old T-cell clone.

Actions of TNF and IFN-gamma on angiogenesis in vitro.

We have developed a model system for studying angiogenesis in which microvascular fragments and myofibroblasts (Mf) isolated from lipid tissues are grown in co-culture. We have found that Mf induce capillary formation by producing an endothelial cell growth factor and by secreting an extracellular matrix that causes endothelial cells to form a cordlike structure. This system appeared to be well suited for examining the effects of vasoactive substances such as the TNF and INF-gamma on capillary growth. TNF-alpha,beta, and IFN-gamma not only significantly inhibited capillary growth induced by Mf, but also blocked capillary development induced by fibroblast growth factors (FGF), well-known potent angiogenic factors. Recently, we have found that platelet-derived growth factor (PDGF) enhances in vitro capillary formation, probably at least in part by acting on Mf. Just as with FGF, capillary growth in the presence of PDGF was almost completely blocked by IFN-gamma. We examined the mode by which IFN-gamma inhibits angiogenesis and found that IFN-gamma inhibits both the proliferation of endothelial cells and collagen(s) synthesis by Mf. These actions of TNF or IFN-gamma could limit vascular formation in solid tumors.

Characterization of extrathymic T cells induced by recombinant murine interferon-beta in the peritoneal cavity of nude mice.

The effects of recombinant murine interferon-beta (rMuIFN-beta) on extrathymic lymphocyte formation in the peritoneal cavity of BALB/c athymic nude mice and tumor-bearing nude mice were examined for comparison with BALB/c normal euthymic mice. The 7 days of administration of 1 x 10(5) IU rMuIFN-beta caused the number of these cells to increase remarkably in the peritoneal cavity as a unique subset of asialoGM1+CD4+CD8-TcR alpha beta+ T cells. The asialoGM1+CD4+ T cells produced IL-2 and IFN-gamma in primary culture without stimulant but did not proliferate. Thus, extrathymic T cells can be induced easily in the peritoneal cavity in addition to the thymus for host defense systems.

Impairment of signal transduction in T cells from old mice.

T cells from old mice showed impaired proliferative response to antigenic stimulation. To understand the mechanism underlying the age-related impairment of T cell functions, the signal transduction pathway was examined and compared between T cells from young and old mice, and between T cell clones established from a young and old mouse. The age-related changes in T cells were as follows: (1) reduction in the expression and the activation of protein tyrosine kinases associated with T cell receptor (TCR) after antigenic stimulation; (2) reduced phosphorylation of phospholipase C gamma 1 (PLC gamma 1); (3) reduced production of second messengers such as inositoltrisphosphate (IP3) and diacylglycerol (DAG); and (4) reduced influx of Ca2+ ion. Thus, a T cell clone established from an old mouse showed impaired proliferation by stimulation with anti-CD3 antibody, but was fully activated to the level of a T cell clone from a young mouse by stimulation with phorbol acetate myristate (PMA) plus ionomycin (INM). However, splenic T cells freshly prepared from old mice did not show full recovery by the same treatment. The results indicate that one major blockade in the signal transduction of T cells from old mice is present in the pathway just after TCR, but besides this, the blockade is also present in multiple sites down-stream, which can not be bypassed by stimulation with PMA plus INM.

Depletion of mycoplasma from infected cell lines by limiting dilution in 6-methylpurine deoxyriboside.

Cells contaminated with mycoplasma were cloned in the presence of 6-methylpurine deoxyriboside. The results showed that a limiting dilution in the culture medium containing the chemical efficiently depletes mycoplasma providing the cells are sensitive to 0.6-1.2 microM 6-methylpurine.

Specificity of an anti-murine B cell serum.

An antiserum (ABS) specific for murine B cells was prepared in rabbits by immunization with lymph node cells from nude mice as reported previously (1). To make the antiserum specific for antibody-forming cell precursors (AFCP) extensive absorption was required with mouse serum, erythrocytes and thymus cells. Even after ABS was no longer cytotoxic for thymus cells, further absorption with thymus cells was necessary to make it ineffective for plaque-forming cells (PFC). The correlation between cells bearing C3- or Fc-receptors and the cells bearing antigen(s) to which ABS reacts was studied. In the residual spleen cells treated with ABS and complement, there were some EAg and EAmCm rosette-forming cells. There were also some spleen cells reacting with ABS even after depletion of C3- and/or Fc-receptor-bearing cells. These results show that the antigen(s) to which ABS reacts could be a marker on B cells different from C3- or Fc-receptors. Spleen cells reacting with ABS were compared with cells bearing C3- or Fc-receptors various days after birth. The cells sensitive to ABS increased in spleens earlier after birth than those with C3- or Fc-receptors.

Enhancement of B cell function as antigen-presenting cell during interplay with Th cells.

We have previously reported that C57BL/6 mouse B cells present Ag to an I-Ab-restricted and OVA-specific T cell clone, 34-7F, to secrete IL-2 but not to proliferate, whereas spleen adherent cells as APC induce a proliferative response of 34-7F cells to OVA. In this T cell response, it was examined whether B cells were stimulated during presenting Ag and acquired the ability as APC to induce proliferative Ag-response of 34-7F cells. For this aim, B cells prepared from unstimulated mouse spleen cells were cultured with 34-7F cells in the presence of OVA and then compared with unstimulated B cells for APC function in the OVA-response of 34-7F cells. Unstimulated B cells proliferated when cultured for 2 days with irradiated 34-7F cells and OVA, and the proliferation was inhibited by an anti-I-Ab mAb. B cells which had been stimulated for 2 days with 34-7F cells and OVA induced an increase in phosphatidyl inositol metabolism in 34-7F cells in the presence of Ag, whereas unstimulated B cells failed to do so. The increase was dependent on the dose of OVA and on the number of the stimulated B cells. Ag presentation by the stimulated B cells (but not by unstimulated ones), also induced IL-2R expression on 34-7F cells, which resulted in IL-2-dependent proliferation. The IL-2R expression does not depend on the possible increase in IL-2 production by the T cells, inasmuch as the activated B cells induced lower expression of IL-2 mRNA or lower secretion of IL-2 by 34-7F cells than that induced by unstimulated B cells. These results suggest that B cells receive signal(s) from 34-7F cells to enhance APC function during Ag-presentation to the T cells which do not proliferate in the response to Ag on unstimulated B cells, and that the stimulated B cells induce the proliferative response of 34-7F cells to OVA.

Role of newly synthesized MHC class II molecules in antigen-specific antigen presentation by B cells.

Using a B lymphoma, A20-HL, bearing IgM receptors for TNP, we have shown that presentation of an Ag taken up through the receptors is highly sensitive, whereas that of an Ag taken up nonspecifically is resistant to inhibition of protein synthesis or protein transport from the endoplasmic reticulum. To analyze the difference, we have examined the effect of protein synthesis inhibition on A20-HL cells in terms of internalization and fragmentation of a specific Ag, TNP-OVA, and distribution of MHC class II molecules. Inhibition of protein synthesis in A20-HL cells with emetine, an irreversible protein synthesis inhibitor, did not decrease the surface expression of anti-TNP receptors, or the kinetics of internalization of 125I-TNP-OVA. To detect fragmentation of TNP-OVA, A20-HL cells were incubated at 37 degrees C in the presence of 125I-TNP-OVA, and the cell lysate was analyzed in SDS-PAGE. The number of detectable fragments increased with the incubation period, and inhibition of protein synthesis did not change the electrophoretic pattern. Expression of MHC class II molecules on the surface of A20-HL cells was not affected by inhibition of protein synthesis. However, intracellular MHC class II molecules markedly decreased in amount in the emetine-treated cells. Thus, presentation of an Ag taken up through Ag receptors seems to be dependent on intracellular MHC class II molecules, whereas that of an Ag taken up nonspecifically does not, suggesting that the Ag-processing pathway in B cells for a specific Ag is different from that for a nonspecific one, at least partly.

B cells as accessory cells in a Con A response of a T cell clone.

Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.

Differential sensitivity to antigenic competition in antigen-specific and -nonspecific antigen presentation by B cells.

We have previously shown that a specific Ag presentation by B cells is different from a nonspecific one in the sensitivity to protein synthesis inhibition. In the present study we have compared the sensitivity of these Ag presentations to antigenic competition. A20-HL cells expressing TNP-specific IgM were pulsed with anti-mouse IgM goat IgG (aMGG) or trinitrophenylated goat IgG (TNP-NGG) as an Ag internalized through Ag receptor or NGG as an Ag internalized by fluid-phase pinocytosis. The pulsed cells induced IL-2 production by NGG-specific cloned T cells. The presence of dog IgG during pulsing A20-HL cells severely inhibited the presentation of NGG but not of aMGG or TNP-NGG. The presence did not decrease the internalization of 125I-NGG into A20-HL cells, suggesting that the inhibition was localized into the complex formation of antigenic peptides with MHC class II molecules. Thus, a specific Ag presentation by A20-HL cells is different from a nonspecific one in its sensitivity to antigenic competition.

A targeting model of boron neutron-capture therapy to hepatoma cells in vivo with a boronated anti-(alpha-fetoprotein) monoclonal antibody.

We described previously that 10B atoms delivered by monoclonal antibody (mAb) exerted a cytotoxic effect on AH66 cells in a dose-dependent manner upon thermal neutron irradiation in vitro. In the present study, the delivering capacity of boronated anti-(alpha-fetoprotein) (AFP) mAb to carry 10B atoms to AFP-producing tumor xenografts in nude mice was determined. Boronated mAb was prepared by conjugating 50 mM 10B compound to an anti-AFP mAb (2 mg/ml) using N-succinimidyl-3-) (2-pyridyldithio) propionate. The number of 10B atoms conjugated directly to the mAb was estimated to be 459/antibody by prompt gamma-ray spectrometry. Boron concentrations in tumor tissue obtained 12, 24, 72, and 120 h after injection of 3.0 mg 10B-conjugated anti-AFP mAb were 11.10 +/- 3.12 (SD, n = 6). 29.30 +/- 5.11, 33.02 +/- 11.8, and 12.91 +/- 5.62 ppm respectively. For control 10B-conjugated anti-dinitrophenol (DNP) mAb, the values were 9.59 +/- 0.99, 10.37 +/- 2.86, 10.00 +/- 2.95, and 8.83 +/- 4.71 ppm respectively. The concentrations in blood were less than 0.40 +/- 0.10 ppm with anti-AFP mAb and less than 0.51 +/- 0.15 ppm with anti-DNP mAb at each sampling time (12, 24, 72, and 120 h). The number of 10B atoms delivered to the tumor cells was calculated to be 0.62 x 10(9), 1.63 x 10(9), 1.84 x 10(9) and 0.72 x 10(9) at each sampling time after injection of 10B-anti-AFP mAb. The amount of 10B atoms necessary for effective boron neutron-capture therapy was estimated to be 10(9)/tumor cell. We were able to carry 1.84 x 10(9) 10B atoms to AH66 tumor cells by using 10B-anti-AFP mAb. The accumulation reached its peak 72 h after injection. These data indicated that the 10B-conjugated antitumor mAb could deliver a sufficient amount of 10B atoms to the tumor cells to induce cytotoxic effects 72 h after injection upon thermal neutron irradiation.

A substrain of NZB mouse as an animal model of autoimmune inner ear disease.

A substrain of an autoimmune-prone mouse, NZB/kl, was found to show spontaneous elevation of the auditory brainstem response (ABR) threshold with age. Morphological examination of the inner ear in NZB/kl mice with high ABR thresholds revealed pathological changes confined to the stria vascularis, including marked thickening of the capillary basement membrane which contained many foamy structures, and vacuolar degeneration of the intermediate cells. Circular or granular IgM deposits and some IgG deposits were found in the stria vascularis in the mice with high ABR thresholds, suggesting that deposits of immune complexes (mainly IgM antibodies) could cause strial damage that resulted in the ABR threshold elevation. Another substrain of NZB mice, NZB/san, showed lower levels of IgM immune complexes and anti-ss DNA antibodies, and did not develop either inner ear morphological changes or a high ABR threshold. NZB/kl mice may provide a useful animal model for studying the mechanism of autoimmune inner ear disease.

[Cultivation and activation of Th1 and Th2 clones].

Murine T helper clones are classified into two subpopulations, Th1 and Th2, according to the cytokines they produce. Immune function is considered to depend which subpopulation is activated by an antigen stimulation. These subpopulations are different from each other in humoral second signal molecules required for their proliferation produced by antigen-presenting cells. Th1 requires IL-12, and Th2 IL-1, indicating that antigen-presenting cells regulate subpopulation responding to some stimulation. These subpopulations are also different in their activation signal transduction pathway mediated by TCR stimulation, suggesting a possibility of a selective activation of these subpopulations.

[Boron neutron capture therapy using 10B entrapped anti-CEA immunoliposome].

A new murine monoclonal antibody (2C-8) was prepared by immunizing mice ip with CEA producing human pancreatic cancer cell line, AsPC-1.SDS-PAGE and Western blot analysis showed that 2C-8 monoclonal antibody recognized CEA and NCA. This anti-CEA monoclonal antibody was conjugated with large multilamellar liposomes incorporated 10B compound (Cs2 10B12H11SH). This immunoliposomes applicated to boron neutron capture therapy. AsPC-1 cells were incubated with the 10B-Lip-MoAb(CEA) for 8 hours. After the irradiation with thermal neutron (1 x 10(11)-1 x 10(13) n/cm2), boronated AsPC-1 cells were showed decreasing uptake of 3H-TdR compared with control group. The numbers of 10B atoms in liposomes bound to an antibody were in proportion to the dose of 10B compounds added and maximum number of 10B atoms was approximatory 1.2 x 10(4)/Ab. These data indicated that the immunoliposomes could deliver highly amount of 10B atoms to the tumor cells and exert cytotoxic effect by thermal neutron. BNCT with immunoliposome may be useful to the non resectable malignant tumors in clinical application.