CD34(+) hematopoietic stem cells exert accessory function in lipopolysaccharide-induced T cell stimulation and CD80 expression on monocytes.

CD34(+) hematopoietic stem cells, which circulate in peripheral blood with very low frequency, exert essential accessory function during lipopolysaccharide (LPS)-induced human T lymphocyte activation, resulting in interferon gamma production and proliferation. In contrast, stimulation of T cells by "conventional" recall antigens is not controlled by blood stem cells. These conclusions are based on the observation that depletion of CD34(+) blood stem cells results in a loss of LPS-induced T cell stimulation as well as reduced expression of CD80 antigen on monocytes. The addition of CD34-enriched blood stem cells resulted in a recovery of reactivity of T cells and monocytes to LPS. Blood stem cells could be replaced by the hematopoietic stem cell line KG-1a. These findings may be of relevance for high risk patients treated with stem cells or stem cell recruiting compounds and for patients suffering from endotoxin-mediated diseases.

Timothy grass pollen major allergen Phl p 1 activates respiratory epithelial cells by a non-protease mechanism.

BACKGROUND: Group 1 allergens from grass pollen (e.g. Phl p 1, the major allergen of timothy grass Phleum pratense) cause IgE reactivity in about 95% of allergic subjects and exist in all grass species. The respiratory epithelium represents a first line of contact of the immune system with airborne allergens, functions as physical barrier and is an important immunological regulation system. OBJECTIVE: The aim of this study was to investigate the interaction of Phl p 1 with human respiratory epithelium to elucidate the contribution of epithelial cells to the development of allergic reactions. METHODS: Purified Phl p 1 was used to stimulate A549 cells and transient transfected HEK293 cells. mRNA level of different mediators were investigated by real-time PCR, release of the mediators was determined by ELISA. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and an ex vivo model of the murine trachea were used to investigate a potential proteolytic activity of Phl p 1. RESULTS: Phl p 1 activates respiratory epithelial cells as measured by induction of IL-6, IL-8 and TGF-beta mRNA and release. Phl p 1, in contrast to Der p 1 from the house dust mite, does not exert proteolytic activity, as investigated by microscopic observation and MTT test. In an ex vivo model of the murine trachea we were able to show that Der p 1, in contrast to Phl p 1, enhances the transportation velocity of particles by the trachea, presumably by ATP released from the injured epithelium. CONCLUSION: We conclude that under physiological conditions Phl p 1 affects tracheal epithelial cells through a non-proteolytic activity. Enhancement of TGF-beta expression induced by Phl p 1 together with the increased release of IL-6 and IL-8 might provide an indirect mechanism through which the allergen may cross the epithelial barrier and attracts immunocompetent cells.

A role for altered TLR gene expression in association with increased expression of CD200R in the induction of mucosal tissue CD4(+) Treg in aged mice following gavage with a liver extract along with intramuscular monophosphoryl lipid A (MPLA) injection.

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice.

Regulation of fetal hemoglobin expression during hematopoietic stem cell development and its importance in bone metabolism and osteoporosis.

We have shown that an altered tissue redox environment in mice lacking either murine beta Hemoglobin major (HgbßmaKO) or minor (HgbßmiKO) regulates inflammation. The REDOX environment in marrow stem cell niches also control differentiation pathways. We investigated osteoclastogenesis (OC)/osteoblastogenesis (OB), in bone cultures derived from untreated or FSLE-treated WT, HgbßmaKO or HgbßmiKO mice. Marrow mesenchymal cells from 10d pre-cultures were incubated on an osteogenic matrix for 21d prior to analysis of inflammatory cytokine release into culture supernatants, and relative OC:OB using (TRAP:BSP, RANKL:OPG) mRNA expression ratios and TRAP or Von Kossa staining. Cells from WT and HgbßmaKO mice show decreased IL-1ß,TNFα and IL-6 production and enhanced osteoblastogenesis with altered mRNA expression ratios and increased bone nodules (Von Kossa staining) in vitro after in vivo stimulation of mRNA expression of fetal Hgb genes (Hgbε and Hgbßmi) by a fetal liver extract (FSLE). Marrow from HgbßmiKO showed enhanced cytokine release and preferential enhanced osteoclastogenesis relative to similar cells from WT or HgbßmaKO mice, with no increased osteoblastogenesis after mouse treatment with FSLE. Pre-treatment of WT or HgbßmaKO, but not HgbßmiKO mice, with other molecules (rapamycin; hydroxyurea) which increase expression of fetal Hgb genes also augmented osteoblastogenesis and decreased cytokine production in cells differentiating in vitro. Infusion of rabbit anti- Hgbε or anti- Hgbßmi, but not anti-Hgbα or anti- Hgbßma into WT mice from day 13 gestation for 3 weeks led to attenuated osteoblastogenesis in cultured cells. We conclude that increased fetal hemoglobin expression, or use of agents which improve fetal hemoglobin expression, increases osteoblast bone differentiation in association with decreased inflammatory cytokine release.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

An altered REDOX environment, assisted by over-expression of fetal hemoglobins, protects from inflammatory colitis and reduces inflammatory cytokine expression.

C5BL/6 female mice receiving dextran sodium sulfate in their drinking water develop an acute inflammatory colitis within 7d, with weight loss, histopathologic signs of inflammation, and colonic expression of inflammatory cytokines. In previous studies we have reported that increased inflammatory cytokine expression in aged mice can be attenuated by oral gavage of a crude fetal extract containing glutathione (GSH), MPLA and fetal hemoglobin, or more specifically by injection of a combination of these purified reagents. We speculated that this combination led to an altered tissue redox environment in which the immune response developed, thus regulating inflammation. Accordingly, we used wild-type (WT) C57BL/6 mice, or mice lacking either murine beta Hemoglobin major (HgbßmaKO) or minor (HgbßmiKO) as recipients of DSS in their drinking water, and followed development of colitis both clinically and by inflammatory cytokine production, before/after oral treatment of mice with a crude fetal liver extract. Mice lacking an intact fetal hemoglobin chain (HgbßmiKO) developed severe colitis, with enhanced colonic expression of inflammatory cytokines, which could not be rescued by extract, unlike WT and HgbßmaKO animals. Moreover, disease in both WT and HgbßmaKO animals could also be attenuated by exposure to 5-hydroxymethyl furfural (5HMF), hydroxyurea or rapamycin. The former has been used as an alternative means of stabilizing the conformation of adult hemoglobin in a manner which mimicks the oxygen-affinity of fetal hemoglobin, while we show that both hydroxyurea and rapamycin augment expression of murine fetal hemoglobin chains. Our data suggests there may be a clinical value in exploring agents which alter local REDOX environments as an adjunctive treatment for colitis and attenuating inflammatory cytokine production.

Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

The LPS receptor (CD14) links innate immunity with Alzheimer's disease.

To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.

Interaction of human lymphocyte subpopulations with interleukin 2 (II-2).

Human peripheral blood T-lymphocytes were fractionated by discontinuous density gradient centrifugation on Percoll. T-cells of low density were capable of proliferation in the presence of the mitogen phytohemagglutinin (PHA) as measured by colony formation, whereas cells of high density were not. Addition of supernatant of PHA-stimulated cultures to cells of high density resulted in proliferation which suggested that these cell fractions lacked interleukin-2 (II-2) producing T-helper cells. This finding was supported by the demonstration that T-cells of high density did not produce II-2 but could be induced to do so by adding II-1 producing monocytes. The data area explained on the basis of the present model of intercellular events leading to activation and proliferation of lymphocytes.

Structural and biological characterisation of a novel tetra-acyl lipid A from Escherichia coli F515 lipopolysaccharide acting as endotoxin antagonist in human monocytes.

We here report on the structural analysis of a novel tetra-acyl lipid A (LA (tetra) ) isolated from Escherichia coli deep rough (Re)-mutant strain F515. In addition to the biologically active hexa-acyl E. coli-type lipid A (compound 506), this incompletely acylated lipid A was found to be also present in the native LPS. Its structure was studied without further derivatisation by chemical analysis, matrix-assisted laser desorption/ionization mass spectrometry, and one- and two-dimensional (1)H- and (13)C-NMR spectroscopy. It was found to be structurally distinct from the tetra-acyl lipid A biosynthetic precursor Ia (compound 406) in lacking the primary (R)-3-hydroxytetradecanoic acid 14:0(3-OH) in position 3' ester-linked to the 'non-reducing' glucosamine (GlcN II). The hydroxyl group at the (R)-3-hydroxytetradecanoic acid attached to position 2' of GlcN II was found to be substituted by dodecanoic acid (12:0), thus forming a dodecanoyloxytetradecanoyl residue 14:0[3-O(12:0)]. The acylation pattern at the 'reducing' GlcN I was identical to that of compound 406 in having two primary (R)-3-hydroxy tetradecanoic acid residues [14:0(3-OH)] attached to positions 3 (ester-linked) and 2 (amide-linked), respectively. In human mononuclear cells (hMNC) the new LA (tetra) antagonized LPS-induced release of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in a dose-dependent manner with identical antagonistic potency as compared with compound 406. Also like compound 406, it was found to be an agonist in murine macrophage-like J774.1 cells.

Decay-accelerating factor (DAF/CD55) is a functional active element of the LPS receptor complex.

Previously, we identified an 80 kDa membrane protein (LMP80) that is capable of binding to LPS and lipid A in the presence of LBP and sCD14. LMP80 could also be detected after immuno-coprecipitation of cell membranes with LPS and lipid A, indicating a physical contact of LMP80 and LPS/lipid A. Further analysis and peptide sequencing revealed that LMP80 is identical to CD55 (decay accelerating factor, DAF), a regulatory molecule of the complement cascade. Transfection of LPS-hyporesponsive Chinese hamster ovary (CHO) cells with human CD55 resulted in the translocation of NF-B upon stimulation with LPS or lipid A. Our results demonstrate a new functional role of CD55 as a molecule able to mediate LPS-induced activation of cells that may be part of a multimeric LPS receptor complex.

Discontinuous density gradient separation of human mononuclear leucocytes using Percoll as gradient medium.

The use of a new commerically available medium (Percoll) for fractionation of human mononuclear leucocytes is described. Cells can be fractionated on the basis of their densities with high reproducibility. The separated cells were characterized by morphological and functional criteria. Monocytes can be obtained in the low density fractions with a purity of 70%--90%. Lymphocytes were found in high density fractions with a purity up to 99%. No separation between E-rosette forming (E-RFC) and surface immunoglobulin-bearing lymphocytes was obtained. However, a reduced number of high avidity E-rosette forming lymphocytes (HAE-RFC) was found within low density lymphocytes. Best spontaneous DNA synthesis and reaction in mixed leucocyte culture (MLC) were obtained with cells isolated from the 1.066/1.068 density interface, whereas the stimulation with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) had a peak response with cells from the 1.064/1.066 density interface. Colony forming myelopoietic stem cells and colony forming T-lymphocytes were detected in fractions of low density.

Application of a new ultra-microculture system. II. Stimulation of human B lymphocytes.

An ultra-microtechnique for culturing human B-lymphocytes in glass capillary tubes using a volume of 2 microliter is described. The advantage of this ultra-microculture system is that only a small number of lymphocytes and minute amounts of culture medium (or test factors) are required. Optimal culture conditions for the formation of Ig-secreting plaque-forming cells (PFC) after stimulation of mononuclear cells with pokeweed mitogen are given. Furthermore it is shown that immunoglobulin secreted into culture supernatants by purified B cells in the presence of T cell subsets can be measured in a microELISA.

A new ultra-microculture system. I. Stimulation of human T lymphocytes by phytohemagglutinin (PHA).

We have developed an ultra-microtechnique for culturing lymphocytes in glass capillary tubes at a final culture volume of 1 microliter or 2 microliter. The advantage of the method is that a substantially lower number of cells and minute amounts of culture medium are required. The cultures are premixed in microtubes, sucked into glass capillary tubes and incubated for an appropriate culture period. For determination of [3H]thymidine ([3H]TdR) incorporation, the cells are transferred into the wells of microtiter plates. Some special accessories have been developed which allow routine use of this system for large numbers of cultures. Optimal culture conditions for stimulation of human T lymphocytes by PHA are described.

T-lymphocyte colony formation of murine spleen cells in a one-stage micro agar culture.

A one-stage agar culture technique for stimulation of T-lymphocyte colony-forming units (TL-CFU) derived from murine spleen cells is described. The cultures are in glass capillaries in a volume of 30 microliter. Both, phytohemagglutinin (PHA) and concanavalin A (Con A) stimulate growth of TL-CFU. The technique does not require prestimulation of lymphocytes in liquid culture or addition of growth factor(s). The most important requirements for optimal colony growth are a low concentration of agar (less than 0.1%) and the appropriate number of seeded cells (60 X 10(3) per culture). This one-stage micro agar culture is economical and easily handled. Many cultures can be prepared and rapidly evaluated. The plating efficiency is about 10 colonies per 10(4) nucleated spleen cells. The T-cells character of the TL-CFU was demonstrated by treatment of the spleen cells with a monoclonal anti-Thy-1.2 antibody (anti-Thy-1.2) and complement before assaying for colony growth and also by staining the cells in the cultures with anti-Thy-1.2-FITC. Growth characteristics of TL-CFU indicate that cell interactions are operative during activation and proliferation. The nature of the cells cooperating in this system is discussed.

Cellular cytotoxicity of human natural killer cells and lymphokine-activated killer cells against bladder carcinoma cell lines.

The cytotoxicity of natural killer (NK) and lymphokine activated killer (LAK) cells against two human bladder tumor cell lines (BT-A and BT-B) was investigated using a fluorometric assay by labeling tumor cell DNA with Hoechst dye No. 33342. Our results demonstrate that BT-A and BT-B cells have low sensitivity to the cytotoxic activity of mononuclear cells (MNC) and NK cells. Cytotoxicity of MNC or NK cells against both tumor cell lines is enhanced during co-culture of the effector cells with the target cells, which suggests that BT-A and BT-B cells provide the signals which could activate MNC to exert cytotoxicity. In contrast to NK cells, IL-2-generated LAK cells showed profound cytotoxicity to BT-A and BT-B within 24 h. In addition to cellular cytotoxicity to bladder tumor cells, we also tested the effect of recombinant interleukin 1 beta (rIL-1 beta), recombinant tumor necrosis factor (rTNF), and the supernatants of co-culture of MNC or LAK cells with bladder tumor cells. The results show no cytotoxic or growth-promoting activity of rIL-1, rTNF, or the crude culture supernatants on bladder tumor cells. We found that LAK cells, but not macrophages or NK cells, may play a major role in cellular cytotoxicity against the two bladder tumor cell lines tested. From this finding we conclude that activation of LAK cells may be one important mechanism induced by adjuvant bacillus Calmette-Guérin (BCG) therapy leading to effective prevention of urothelial bladder carcinoma reappearance.

Effect of human recombinant interleukin 2 on natural killing of low density Percoll fraction cells.

Human recombinant IL-2 (R IL-2) was used to validate the ability of this lymphokine to increase NK cell activity. It was found that R IL-2 was able to augment the K562 lytic activity of phagocyte-depleted mononuclear cells of low density in a dose dependent manner. This phenomenon was detectable after 24 h of incubation. Low concentrations of R IL-2 were sufficient for the activation of natural killing when the incubation time was longer than 24 h. Furthermore, IL-1 exerted an additive effect on R IL-2 induced augmentation of natural killing after 24 h, but not after longer incubation periods. Lymphocyte culture derived (L) and R IL-2 had similar effects on the K562 cytolytic potential of cells contained in low density fractions. The highest cytolytic activity was observed when Leu11a+ cells were incubated with IL-2. IL-2 induced K562 cytolytic activity was also seen after incubation of low density Leu11- cells for 48 h, 72 h, or 7 days. In cultures of low density Leu11- cells incubated with IL-2 for 7 days, a proportion of cells became Leu11+. From these findings we conclude that low density Leu11- cells are pre-NK cells which acquire natural killing potential under the influence of IL-2.

Interleukin 2 induces appearance of LGL/Leu11+/K562 lytic cells in Leu11- low density peripheral blood mononuclear cell population.

Low density Percoll fraction cells cultured with interleukin 2 (IL-2) showed a higher proportion of large granular lymphocytes (LGL) and higher K562 cytolytic activity, as compared to a culture lacking IL-2. Furthermore, in a negatively selected Leu11- population, derived from low density cells, cultured for 7 days in medium supplemented with lymphocyte (L) or recombinant (R) IL-2, there appeared LGL and Leu11+ cells. Moreover, some level of K562 lytic activity and higher proportion of DR+ and Tac+ cells was found as compared to lacking IL-2 culture. Cytofluorograph analysis of cells labelled with propidium iodide revealed that a proportion of the low density Leu11- starting cell population entered the growth cycle while cultured with IL-2. In addition was found that Leu11+ cells evolve during culture with IL-2 into population lacking in part this phenotype marker. The present work shows that precursors of K562 cytolytic cells lacking Leu11 antigen reside in low density cell fraction, and that they may differentiate in LGL/Leu11+ cells.

The so-called interleukin-2 inhibitory activity of human serum is largely cytotoxic to mouse cells.

No specific interleukin-2 (IL-2) inhibitor has ever been demonstrated in human, mouse, or any other animal serum. Native mouse serum contains activities which completely inhibit IL-2-dependent and IL-2-independent in vitro proliferation of cells of different animal species by a non-cytotoxic mechanism. The decisive inhibitory component of mouse serum has a molecular weight of about 80,000, is heat-labile and has not been found in other animal sera. Also, native human serum completely suppresses the proliferation of various mouse cell types, predominantly by a cytotoxic effect caused by natural IgM antibodies and complement. Heat-inactivated human serum is no longer cytotoxic to mouse cells, and inhibits the proliferation of mouse cells much less than native serum. There is thus no evidence for a specific IL-2 inhibitor in mouse, human or other serum.

Lissencephaly, abnormal lymph nodes, and T-cell deficiency in one patient.

We report on a child with lissencephaly type I, abnormal lymph nodes, and immunodeficiency, associated with recurrent infections, autoimmune disease, spastic tetraplegia, and psychomotor retardation. Diagnostic measures included cranial computer tomography (CT) and magnetic resonance imaging (MRI) scanning, several in vivo and in vitro immunological tests, and histology of skin, lymph nodes, and liver including electron microscopy and immunohistology. Despite medical supervision, the child died at age 4 years. A common pathogenetic mechanism of defective migration of neurons and the dysmaturation of lymph nodes is most probable. The T-cell deficiency may represent a common defect of the development of both neuronal and lymphatic tissue, as the six-layered cerebral cortex and the B-cell areas in lymph nodes develop at about the same gestational age. A common defect could also be assumed involving genetically determined cell surface proteins.