A role for Ia antigens in thymocyte binding by macrophages

To investigate the membrane structures involved in cellular interactions between thymocytes and macrophages, the relative ability of different murine macrophage populations to spontaneously bind thymocytes was compared. Macrophages derived from the spleen or thymus bound three to four times the number of thymocytes than macrophages from peripheral blood, peritoneum, or bone marrow. This reflects differences both in the number of macrophages binding thymocytes and in the number of thymocytes bound per macrophage. The extent of binding seems to positively correlate with the number of Ia-positive macrophages contained in these populations, as based on previously published values. This was confirmed by showing that elimination of splenic Ia-positive macrophages with anti-Ia and complement treatment dramatically reduced thymocyte binding. In addition, mouse peritoneal washout macrophages incubated for several days with supernatant fluid from concanavalin A-stimulated spleen cells, which induce Ia-antigen expression, exhibited a marked increase in the number of macrophages that bound thymocytes and the number of thymocytes bound per macrophage. To determine if Ia antigens were directly involved in binding, spleen, thymus, or Ia-induced peritoneal macrophages were treated with a monoclonal anti-Ia antibody prior to the addition of thymocytes. Treatment with anti-Ia reduced binding by around 50%, whereas treatment with anti-H-2D antibody had no effect. Monoclonal anti-I-A and anti-I-E antibody treatments of macrophages both inhibited thymocyte binding to similar extents, and treatment of macrophages with both reagents together reduced thymocyte binding by 80%. These results indicate that thymocyte binding is in part dependent on macrophage Ia expression.     

Requirement for macrophages in lipopolysaccharide-stimulated mixed thymocyte cultures.

Cortisone-resistant thymocytes are stimulated by allogeneic thymocytes in the presence of lipopolysaccharide or small numbers of syngeneic, allogeneic or third party macrophages, as measured by ³H-thymidine uptake and by the generation of specifically cytotoxic effector cells. Lipopolysaccharide (LPS) has no stimulating effects on macrophage-depleted mixed thymocyte cultures. It also has no stimulating effect on syngeneic thymocytes in the absence of allogeneic thymocytes.Macrophages and lipopolysaccharide are only required in the induction phase of mixed thymocyte reactions. The possible role of both lipopolysaccharide and macrophages in lymphoproliferative reactions involving pure thymocyte populations is discussed.     

The role of EGF receptor transmodulation in embryonal carcinoma-derived growth factor-induced mitogenesis.

Exposure of quiescent 10T1/2 fibroblast cells to embryonal carcinoma-derived growth factor (ECDGF) results in a rapid temperature and ECDGF concentration-dependent inhibition of [125I]EGF binding to the epidermal growth factor (EGF) receptor (transmodulation). ECDGF predominantly inhibits the association of [125I]EGF with a high affinity subclass of EGF receptors, and induces increased phosphorylation of the EGF receptor on serine and threonine residues. No mitogenic effect of EGF can be detected in the presence of ECDGF concentrations which induce maximal EGF receptor transmodulation. ECDGF-induced EGF receptor transmodulation is sensitive to phorbol ester-induced desensitization whereas ECDGF-induced DNA synthesis is unaffected by prolonged pre-treatment with biologically active phorbol ester. These findings suggest that EGF receptor transmodulation is not essential for ECDGF mitogenicity but may inhibit EGF-induced DNA synthesis.     

Proteasomes play an essential role in thymocyte apoptosis.

Cell death in many different organisms requires the activation of proteolytic cascades involving cytosolic proteases. Here we describe a novel requirement in thymocyte cell death for the 20S proteasome, a highly conserved multicatalytic protease found in all eukaryotes. Specific inhibitors of proteasome function blocked cell death induced by ionizing radiation, glucocorticoids or phorbol ester. In addition to inhibiting apoptosis, these signals prevented the cleavage of poly(ADP-ribose) polymerase that accompanies many cell deaths. Since overall rates of protein degradation were not altered significantly during cell death in thymocytes, these results suggest that the proteasome may either degrade regulatory protein(s) that normally inhibit the apoptotic pathway or may proteolytically activate protein(s) than promote cell death.     

The role of calcium ions in the regulation of rat thymocyte pyruvate oxidation by mitogens.

1. Calcium concentrations in the nanomolar range cause a specific stimulation of the oxidation of pyruvate by isolated mitochondria from rat thymus that is sufficient to account precisely for the stimulation of pyruvate oxidation observed when rat thymocytes are incubated with the mitogens concanavalin A or ionophore A23187. 2. Higher concentrations of Ca2+ (more than 50 nM) inhibit the oxidation of NAD+-linked substrates by rat thymus mitochondria without affecting the oxidation of succinate or ascorbate+ NNN'N'-tetramethyl-p-phenylendiamine. 3. The addition of Ni2+ or Co2+ (2mM) to rat thymocytes prevents the response to concanavalin A at the level of pyruvate oxidation without affecting the stimulation of glycolysis induced by this mitogen. In contrast, the complete metabolic response to the ionophore A23187 is abolished by these ions. Ni2+ and Co2+ interfere with the ability of the ionophore to transport Ca2+ across the plasma membrane. 4. Concanavalin A, but not ionophore A23187, increases the respiratory inhibition induced by Ni2+ and Co2+. 5. These results support the view that mitogens stimulate lymphocyte pyruvate oxidation through an increase in cellular Ca2+ uptake.     

Concanavalin A-induced chemiluminescence in rat thymus lymphocytes. Its origin and role in mitogenesis.

Incubation of a particulate preparation from potato tissue culture cells with UDP-beta-L-[1-3H] arabinose yielded a glycoprotein fraction containing labelled material with the characteristics of hydroxyproline arabinosides. The sugar-protein linkage was resistant to hot alkaline hydrolysis, and the hydrolytic products showed similar electrophoretic and chromatographic behavior to authentic hydroxyproline-arabinosides prepared from potato tissue culture cell walls. Incorporation of arabinose into glycoprotein was stimulated by the addition of de-arabinosylated potato lectin. The product of the incubation co-migrated with native potato lectin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The subcellular distribution of the arabinosyl-transferase was investigated by fractionating potato tissue culture membranes on a discontinuous sucrose gradient in the presence or absence of Mg2+. Under both fractionation conditions the highest specific activity of the enzyme was found in the Golgi-enriched fraction. The results are discussed in relation to the synthesis of the hydroxy-proline-rich glycoprotein component of plant cell walls.     

A role for CD8 in limiting degeneracy of thymocyte selection.

The introduction of a soluble TCR (sTCR) recognizing class I major histocompatibility complex (MHC) in the fetal thymic microenvironment in vitro produces the selection of thymocytes with enhanced avidity for self class I MHC (8). The sTCR was supposed to impose enhanced avidity for self MHC at an early degenerate phase of TCR-driven selection. This could determine increased reactivity to self at later stages of differentiation when specificity of TCR-ligand interaction augments and the effect of sTCR vanishes. This hypothesis was based on the observed deletion of CD4+8+ thymocytes upon upregulation of TCR and the increase in cell size of some CD8+ cells which are expanded in long-term fetal thymus organ cultures (FTOC) as well as in the periphery of adoptively transferred nude mice. Here we show that the developing alphabeta thymocyte which does not express CD8 at the cell surface has a selective advantage in FTOC with sTCR, thus suggesting that participation of CD8 in self peptide/MHC recognition confers specificity to T-cell selection and results in excessive signaling in thymocytes in spite of the presence of sTCR.     

Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore.

Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH)2D3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH)2D3 of phytohemagglutinin. Stimulation of thymocytes with either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH)2D3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH)2D3 but in the absence of both TPA and A23187, was inhibited by 1,25(OH)2D3. The extent of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH)2D3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. In conclusion: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH)2D3 enhances or inhibits thymocyte stimulation. (2) Both stimulatory and inhibitory actions of 1,25(OH)2D3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C.     

The role of microtubules in the response of macrophages to MIF.

The role of the microtubular system in the response of macrophages to migration inhibitory factor (MIF) was investigated using agents which affect microtubule assembly. Microtubule-disrupting agents such as colchicine or vinblastine decrease or abolish the effect of MIF on macrophages. On the other hand, deuterium oxide enhances the effect of MIF on the macrophage. The presence of deuterium oxide during the first hour of interaction of MIF with macrophages is sufficient to increase the MIF response, whereas a pulse of D₂O during the first hour and subsequent addition of MIF had no effect. These results indicate that the state of the microtubule system during the initial period of macrophage-MIF interaction determines the response to MIF.     

Matrix metalloproteinase-9 in macrophages induces thymic neovascularization following thymocyte apoptosis

Matrix metalloproteinase-9 (MMP-9) has been implicated in the degradation of the extracellular matrix in a variety of physiological and pathological processes. We found that MMP-9 expression in thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis was increased both at mRNA and protein levels. Macrophages are shown to be the principal stromal cells responsible for phagocytosis of dying thymocytes, and macrophages were found to constitutively express MMP-9. The activity of plasmin, which is known as one of the activators for MMP-9, was increased in the thymuses with MMP-9 activation. Binding of Ab HUIV26, which recognizes a cryptic epitope on collagen type IV following proteolytic cleavage, was found to be reduced in MMP-9 knockout mice, suggesting that collagen type IV is a substrate of MMP-9. Although the formation of thymic neovessels was found following thymocyte apoptosis, it was diminished in anti-CD3 Ab-injected MMP-9 knockout mice. In vivo administration of Ab HUIV26 resulted in a reduction of thymic neovascularization. After clearance of apoptotic thymocytes, the number of macrophages in the thymuses was decreased, and this decrease was delayed by blocking of HUIV26 epitope. Taken together, our results suggest that MMP-9 expression in macrophages mediates degradation of collagen type IV and facilitates their migration from the thymus after clearance of apoptotic thymocytes. These studies demonstrate a potential role of macrophage MMP-9 in the remodeling of thymic extracellular matrix following thymocyte apoptosis.     

Spontaneous regression of Friend virus-induced erythroleukemia. III. The role of macrophages in regression.

The spontaneous regression of erythroleukemia induced by the RFV strain of Friend virus is a macrophage-dependent process. Functional suppression or elimination of the macrophage population in leukemic mice with silica, carrageenan, anti-macrophage serum, or trypan blue inhibited regression. Prior protection of the macrophages with PVNO allowed regression in silica or carrageenan-treated mice. Macrophage phagocytic activity was inhibited in about half the RFV-induced leukemic mice at 25 to 30 days post virus inoculation. Those animals with normal macrophages regressed, whereas whereas those with inhibited macrophages did not. Progressor mice could be induced to regress by inoculation with normal syngeneic macrophages; other cell types were ineffective. The inhibition of macrophage function in leukemic mice was the result of infection of the macrophages by virus. Removal of the infected cells by cytolysis with anti-virus antiserum and C restored the phagocytic activity of the population. Inhibited macrophages were less capable of responding to immobilized antigen-antibody complexes than normal macrophages, suggesting that the loss of function was due to a change in their Fc receptor.     

Direct role of viral hemagglutinin in B-cell mitogenesis by influenza viruses

The mitogenic activity of influenza virus is a function of the hemagglutinin (HA) molecule. Purified HA is mitogenic for murine B lymphocytes but not T lymphocytes. Furthermore, like the intact virus, HA of the H2 (but not H3) subtype is mitogenic only for B cells expressing the class II major histocompatibility complex glycoprotein I-E. Since virus bearing uncleaved HA is as mitogenic as virus bearing cleaved HA, the membrane fusion activity of the HA molecule is not involved.     

The role of phospholipases from inflammatory macrophages in demyelination

Activated macrophages harvested from rat peritoneum were shown to contain phospholipase A₁, A₂ and lysophospholipase activities which were defined on a series of radiolabelled phospholipid substrates. During in vitro culture of these elicited macrophage populations, phospholipase enzymes were secreted into the culture medium. Radiolabelled myelin, prepared from young rats after intracerebral injection of¹⁴C acetate, was used as a substrate to analyze the susceptibility of central nervous system (CNS) myelin to attack by cell-associated and secreted macrophage enzymes. Homogenates of peritoneal macrophages degraded the myelin lipids at acid pH; phosphatidyl choline (PC) and ethanolamine phosphatide (EP) were both degraded with liberation of free fatty acid and small amounts of lysolipids. The ethanolamine lipids were most vulnerable; up to 20% of this fraction was degraded in six hours. Selected batches of macrophage culture supernatant similarly degraded the myelin EP at acid pH. These results suggest that phospholipase enzymes, released from activated macrophages in close proximity to the myelin sheath, may participate in primary demyelination in inflammatory CNS lesions.     

The role of macrophages in the uptake of endotoxin by the mouse liver.

The purpose of this investigation was to analyse the macrophage subpopulations involved in the uptake of endotoxin in the liver. The results show that in normal B10.D2 mice the liver macrophages constitute a heterogeneous population of cells which, depending on their state of differentiation, are distinguished by their differential distribution in the liver acinus and by their ability to phagocytose latex. Following the intravenous administration of endotoxin (lipopolysaccharide = LPS) from Salmonella abortus equi, endotoxin-carrying non-parenchymal cells of the liver (NPLC) were investigated immunohistochemically (in situ) and immunocytochemically (after isolation) between 1 h and 14 days after the injection. The endotoxin content of the blood and of isolated NPLC was also determined, using radioactivity labeled LPS. Following LPS injection, the total number of macrophages in the liver increased, reaching a maximum after 3 days. There was a striking increase in the ratio of mature to immature macrophages. After day 3, the number of macrophages decreased again, returning to the pre-injection values by day 14. 1 h after the administration of LPS, 41% of the isolated NPLC were already endotoxin-positive, a percentage which remained constant until the 3rd day. Thereafter, the number of LPS-bearing cells increased to a maximum of about 52% on the 5th day. This increase mostly involved macrophages which had taken up endotoxin. Concurrent with these changes there was a threefold increase in radioactivity-labeled LPS from the 7th h to the 5th day after injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of p23,30-bearing human macrophages in antigen-induced T lymphocyte responses.

The effect of anti-p23,30, a rabbit antiserum to the human Ia-like antigen p23,30, on two macrophage-dependent T-cell functions, proliferation in response to soluble antigens, and production of lymphocyte mitogenic factor (LMF) was studied. T cells depleted of macrophages neither proliferate nor secrete LMF, and these functions are restored by addition of as few as 0.5% adherent macrophages. Treatment of macrophages with anti-p23,30 and C, however, abolishes their capacity to reconstitute these T-cell functions. In contrast, treatment of T cells with anti-p23,30 and C did not affect their capacity to respond in the presence of untreated adherent cells. We conclude that the presence of p23,30-bearing macrophages is critical for the expression of these antigen-induced T-cell responses.