Selective release of lysosomal hydrolases from phagocytic cells by cytochalasin B

1. Cytochalasin B (10mug/ml) enhances the release of rabbit polymorphonuclear leucocyte lysosomal acid hydrolases induced by retinol (vitamin A alcohol). 2. This effect is seen at doses of the vitamin that cause selective release of acid hydrolases and those causing more general enzyme release indicated by the loss of lactate dehydrogenase. 3. Cytochalasin B (2-50mug/ml) has no effect on the release of sedimentable acid hydrolases of intact granules obtained from disrupted polymorphonuclear leucocytes. 4. Cytochalasin B (2-10mug/ml) causes a time- and dose-dependent release of mouse peritoneal macrophage acid hydrolases. 5. This effect is selective at all doses of cytochalasin B used, since no release of lactate dehydrogenase, malate dehydrogenase and leucine 2-naphthylamidase was detected. 6. Treatment with cytochalasin B at doses of up to 10mug/ml for as long as 72h did not significantly change the total activities of any of the enzymes measured. 7. The lack of toxicity of cytochalasin B was shown by dye-exclusion tests and its failure to release radioactive colloidal gold stored in secondary lysosomes.     

Free fatty acids,lipid peroxidation,and lysosomal enzymes in experimental focal cerebral ischemia in primates: Loss of lysosomal latency by lipid peroxidation

Experimental focal cerebral ischemia was produced in monkeys (Macaca radiata) by occlusion of the right middle cerebral artery (MCA). The release of the lysosomal glycosidases, -d-hexosaminidase, -l-fucosidase and -d-mannosidase into the soluble fraction in the right basal ganglia of the experimental animals was measured at different periods from 30 min to 12 hr after occlusion and compared with the corresponding sham operated control animals. There was a significant increase in the released lysosomal enzymes in the MCA occluded animals at all periods and particularly at 4 hr after occlusion. The CSF from the experimental animals also showed elevated levels of hexosaminidase and fucosidase. The free fatty acids (FFA) measured in the basal ganglia at 30 min and 2 hr after occlusion showed a 100 fold increase in the experimental animals. The predominant fatty acid released was linoleic acid (18:2) followed by arachidonic acid (20:4). Lipid peroxidation in the basal ganglia measured by the thiobarbituric acid (TBA) reaction in the presence or absence of ascorbic acid also showed a significant increase in the experimental animals at all periods with a maximum at 30 min to 2 hr after occlusion. In order to assess whether lipid peroxidation causes damage to the lysosomes and release of the enzymes, a lysosome enriched P₂ fraction from the normal monkey basal ganglia was prepared and the effect of peroxidation studied. Maximum peroxidation in the P₂ fraction was observed in the presence of arachidonic acid, ascorbic acid and Fe²⁺. There was a good correlation between the extent of lipid peroxidation and the in vitro release of lysosomal hexosaminidase from the P₂ fraction. Anti-oxidants which strongly inhibited lipid peroxidation in the P₂ fraction prevented the release of hexosaminidase. The results suggested that in ischemia produced by MCA occlusion lipid peroxidation which damages the lysosomal membrane causes the release of lysosomal hydrolytic enzymes.Abbreviations used BHA butylated hydroxyanisole - BHT butylated hydroxytoluene - FFA free fatty acids - MCA middle cerebral artery - MDA malonaldehyde - PUFA polyunsaturated fatty acids - TBA thiobarbituric acid     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E2 and 6-keto-prostaglandin F1 alpha. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 microgram/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 microgram/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolases are independently regulated.     

Induction of macrophage lysosomal hydrolase synthesis and secretion by beta-1,3-glucan

Studies were undertaken to elucidate the active component in zymosan necessary to induce the delayed-onset synthesis and secretion of representative lysosomal hydrolases, hexosaminidase, and beta-glucuronidase in macrophages. Resident mouse peritoneal macrophages were challenged with zymosan particles and particulate beta-1,3-glucan, the major subcomponent of zymosan. Zymosan was found to induce a rapid secretion of preformed hexosaminidase with maximal release (75%) occurring 6 hr after the addition of zymosan. By contrast, beta-1,3-glucan was totally inactive in this respect. However, both zymosan and beta-1,3-glucan were found to induce the delayed-onset synthesis and secretion of hexosaminidase and beta-glucuronidase while maintaining constant cellular enzyme levels over a 5-day period following the addition of stimulus. These late responses were almost totally blocked by a noncytolytic concentration of cycloheximide, indicating their dependence on de novo protein synthesis. Mannan, the second major subcomponent of zymosan, had no effect on either immediate secretion or delayed-onset synthesis and secretion of hexosaminidase. These results suggest that the induction of the delayed-onset synthesis and secretion of the lysosomal hydrolases by zymosan may be dependent on the glucan subcomponent of zymosan. Moreover, it would also appear that the release of preformed lysosomal enzymes is not the trigger for the delayed-onset synthesis and secretion of hexosaminidase.     

Stimulation of macrophage tumoricidal activity by the growth and differentiation factor CSF-1

The effect of the macrophage growth and differentiation factor CSF-1 on the tumoricidal capacity of murine peritoneal exudate macrophages was investigated. Pretreatment of peptone-elicited macrophages 1 day with 300-1200 U/ml CSF-1 induced moderate killing and greatly stimulated lymphokine (LK)-induced killing of [3H]thymidine-labeled TU5 sarcoma cells to levels above that seen with fresh macrophages. Further addition of CSF-1 at Day 1 at the time of the tumor lysis assay promoted moderate increases in spontaneous and LK-induced activity. CSF-1 did not stimulate freshly harvested exudate macrophages to lyse TU5 targets in the presence or absence of lymphokine (LK) activators. Lipopolysaccharide (LPS) at 0.1-1000 ng/ml did not stimulate cytotoxicity, and the low endotoxin content and the use of polymyxin B and C3H/HeJ mice excluded a role for LPS in these experiments. Incubation of the macrophages with IFN and the myeloid growth factors IL-3 and GM-CSF did not stimulate tumoricidal activity. CSF-1 has been proposed as a therapeutic agent to restore myeloid cell numbers in induced (cancer chemotherapy, bone marrow transplantation, etc.) and natural aplastic anemias. These studies show that CSF-1 also may be useful in combination with LK activators to promote resistance to cancer in mature mononuclear cells. CSF-1 may have similar effects in LK-activated macrophages to enhance resistance to infectious diseases.     

Amphotericin B stimulates secretion of beta-hexosaminidase from mouse adherent spleen cells.

Secretion of the lysosomal enzyme hexosaminidase is induced by amphotericin B in mouse spleen adherent cells that show a significant increase in their candidacidal activity. The stimulation of beta-hexosaminidase is both time and dose dependent. Amphotericin B treatment did not change hexosaminidase expression that is represented mainly by "A-type" hexosaminidase in macrophages, on the basis of its biochemical properties.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E₂ and 6-keto-prostaglandin F_1α. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 μg/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 μg/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolyses are independently regulated.     

Cytotoxicity of resident and lymphokine-activated mouse peritoneal macrophage against Trichomonas vaginalis

This study was aimed to observe the direct and lymphokine-activated cell mediated cytotoxic effects against Trichomonas vaginalis by mouse peritoneal macrophages. Cytotoxicity was measured as release of 3H-thymidine from prelabeled protozoa, and tested in U-bottom microtiter plates. A 0.1 ml suspension of labeled protozoa (2 x 10(5)/ml) was placed in each well, followed by 0.1 ml of a suspension containing increasing numbers of peritoneal cells. After a 24 hr incubation at 37 degrees C, 0.1 ml of the supernatant was collected and counted in liquid scintillation counter. Mouse peritoneal macrophages had appreciable level of spontaneous cytotoxicity against T. vaginalis at the effector to target cell ratios from 5:1 to 50:1. Treatment of macrophages with lymphokine, produced by PHA-stimulated spleen cells, increased the cytotoxicity in comparison with resident macrophages against T. vaginalis. The degree of macrophage activation for the killing was not dependent upon the lymphokine concentration. Peritoneal cells adherent to plastic displayed significant levels of cytotoxicity against T. vaginalis. This study indicates that mouse peritoneal macrophages are spontaneously cytotoxic for T. vaginalis and lymphokine increases the cytotoxicity by activating macrophages to kill T. vaginalis.     

Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells

Mast cells are important players in innate immunity and mediate allergic responses. Upon stimulation, they release biologically active mediators including histamine, cytokines and lysosomal hydrolases. We used permeabilized rat basophilic leukaemia cells as model to identify R-SNAREs (soluble NSF (N-ethylmaleimide-sensitive fusion protein)) mediating exocytosis of hexosaminidase from mast cells. Of a complete set of recombinant mammalian R-SNAREs, only vesicle associated membrane protein (VAMP8)/endobrevin consistently blocked hexosaminidase release, which was also insensitive to treatment with clostridial neurotoxins. Thus, VAMP8, which also mediates fusion of late endosomes and lysosomes, plays a major role in hexosaminidase release, strengthening the view that mast cell granules share properties of both secretory granules and lysosomes.     

Demonstration of multiple forms of acid phosphatase in hemolymph of the African snail, Archachatina marginata.

1. Hemolymph from the giant African snail Archachatina marginata has been analyzed for its content of certain lysosomal hydrolases and shown to contain substantial quantities of acid phosphatase (285 units/ml) hexosaminidase (512 units per ml) and beta-glucuronidase (28 units/ml). 2. Hemolymph acid phosphatase can be fractionated into 6 active components by DEAE-Sephadex chromatography. 3. Some of the acid phosphatase species can be distinguished on the basis of heat stability, pH dependency and sensitivity to inhibitors including phosphate, L(+) tartrate, fluoride, formaldehyde and 1.10 phenanthroline.     

Colony-stimulating factor-induced monocyte survival and differentiation into macrophages in serum-free cultures

The role of mononuclear phagocyte-specific colony-stimulating factor (CSF-1) in human monocyte to macrophage differentiation was investigated. The addition of 1000 U/ml of CSF-1 to serum-free monocyte cultures resulted in monocyte survival comparable to that in cultures containing 5% AB serum, whereas cells in serum- and CSF-1-free medium lost their viability in 3 to 5 days. The requirement for CSF-1 coincided with the time (40 to 64 hr of culture) when the major changes in morphology and biochemical function took place in monocytes undergoing differentiation into macrophages. If CSF-1 was removed from the cultures before this time, death of the monocytes resulted. In cultures containing CSF-1, as in serum containing cultures, the lysosomal enzyme acid phosphatase was enhanced 10- to 20-fold by day 4 to 5. Superoxide production in response to phorbol myristic acetate was maintained in CSF-1 cultured monocytes, but declined with time in monocytes cultured in serum. The expression of monocyte-macrophage antigens p150.95 (LeuM5), OKM1, LeuM3, Fc receptors (32.2), and HLA-DR had increased in CSF-1 containing cultures at day 4. When antigen expression was analyzed at day 2 to 3, when cell size and 90 degrees scatter characteristics were still identical to control serum-free cultures, only p150.95, HLA-DR and FcR expression were enhanced by CSF-1. Low amounts of lipopolysaccharide (0.1 ng/ml) were found to enhance monocyte survival in the absence of added CSF-1. Lipopolysaccharide-containing cultures were found to produce CSF-1 (up to 450 U/ml, as detected by radioimmunoassay). Lipopolysaccharide (1 microgram/ml), however, did not induce enhanced expression of the maturation-related antigens. Based on these observations we conclude that CSF-1 is enhancing human monocyte survival and is involved in the events leading to the differentiation of monocytes into macrophages.     

Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis

Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, β-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.     

Alveolar macrophages differ from blood monocytes in human IL-1 beta release. Quantitation by enzyme-linked immunoassay

Fresh human alveolar macrophages and blood monocytes were stimulated with LPS and assessed for their ability to produce and release antigenic IL-1 beta. Using a sensitive and specific ELISA for IL-1 beta, monocytes released 13.3 +/- 3.1 ng/10(6) cells compared to 3.5 +/- 0.8 ng/10(6) cells for alveolar macrophages (p less than 0.01). To investigate the reason for this difference in IL-1 beta release, monocytes were compared to alveolar macrophages for total IL-1 beta production (i.e., the amount released plus that detected in the lysates). Monocytes produced a total of 19.0 +/- 3.2 ng/10(6) cells whereas alveolar macrophages produced 24.8 +/- 5.6 ng/10(6) cells (p = 0.37). The relative increase in alveolar macrophage intracellular IL-1 beta was confirmed by Western blot analysis of cell lysates. Thus, the limitation in IL-1 release from alveolar macrophages appears to be due to a decrease in the processing and release of the IL-1 beta precursor. In addition, TNF production studies demonstrated that the limitation in IL-1 release was not a generalized defect. In contrast to the IL-1 beta data, when TNF was measured from monocytes and macrophages, monocytes released only 14.6 +/- 3.4 ng/10(6), whereas macrophages released 101 +/- 30 ng/10(6) (p less than 0.02). In this same context, when fresh monocytes were allowed to mature in vitro they took on monokine production characteristics similar to alveolar macrophages. In vitro matured monocytes had a greater than 20-fold decrease in their ability to release IL-1 beta and a 6- to 8-fold increase in their ability to release TNF. Taken together, these studies suggest that IL-1 beta release is limited in mature mononuclear phagocytes as compared to fresh blood monocytes, and furthermore, that IL-1 beta regulation differs significantly from that of TNF-alpha.     

The effects of an anti-I-Ab antibody on murine host resistance to Listeria monocytogenes

Infection with Listeria monocytogenes stimulates T cell proliferation and T cell-derived lymphokine production. The release of lymphokines, in turn, "activates" macrophages, enhancing their bactericidal capacity. Because prior studies suggest that I-A+ accessory cells play a critical role in this pathway, we assessed the effects of an anti-I-A antibody on the murine host resistance to listerial infection. To this end, we infused Listeria into control C57BL/6 mice (I-Ab haplotype) and mice of the same strain which had been pretreated 18 hr earlier with D3137 (a monoclonal IgG2a anti-I-Ab,d antibody). Preliminary studies demonstrated that this antibody can markedly inhibit antigen-induced proliferation of Listeria-dependent T cells in vitro and (at a dose of 1 mg/animal) can markedly reduce I-A expression on splenocytes in vivo. Even though D3137 pretreatment prevented the splenomegaly normally observed after Listeria infusion into mice, it protected animals infused with otherwise lethal concentrations of Listeria. Because antibody-treated animals had sevenfold fewer organisms in their spleens 18 hr after infection and 1000-fold fewer organisms than control animals 3 days after infection, improved survival resulted from an antibody-induced increase in the bactericidal capacity of the MPS. Protection was not noted when C1.18.4 (an IgG2a myeloma protein without known antibody activity) was infused into C57BL/6 mice or when D3137 was infused in B10.BR (I-Ak) mice. D3137 also protected (B10 X B10.BR)F1 mice (which are hybrids bearing I-Ab and I-Ak), suggesting that complete blockade of antigen presentation is not a prerequisite for its protective action. Further studies into the mechanism for these effects may provide new insights into the pathophysiology of MPS activation in response to immunologic challenge.     

Interleukin 1 stimulates proliferation of a nontransformed T lymphocyte line in the absence of a co-mitogen

Although interleukin (IL) 2-responsive T cell lines provide an opportunity to study the cellular effects of this lymphokine on homogeneous T lymphocyte populations, T cell clones which proliferate in response to IL-1 alone have not been available. We have isolated from cultures of the nontransformed murine T helper cell line, D10 . G4 . 1, a variant (MD10 cells) which proliferates (no lectin or antigen needed) in response to IL-1 alone. The MD10 cells are markedly sensitive to either murine or human recombinant IL-alpha (HrIL-1 alpha) with half-maximal responses observed at monokine concentrations as low as 0.4 X 10(-12) M or 0.8 U/ml, respectively. MD10 cells show the maximal IL-1 effect at 72 hr where the response exceeds the base line by 100-fold (approximately 3,000----300,000 cpm of [3H]thymidine). Whereas both HrIL-2 and purified murine B cell-stimulatory factor 1 (MpBSF-1) induce MD10 proliferation, the maximal response to either is much lower (HrIL-2: 50X baseline; MpBSF-1: less than 20X base line) than to IL-1. Conditioned media from control, concanavalin A-, or IL-1-treated MD10 cells fail to stimulate CTLL or HT-2 cell proliferation alone or inhibit CTLL mitogenesis in the presence of added HrIL-2. Furthermore, monoclonal antibodies to BSF-1 fail to inhibit IL-1-stimulated MD10 replication, and neither HT-2 nor CTLL cells proliferate despite direct cell-to-cell contact with IL-1-treated MD10 cells. When combined, IL-1 (10(-13), 10(-12) M) and IL-2 (10(-13) to 10(-10) M) act synergistically in their MD10 cell growth-promoting effects. MD10 proliferation induced by either IL-1 or IL-2 is relatively resistant to cyclosporine A, with the ID50 of cyclosporine for both IL-1- and IL-2-exposed MD10 cells (ID50 5000 ng/ml) exceeding that for concanavalin A-activated splenocytes (ID50 20 ng/ml) by 2 to 3 orders of magnitude. Finally, MD10 cells bear the L3T4 antigen, IL-2 receptors, and the same clonotypic antigen receptor as the parent clone as recognized by monoclonal antibody 3D3. These data suggest that, in respect to this particular T cell line, IL-1 is directly growth-promoting or, alternatively, induces the production of undetectable, intermediate growth factor(s) resistant to inhibition by cyclosporine A.     

Cholesterol delivered to macrophages by oxidized low density lipoprotein is sequestered in lysosomes and fails to efflux normally

Oxidized low density lipoprotein (LDL) has been found to exhibit numerous potentially atherogenic properties, including transformation of macrophages to foam cells. It is believed that high density lipoprotein (HDL) protects against atherosclerosis by removing excess cholesterol from cells of the artery wall, thereby retarding lipid accumulation by macrophages. In the present study, the relative rates of HDL-mediated cholesterol efflux were measured in murine resident peritoneal macrophages that had been loaded with acetylated LDL or oxidized LDL. Total cholesterol content of macrophages incubated for 24 h with either oxidized LDL or acetylated LDL was increased by 3-fold. However, there was no release of cholesterol to HDL from cells loaded with oxidized LDL under conditions in which cells loaded with acetylated LDL released about one-third of their total cholesterol to HDL. Even mild degrees of oxidation were associated with impairment of cholesterol efflux. Macrophages incubated with vortex-aggregated LDL also displayed impaired cholesterol efflux, but aggregation could not account for the entire effect of oxidized LDL. Resistance of apolipoprotein B (apoB) in oxidized LDL to lysosomal hydrolases and inactivation of hydrolases by aldehydes in oxidized LDL were also implicated. The subcellular distribution of cholesterol in oxidized LDL-loaded cells and acetylated LDL-loaded cells was investigated by density gradient fractionation, and this indicated that cholesterol derived from oxidized LDL accumulates within lysosomes. Thus impairment of cholesterol efflux in oxidized LDL-loaded macrophages appears to be due to lysosomal accumulation of oxidized LDL rather than to impaired transport of cholesterol from a cytosolic compartment to the plasma membrane.     

Regulation of prostaglandin synthesis and of the selective release of lysosomal hydrolases by mouse peritoneal macrophages.

Macrophages isolated from the peritoneal cavity of untreated mice and maintained in tissue culture synthesize and release prostaglandins when challenged with zymosan. These cells also selectively release lysosomal acid hydrolases under the same conditions. The major prostaglandins released into the media are found to be prostaglandins E1, E2 and 6-oxoprostaglandin F1a, whereas prostaglandin F2a is not detected. Macrophages isolated from mice that have received an intraperitoneal injection of thioglycollate broth are far less responsive to zymosan challenge. These cells require 300 microgram of zymosan to synthesize and release one-third the amount of prostaglandins released from non-stimulated macrophages exposed to 50 microgram of zymosan. In addition, thioglycollate-stimulated macrophages release less than 10% of their lysosomal acid hydrolases when exposed to 300 microgram of zymosan whereas non-stimulated cells release approximately 50% of these enzymes after treatment with 50 microgram of zymosan. The zymosan-stimulated synthesis and release of prostaglandins are completely inhibited by indomethacin, whereas the increased selective release of lysosomal acid hydrolases is not affected. Macrophages, unlike fibroblasts, do not synthesize and release prostaglandins when exposed to serum or to bradykinin.     

Differential endotoxin sensitivity of lymphocytes and macrophages from mice with an X-linked defect in B cell maturation.

The in vitro sensitivity of B lymphocytes and macrophages derived from (CBA/N X DBA/2N) F1 male mice, which carry an X-linked recessive gene that produces defective B cell maturation, was compared to phenotypically normal F1 female mice. B lymphocytes of F1 males exhibit an abnormal mitogenic response to LPS in serum-free culture conditions, which is partially reversed in the presence of serum. In contrast, both resident and thioglycollate-induced peritoneal macrophages of F1 male mice respond normally to LPS. In response to LPS in vitro, F1 male macrophages produce the monokine, lymphocyte-activating factor (LAF) and release prostaglandins. Furthermore, F1 male macrophages are sensitive to the lethal effects of LPS. Therefore, the defective CBA/N gene appears to be expressed only in B lymphocytes and not in macrophages. Since F1 male mice are normally sensitive to the lethal and adjuvant effects of LPS in vivo, these findings suggest that a mature B lymphocyte population is not required for these effects and support the role of the macrophage in the mediation of LPS-induced lethality and adjuvanticity.     

Intercellular exchange of lysosomal hydrolases between mutant human fibroblasts and other cell types

Human fibroblasts with a genetic deficiency of a single lysosomal enzyme and fibroblasts from a patient with ‘I-cell’ disease with a multiple deficiency of lysosomal hydrolases were used as recipient cells in studies on recognition and uptake of β-N-acetylhexosaminidase (hexosaminidase), β-glucuronidase and β-galactosidase. Normal human fibroblasts, and fibroblasts, hepatocytes and hepatoma cells from the rat were used as donor cells. The release of hexosaminidase was found to be similar among these different cell types, but the extracellular activities of β-glucuronidase and β-galactosidase were much higher in the rat cell cultures than in cultures of normal human fibroblasts. The enzymes released by rat fibroblasts were ingested by deficient human fibroblasts; enzyme from normal human fibroblasts was shown to be taken up by rat fibroblasts by means of electrophoresis. This indicates that reciprocal transfer of lysosomal hydrolases occurs between human and rat fibroblasts. Rat hepatocytes released hydrolases that were poorly taken up by human recipient fibroblasts and uptake of human fibroblast enzyme was not detected in the hepatocytes. Rat hepatoma cells, on the other hand, released lysosomal enzymes that were taken up by human deficient cells with a higher efficiency than those from fibroblasts. The uptake was subject to competitive inhibition by mannose 6-phosphate, the kinetics of which were comparable with those reported for ‘high-uptake’ forms of lysosomal enzymes [1–2]. Electrophoretic studies showed that rat hepatoma cells were not only capable of ingesting hexosaminidase from normal human fibroblasts, but also defectively processed enzyme [4–5] released by ‘I-cells’. These findings make rat hepatoma cells a useful model for the study of recognition and uptake of lysosomal enzymes.     

Dexamethasone increases expression of mannose receptors and decreases extracellular lysosomal enzyme accumulation in macrophages

Macrophages express a mannose-specific pinocytosis receptor that binds and internalizes lysosomal hydrolases. Treatment of rat bone marrow-derived macrophages with dexamethasone resulted in a concentration- and time-dependent increase in mannose-receptor activity. The dexamethasone effect was maximal at 24 h. Half-maximal effects were observed at a dexamethasone concentration of 2.5 X 10(-9) M. With 125I-beta-glucuronidase as ligand, a 2.5-fold increase in uptake rate was observed in dexamethasone-treated cells, with no change in Kuptake (2.5 X 10(-7) M beta-glucuronidase). Cell surface binding (4 degrees C) was elevated 2.6-fold following dexamethasone treatment. The increase in ligand binding appeared to be due to an increase in number of sites with no change in affinity. Cycloheximide suppressed the dexamethasone-mediated rise in receptor number, while cycloheximide alone had little effect on receptor activity over 16 h. These results suggest that dexamethasone stimulates synthesis of mannose receptors in macrophages. Extracellular accumulation of hexosaminidase was sharply reduced by dexamethasone treatment, and corresponded with the rise in mannose-receptor activity. Extracellular levels of hexosaminidase from untreated macrophages were modestly increased by the presence of mannan, while the extracellular activity from dexamethasone-treated cells was increased significantly by mannan. Extracellular hexosaminidase, released from zymosan-treated macrophages, was dramatically reduced by dexamethasone pretreatment. Enzyme released from zymosan-stimulated macrophages was efficiently endocytosed by dexamethasone-treated cells in co-culture experiments, and this endocytosis was blocked by the addition of mannan. These results suggest that the mannose receptor of macrophages may play a role in regulating extracellular levels of lysosomal enzymes via a secretion-recapture mechanism.