The fine structure of the cells of the mouse peritoneum

Summary The cells of the peritoneum of the mouse have been examined with the electron microscope both by studying the gastro-splenic omentum and by washing the cells out of the peritoneal cavity. They comprise mesothelial cells, mast cells, lymphocytes and macrophages. The mesothelial cells were probably nearly all degenerate. The mast cells released granules which were phagocytosed by the other cells. The lymphocytes were either classical small lymphocytes, or rather larger cells similar to previously described immunoblasts. The macrophages varied considerably in size. Some were probably derived from the covering cells of the milk spots. They contained varying numbers of dense bodies, with the structure of lysosomes. A series of appearances was seen which suggested that these were synthesized in the granular endoplasmic reticulum. A gradation of structure was seen between lymphocytes and small macrophages.The gastro-splenic omentum consisted of two layers of mesothelium, in places fenestrated. The milk spots which were scattered throughout this structure were covered by cells similar to macrophages, and had a core of lymphoid cells in which ran a small blood vessel. The most notable difference between the mesothelial cells and the macrophages was the presence of many small caveolae at the surface of the mesothelial cells, and of larger vacuoles and indentations at the surface of the macrophages. Acknowledgements. I am grateful to Professor R. Barer for much advice and criticism, to Dr. G. A. Meek for guidance on electron microscopy, and to Miss M. Tune and Mr. M. Turton for photographic assistance.This work was supported by a grant from the Medical Research Council and by grants to the Department from the S.R.C., Nuffield Foundation and Unilever Limited.     

Immunocytochemical staining of Histoplasma capsulatum at the electron microscopic level

Summary Rabbits were immunized with histoplasmin emulsified in Freund's complete adjuvant. Antibody raised in these rabbits was exposed to Histoplasma capsulatum yeast cells, either in tissue culture medium, or after in vitro or in vivo phagocytosis by mouse macrophages. The sites of antibody binding were identified using an immunoperoxidase technique. At least two sites of antibody binding were identified, one to the fungal cell wall and the other to the outer cell membrane. Within 6 h after phagocytosis by macrophages, fungal cell walls appeared roughened, with what appeared to be cell wall antigen released into the phagolysosome, appearing associated with the phagolysosome membrane, and possibly adjacent macrophage cytoplasm. Similar staining of fungal antigen was noted in alveolar macrophages which had ingested Histoplasma capsulatum after a respiratory challenge. This method may be useful in detailing the host/pathogen interactions which occur in human pulmonary histoplasmosis.     

Pulmonary collectins enhance phagocytosis of Mycobacterium avium through increased activity of mannose receptor

Collectins, including surfactant proteins A (SP-A) and D (SP-D) and mannose binding lectin (MBL), are the important constituents of the innate immune system. Mycobacterium avium, a facultative intracellular pathogen, has developed numerous mechanisms for entering mononuclear phagocytes. In this study, we investigated the interactions of collectins with M. avium and the effects of these lectins on phagocytosis of M. avium by macrophages. SP-A, SP-D, and MBL exhibited a concentration-dependent binding to M. avium. The binding of SP-A to M. avium was Ca(2+)-dependent but that of SP-D and MBL was Ca(2+)-independent. SP-A and SP-D but not MBL enhanced the phagocytosis of FITC-labeled M. avium by rat alveolar macrophages and human monocyte-derived macrophages. Excess mannan, zymosan, and lipoarabinomannan derived from the M. avium-intracellular complex, significantly decreased the collectin-stimulated phagocytosis of M. avium. Enhanced phagocytosis was not affected by the presence of cycloheximide or chelation of Ca(2+). The mutated collectin, SP-A(E195Q, R197D) exhibited decreased binding to M. avium but stimulated phagocytosis to a level comparable to wild-type SP-A. Enhanced phagocytosis by cells persisted even after preincubation and removal of SP-A or SP-D. Rat alveolar macrophages that had been incubated with SP-A or SP-D also exhibited enhanced uptake of (125)I-mannosylated BSA. Analysis by confocal microscopy and flow cytometry revealed that the lung collectins up-regulated the cell surface expression of mannose receptor on monocyte-derived macrophages. These results provide compelling evidence that SP-A and SP-D enhance mannose receptor-mediated phagocytosis of M. avium by macrophages.     

Nuclear membranous whorls

Summary Membranous whorls have been seen in the nuclei of peritoneal and testicular cells which had been subjected to various experimental manoeuvres. It seems likely that this is an early manifestation of cell degeneration which is demonstrated readily only by glutaraldehyde fixation, and to that extent can be regarded as a glutaraldehyde artifact. Acknowledgements. This work was supported by grants from the Medical Research Council, and the University of Sheffield Tuberculosis Research Fund, and by a grant to the Department from Unilever Ltd.I am grateful to Professor R. Barer for his advice and criticism, to Dr. G. A. Meek for guidance on electron microscopy, to Dr. E. J. Clegg for permission to use material from joint experiments. Technical and photographic assistance was provided by Messrs. P. GarLick and L. Murgatroyd and by Miss M. Tune.     

Determination of quantitative parameters of Escherichia coli phagocytosis by mouse peritoneal macrophages

Quantitative parameters of phagocytosis of fluorescein-labeled Escherichia coli cells by mouse peritoneal macrophages were studied using a fluorimetric method. E. coli cells were conjugated with fluoresceinisothiocyanate (FITC) and then incubated with macrophages. At the end of incubation, phagocytosis was stopped by the addition of a lysing solution (0.5% Triton X-100 in 0.01 M phosphate buffer in 0.15 M saline, pH 7.4). Trypan blue at a concentration of 0.04% was used as a quenching agent to differentiate between attached and ingested E. coli cells. It was shown that phagocytosis of E. coli cells depended on temperature and opsonization of bacteria. The number of E. coli cells ingested by macrophages increased rapidly for the initial 60 min of incubation at 37°C. To achieve optimal uptake of E. coli cells, their opsonization with 5% native serum was needed. The uptake of nonopsonized bacteria by macrophages was significantly lower than that of the opsonized ones (p < 0.05). Sodium azide was shown to produce a dose-dependent suppression of phagocytosis of E. coli cells by mouse peritoneal macrophages.     

Nitric oxide control of large veins in the toad <Emphasis Type="Italic">Bufo marinus</Emphasis>

This study examined the nitric oxide (NO) control of the vascular smooth muscle of the ventral abdominal vein and vena cava of the toad, Bufo marinus, by using anatomical and physiological approaches. Nicotinamide adenine di-nucleotide phosphate-diaphorase histochemistry and immunohistochemistry using endothelial nitric oxide synthase (NOS) and neural NOS antibodies produced no evidence for endothelial NOS in the veins, but, neural NOS-immunoreactive perivascular nerves were present. Acetylcholine (10^–5 M) caused a vasodilation in both veins that was endothelium-independent, and which was blocked by the soluble guanylyl cyclase inhibitor, ODQ (10^–5 M). The NOS inhibitors, L-NNA (10^–4 M) and L-NAME (10^–4 M), did not significantly reduce the vasodilatory effect of acetylcholine in the veins; this suggested that the vasodilation was not due to NO. However, in the presence of phenoxybenzamine (10^–7–10^–8 M), L-NNA significantly reduced the vasodilatory effect of acetylcholine in the veins. This unusual response is due to phenoxybenzamine partially inactivating the muscarinic receptor pool in the veins. In addition, the neural NOS inhibitor, vinyl-L-NIO (10^–5 M), significantly reduced the acetylcholine-mediated vasodilation in the presence of phenoxybenzamine. The results show that in toad veins, nitrergic nerves rather than an endothelial NO system are involved in NO-mediated vasodilation.     

Sodium channels in cultured neuroblastoma cells grown in high glucose or l-Fucose

Patch clamp techniques were used to record whole cell and single channel Na⁺ currents from NB41A3 neuroblastoma cells grown in culture. Cells were grown for two weeks in control medium or medium supplemented with 30 mm d-glucose of 30 mm l-fucose.Cells exposed to glucose or l-fucose had smaller whole cell Na⁺ currents than cells grown in unsupplemented medium, consistent with earlier studies (Yorek, Stefani & Wachtel, 1994). Whole cell macroscopic currents showed no change in activation or inactivation kinetics. Single channel current properties and opening probability were also unchanged.The number of [³H]saxitoxin binding sites, and therefore the total number of Na⁺ channels, was not reduced in cells grown in glucose or l-fucose (Yorek et al., 1994). Therefore, we conclude that some of the channels must have been rendered nonfunctional by the conditioning media. The finding that single channel properties are not altered suggests that channels become nonfunctional in an all-or-none manner.This work was supported by Merit Review Awards to M.A. Yorek and R.E. Wachtel from the Department of Veterans Affairs and by National Institutes of Health grant DK45453 to M.A. Yorek.     

Fine structure of synapses in the dorsal nucleus of the lateral geniculate body of normal and blinded rats

Summary Degenerating boutons, observed from 2 to 60 days after eye enucleation, displayed decreased plasma membrane density, increased axoplasmic density, and enlarged mitochondria with deformed cristae when compared with boutons from normal animals. There was also a loss of synaptic plasma membrane specialization and the boutons abnormally indented contiguous dendrites. The number and appearance of synaptic vesicles in some degenerating boutons were notably altered. Phagocytosis of boutons in most instances appeared to be accomplished by astrocytes. When degeneration was first apparent in some boutons, the subsynaptic organelle in the adjacent dendritic cytoplasm was enlarged, somewhat less dense and was associated with small granular and circular profiles. Subsynaptic organelles in experimental animals were absent from contiguities between dendrites and other cell processes, except in a few instances when only small portions of boutons remained at their synaptic sites, suggesting that the organelles disappeared when boutons had been completely phagocytized.Degenerating myelinated axons, observed from 2 to 300 days after enucleation, exhibited the same triad of features as degenerating boutons. They appeared to be phagocytized in most instances by dense glial processes, presumably oligodendrocytic, which were normally situated between the axon and its myelin sheath and were related to the inner mesaxon.This investigation was supported by U.S.P.H.S. Training Grants Nos. 2 T1 GM 202 T1 CA 505506, and 2RO 1 AM 368806.The author expresses his appreciation to Dr. A. J. Ladman for acquainting him with the techniques used in the study and to Dr. R. J. Barrnett for valuable criticism of this report. Gratitude is also extended to Mr. E. Z. Rutkowski for making the drawing.     

The role of Rab6 GTPase in the maturation of phagosome against Staphylococcus aureus

Phagocytosis, an evolutionarily conserved process in animals, plays a central role in host defense against pathogens. As reported, Rab6 GTPase was involved in the regulation of hemocytic phagocytosis in invertebrates. However, the role of Rab6 GTPase in mammalian phagocytosis remains to be addressed. In this study, the results showed that Rab6 GTPase took great effects on phagocytosis of mouse leukemic monocyte macrophages (RAW 264.7 cells). It was revealed that Rab6 GTPase was required during the phagosome maturation by its interaction with bicaudal-D1 (BICD1) protein. Further data presented that the Rab6 GTPase-regulated phagocytosis could influence the proliferation of Staphylococcus aureus in macrophages. Therefore, our study demonstrated a novel insight into the mechanism of regulation of mammalian phagocytosis by Rab6 GTPase and a novel strategy for the control of Staphylococcus aureus.     

The subcellular fractionation of the electric organ of Torpedo

Summary The acetylcholine-rich electric organ of Torpedo has been submitted to subcellular fractionation in an attempt to isolate nerve endings and synaptic vesicles derived from cholinergic neurones. Fractions containing small vesicles and granules as their only morphologically identifiable components also contained appreciable amounts of bound acetylcholine; however, it was not possible to demonstrate a specific enrichment of any one fraction with respect to bound acetylcholine as has been possible in brain. The tissue proved difficult to homogenize and few detached nerve endings (synaptosomes) were formed. A low-speed fraction rich in Na, K- activated adenosine triphosphatase contained numerous membrane fragments with tubular appendages derived from the non-innervated surface of electroplaques. Homogenization in media isotonic with elasmobranch plasma (e.g. 0.5 M sucrose + 0.33 M urea) was essential to preserve the structure of osmotically sensitive organelles (e.g. mitochondria).We wish to express our gratitude to Dr. R. D. Keynes who arranged the supply of Torpedos and to Mr G. H. C. Dowe and Miss L. Swales for skilled technical assistance. The electron microscopic facilities were provided by a grant from the Wellcome Trust and the work was supported by a grant no. NB-03928-02 (to V.P.W.) from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. During the period of this work Dr. Sheridan held a Postdoctoral Fellowship of the U.S. Public Health Service and Dr. Israël was an Exchange Scholar of the Medical Research Council.We are also most grateful to Professor Sir Bryan Matthews, C.B.E., Sc. D., F.R.S., for providing aquarium facilities in the Physiological Laboratory of Cambridge University.     

The Inhibitory Effect of Staphylococcus epidermidis Slime on the Phagocytosis of Murine Peritoneal Macrophages Is Interferon-Independent

The extracellular slime produced by Staphylococcus epidermidis has been shown to interfere with several human neutrophil functions in vitro, such as chemotaxis, degranulation and phagocytosis. Slime production has been suggested as a useful marker for clinically significant infections with coagulase-negative Staphylococcus. Since the main role of macrophages in defense mechanisms is phagocytosis, the effect of slime on the phagocytic activity of macrophages was investigated. The phagocytic activity of murine peritoneal macrophages treated with slime in vitro decreased in a dose-dependent fashion. A similar decrease was also observed in macrophages isolated from mice that had previously received intraperitoneal injection of slime. To investigate whether interferon also plays a role in this process, mice were treated with interferon or an interferon inducer, polyinosinic-polycytidylic acid (poly I:C), together with slime before macrophage isolation. The slime-suppressed phagocytic activity of macrophages was partially relieved by both agents, and the recovery effect of poly I:C in slime-suppressed phagocytosis of macrophages in vivo might be attributed to the increased interferon level in peritoneal fluid and sera. However, when slime was given to poly I:C-pretreated mice, the phagocytic activity remained suppressed. Thus, it appears that slime is able to suppress the phagocytic activity of macrophages regardless of the state of macrophage activation by poly I:C. The results suggest that the inhibition of phagocytosis by S. epidermidis slime may be independent from the activation of interferon.     

Ultrastructural distribution of endogenous IgGs in the glomerular wall of control and diabetic rats

Summary Endogenous IgG molecules were revealed with high resolutionem over the glomerular wall in renal tissues sampled from short and longterm control and streptozotocin induced diabetic rats by applying the protein A-gold immunocytochemical approach. In tissues from control animals, IgG antigenic sites were revealed on the subendothelial side of the basement membrane, the epithelial side being only weakly labelled. In contrast, in longterm diabetic animals IgG antigenic sites were present throughout the entire thickness of the basement membrane, and in patches closely associated with the plasma membrane of the epithelial cells. Deposits of basement membrane-like material present in the mesangial area were also highly labelled for IgG. Numerous intensely labelled lysosome-like structures were present in the epithelial cells. Morphometrical evaluation of the distribution of the labelling over the basement membrane confirmed these observations. In control animals a peak of labelling was found at 30 nm from the endothelial cell region corresponding to the subendothelial side of the lamina densa. In longterm diabetic animals the labelling was more uniformly distributed throughout the entire thickness of the basement membrane. These data were correlated to biochemical determinations of proteinuria and IgG excretion in urine samples from the control and the diabetic animals. These results suggest that in normal conditions the lamina densa may represent the main barrier for the restriction of the passage of IgGs through the glomerular wall. Modifications at that level occur during diabetes leading to or participating in the loss of the selective permeability of the basement membrane.     

Studies by negative staining on the structure of collagen fibrils in normal and lathyritic rats

Summary Electron microscope studies on collagen from rat-tail tendon using the negative staining technique indicate the presence of fine filaments 15–30 Å in diameter within the fibres. The banding of the fibrils was only slightly affected by aminoacetylnitrile bisulphate, but an increased amount of fine fibrous material was present in preparations from experimental animals. It is suggested that this material represents a soluble form of collagen which is known to predominate after administration of the drug. Despite the fact that this would be a chemically abnormal collagen, its structure by electron microscopy corresponds remarkably well with the form suggested from X-ray diffraction and physico-chemical studies on normal soluble collagen.The author would like to thank Mr. J. Clements for technical assistance and Mr. R. Thomas (Dental School, Bristol) for discussion on the effects of aminoacetylnitriles in rats.     

Bidirectional movements of mitochondria along axons of an isolated nerve segment

Summary The sciatic nerve of the rat was constricted at two sites about 10 mm apart. Three and seventeen hours later, a part of the nerve including the intermediate segment between the two crushed regions was dissected out and studied electron microscopically. Mitochondria and other organelles accumulated at the ends of interrupted fibres both proximally and distally of either crushed region. In the nerve segment between the two constrictions mitochondria collected at the fibre ends, whereas their number became significantly reduced in axons of the middle part of the segment. It is inferred that this translocation of mitochondria is brought about by a bidirectional shift of particles towards the fibre ends at which they become deposited.Skillful technical assistance of Mrs. M. Sobotková and Mr. M. Doubek is gratefully acknowledged.     

Effects of cyclooxygenase inhibitors and prostaglandin E2 on macrophage activation in vitro

The effect of the cyclooxygenase inhibitors, indomethacin and diclofenac, and of PGE₂ on either resting or stimulated macrophages was investigated. Peritoneal macrophages were obtained from untreated mice and cultured for 10 days. Macrophage activation was induced by zymosan phagocytosis and was monitored by testing for plasminogen activator secretion and the cellular levels of lactate dehydrogenase, β-glucoronidase and alkaline phosphodiesterase I.It was found that cyclooxygenase inhibitors activate resting macrophages and enhance the degree of activation obtained after zymosan phagocytosis. Addition of exogenous PGE₂, on the other hand, had the opposite effect, it suppressed activation induced either by cyclooxygenase inhibitors, phagocytosis or a combination of both. Cyclooxygenase inhibitors and PGE₂ did not affect the hexose monophosphate shunt activity of resting macrophages and had only a minor effect on the respiratory on the respiratory macrophages and had only a minor effect on the respiratory burst occuring during zymosan phagocytosis. It appears, therefore, that the observed changes in the state of activation of the machrophages are not related to hexose monophosphate shunt activity.The described effects suggest that PGE₂ and possibly other cyclooxygenase products may function as inhibitory feed-back regulators of macrophage activation.     

Specialization of the macrophage plasma membrane at sites of interaction with opsonized erythrocytes

We incubated mouse peritoneal macrophages for 3-8 min at 37 degrees C with antibody-coated sheep erythrocytes and examined regions of close interaction between the two cell types by electron microscopy. At sites of focal macrophage-erythrocyte contact we observed a distinctive specialization of the macrophage plasma membrane consisting of a prominent subplasmalemmal band of electron-dense material, approximately 25-35 nm in thickness. In many instances, this band showed a periodic substructure similar to that seen in clathrin coats. Moreover, many slender erythrocyte processes penetrated into invaginations of the macrophage surface which were bristle-coated at their blind extremity. As previously shown for clathrin-coated pits, the segments of the macrophage plasma membrane beneath which the defense material was found were selectively resistant to the membrane- perturbing effect of the antibiotic, filipin. This structural specialization of the macrophage plasma membrane at sites of ligand- receptor interaction during immune phagocytosis of antibody-coated erythrocytes may represent the morphological counterpart of the zipper mechanism of phagocytosis previously demonstrated by functional studies.     

Macrophages containing langerhans cell granules in normal lymph nodes of the rabbit

Summary Light and electron microscopic studies on macrophages of normal rabbit lymph nodes showed two types, one with little phagocytic activity and many features similar to those of epidermal Langerhans cells. Among these are characteristic Langerhans cell granules. From these findings it is concluded that the Langerhans cells may be derived from lymph node macrophages.The helpful advice and criticism of Dr. Toshio Nagano, Department of Anatomy, are gratefully acknowledged. The discussion with Dr. Mitsumasa Itoh, Department of Dermatology, was also helpful.     

Modulation of Adherence and Chemotaxis of Macrophages by Norepinephrine. Influence of Ageing

We evaluated thein vitro effect of norepinephrine (NE), over the range of concentrations between 10^-12 M and 10^-3 M, on adherence (to plastic surfaces) and chemotaxis (in a Boyden chamber) of peritoneal macrophages from BALB/c mice of different ages: young (12 weeks), adult (22 weeks), mature (48 weeks) and old (72 weeks). Increased adherence was induced by 10^-12 M of NE in macrophages from young, adult, mature and old mice. Also, 10^-9 M stimulated adherence in old animals, 10^-5 M in mature mice, and 10^-3 M in both young and old mices. With respect to chemotaxis, the low concentration of NE (10^-12 M) was stimulatory only in young and adult animals, higher concentrations (10^-5 M and 10^-7 M) were inhibitory for macrophages from mature and old animals, and the highest concentration of NE (10^-3M) stimulated this capacity of macrophages only in young and mature animals. The conclusion is that while the mobility of macrophages to the focus of infection (i.e. chemotaxis) is stimulated by low concentrations of NE (10^-12, M) only in young-adult animals, this neurotransmitter induces a decline in this capacity in mature and old mice at high concentrations (10^-5 M - 10^-7 M). Also, macrophages from old animals have lost the capacity to respond to pharmacological (10^-3 M) concentrations of NE. The lower capacity of response to NE by macrophages from old animals possibly contributes to immunosenescence.     

l-Arginine reduces thioflavin T fluorescence but not fibrillation of bovine serum albumin

This work examines the effects of l-arginine (l-Arg) on the aggregation and amyloid fibrillation of bovine serum albumin (BSA). We demonstrate that l-Arg dose-dependently reduces thioflavin T (ThT) fluorescence of BSA within the l-Arg concentration range used (0–1.4 M). However, as revealed by electron microscopy, size exclusion chromatography, and dynamic light scattering results, l-Arg does not prevent amyloid-like fibril formation by BSA. We conclude that l-Arg competes against ThT for binding sites on BSA amyloid-like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.     

Tyrpanosoma gambiense: phagocytosis in vitro

When Trypanosoma gambiense was exposed in vitro to rat macrophages, the addition of homologous antiserum resulted in immediate adherence of the parasites to the surface of macrophages.The antigenic components of the parasite were fractionated by Sephadex G-200, DEAE-cellulose, and block electrophoresis. Each fraction was used for immunizing rats, and the antisera obtained were each treated for agglutinating antibodies and for their effect on in vitro phagocytosis by rat macrophages. Only the antiserum capable of agglutination could enhance phagocytosis. A strong possibility that the agglutination antigen is mostly responsible for phagocytosis, hence protection, is indicated.Scanning electron micrographs showed that adherence to the macrophage is mostly by the anterior part of the parasite.