Membrane glycoprotein changes in primary mammary tumors associated with autonomous growth

Primary and transplanted mammary tumors of the GR mouse were explanted in tissue culture and grown in the presence of radioactive fucose. Labelled membrane glycopeptides were isolated and compared by cochromatography with differentially labelled glycopeptides from normal mammary gland tissue. Differences with controls in the glycopeptide elution profiles were observed in autonomous, hormone-independent tumors but were absent in histologically similar tumors which required a continuous hormonal stimulus for growth. The results suggest that alterations in membrane glycopeptides are associated with the capacity of autonomous, hormone-independent growth of murine adenocarcinoma cells.     

MacMARCKS is not essential for phagocytosis in macrophages

MacMARCKS (also known as myristoylated alanine-rich protein kinase C substrate (MARCKS)-related protein) is a member of the MARCKS family of protein kinase C substrates. MacMARCKS contains within it a basic effector domain that contains the serine residues that are phosphorylated by protein kinase C, as well as a calcium/calmodulin and actin-binding site. Two previous reports demonstrated that a macrophage cell line expressing a mutant form of MacMARCKS that lacks the effector domain is defective in phagocytosis and cell adhesion (Zhu, Z., Bao, Z., and Li, J. (1995) J. Biol. Chem. 270, 17652-17655; Li, J., Zhu, Z., and Bao, Z. (1996) J. Biol. Chem. 271, 12985-12990). We report here that macrophages from MacMARCKS null mice phagocytose and spread normally. Thus, although MacMARCKS is recruited to phagosomes, it is not absolutely required for phagocytosis.     

Arthroplasty implant biomaterial particle associated macrophages differentiate into lacunar bone resorbing cells

OBJECTIVE: To study the pathogenesis of aseptic loosening: in particular, to determine whether macrophages responding to particles of biomaterials commonly used in arthroplasty surgery for arthritis are capable of differentiating into osteoclastic bone resorbing cells, and the cellular and hormonal conditions required for this to occur. METHODS: Biomaterial particles (polymethylmethacrylate, high density polyethylene, titanium, chromium-cobalt, stainless steel) were implanted subcutaneously into mice. Macrophages were isolated from the foreign body granulomas that resulted, cultured on bone slices and coverslips, and assessed for both cytochemical and functional evidence of osteoclast differentiation. RESULTS: Tartrate resistant acid phosphatase (TRAP) negative macrophages isolated from granulomas containing particles of all types of biomaterial composition were capable of differentiating into TRAP positive cells capable of extensive lacunar bone resorption (assessed by scanning electron microscopy). The presence of both UMR106 rat osteoblast-like cells and 1,25-dihydroxy vitamin D3 was necessary for this to occur. CONCLUSION: All implant materials produce wear particles that are the focus of a heavy foreign body macrophage response in the fibrous membrane between a loose implant component and the host bone undergoing resorption. These findings underline the importance of biomaterial wear particle generation and the macrophage response to different types of biomaterial wear particles in the pathogenesis of aseptic loosening.     

A requirement for ARF6 in Fcgamma receptor-mediated phagocytosis in macrophages

Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members of the ARF family of GTPases are essential for many aspects of membrane trafficking. To test a role for this family of proteins in Fcgamma receptor-mediated phagocytosis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 microM BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Golgi apparatus as indicated by altered immunofluorescent distribution of beta-COP and reduced efflux of BODIPY C5-ceramide, a phospholipid that normally accumulates in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis of IgG-coated erythrocytes. These results suggested that activation of BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particles. These results indicate a requirement for ARF6 in Fcgamma receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fcgamma receptor activation.     

Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages

Although Fc receptor-mediated phagocytosis is accompanied by a variety of transmembrane signaling events, not all signaling events are required for particle ingestion. For example, Fc receptor-mediated phagocytosis in mouse inflammatory macrophages (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106:657; Greenberg, S., J. El Khoury, F. Di Virgilio, and S. C. Silverstein. 1991. J. Cell Biol. 113:757) and neutrophils (Della Bianca, V., M. Grzeskowiak, and F. Rossi. 1990. J. Immunol. 144:1411) occurs in the absence of cytosolic calcium transients. We sought to identify transmembrane signaling events that are essential for phagocytosis. Here we show that tyrosine phosphorylation is an early event after Fc receptor ligation in mouse inflammatory macrophages, and that the formation of tyrosine phosphoproteins coincides temporally with the appearance of F-actin beneath phagocytic cups. The distribution of tyrosine phosphoproteins that accumulated beneath phagocytic cups was punctate and corresponded to areas of high ligand density on the surface of the antibody-coated red blood cells, which provided the phagocytic stimulus. A tyrosine kinase inhibitor, genistein, but not several inhibitors of protein kinase C, blocked the appearance of tyrosine phosphoproteins as assessed by immunofluorescence, the focal accumulation of F-actin beneath immunoglobulin G-opsonized particles, and the ingestion of these particles as well. We suggest that tyrosine phosphorylation is a critical signaling event that underlies Fc receptor-mediated phagocytosis in mouse macrophages, and is necessary for the engulfment per se.     

Essential role of phosphatidylserine externalization in apoptosing cell phagocytosis by macrophages

In many apoptotic cells, phosphatidylserine (PS), that is normally restricted to the inner membrane layer, is externalized and subsequently recognized by phagocytes. However, it has been unclear whether PS externalization is sufficient for phagocytosis induction. In a cultured cell line undergoing Fas-mediated apoptosis, PS externalization preceded other apoptotic events. When transbilayer movement of membrane phospholipids was analyzed, a decrease of the uptake of PS and phosphatidylethanolamine and an increase of phosphatidylcholine incorporation were observed upon apoptosis induction. Apoptotic cultured cells were phagocytosed by macrophages in a manner dependent on externalized PS before plasma membrane permeability increased. Moreover, a N-ethylmaleimide treatment caused PS externalization independent of apoptosis, and such cells underwent PS-mediated phagocytosis. These results suggested that PS is externalized as a result of membrane phospholipid redistribution and externalized PS by itself induces apoptosing cell phagocytosis.     

Respiratory changes associated with rapid eye movements in normo- and hypercapnia during sleep

Rapid eye movements during rapid-eye-movement (REM) sleep are associated with rapid, shallow breathing. We wanted to know whether this effect persisted during increased respiratory drive by CO2. In eight healthy subjects, we recorded electroencephalographic, electrooculographic, and electromyographic signals, ventilation, and end-tidal PCO2 during the night. Inspiratory PCO2 was changed to increase end-tidal PCO2 by 3 and 6 Torr. During normocapnia, rapid eye movements were associated with a decrease in total breath time by -0.71 +/- 0.19 (SE) s (P < 0.05) because of shortened expiratory time (-0.52 +/- 0.08 s, P < 0.001) and with a reduced tidal volume (-89 +/- 27 ml, P < 0.05) because of decreased rib cage contribution (-75 +/- 18 ml, P < 0.05). Abdominal (-11 +/- 16 ml, P = 0.52) and minute ventilation (-0.09 +/- 0.21 ml/min, P = 0.66) did not change. In hypercapnia, however, rapid eye movements were associated with a further shortening of total breath time. Abdominal breathing was also inhibited (-79 +/- 23 ml, P < 0.05), leading to a stronger inhibition of tidal volume and minute ventilation (-1.84 +/- 0.54 l/min, P < 0.05). We conclude that REM-associated respiratory changes are even more pronounced during hypercapnia because of additional inhibition of abdominal breathing. This may contribute to the reduction of the hypercapnic ventilatory response during REM sleep.     

Influence of sodium concentration on changes of membrane capacitance associated with the electrogenic ion transport by the Na,K-ATPase

Electrogenic ion transport by the Na,K-ATPase was investigated in a model system of protein-containing membrane fragments adsorbed to a lipid bilayer. Transient Na+ currents were induced by photorelease of ATP from inactive caged ATP. This process was accompanied by a capacitance change of the membrane system. Two methods were applied to measure capacitances in the frequency range 1 to 6000 Hz. The frequency dependent capacitance increment, delta C, was of sigmoidal shape and decreased at high frequencies. The midpoint frequency, f0, depended on the ionic strength of the buffer. At 150 mM NaCl f0 was about 200 Hz and decreased to 12 Hz at high ionic strength (1 M). At low frequencies (f < f0) the capacitance increment became frequency independent. It was, however, dependent on Na+ concentration and on the membrane potential which was generated by the charge transferred. A simple model is presented to analyze the experimental data quantitatively as a function of two parameters, the capacitance of the adsorbed membrane fragments, Cp, and the potential of maximum capacitance increment, psi 0. Below 5 mM Na+ a negative capacitance change was detected which may be assigned to electrogenic Na+ binding to cytoplasmic sites. It could be shown that the results obtained by experiments with the presented alternating current method contain the information which is determined by current-relaxation experiments with cell membranes.     

Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus

OBJECTIVE: To investigate whether the established impaired phagocyte function in systemic lupus erythematosus (SLE) patients also affects apoptotic cell clearance. Accumulation of apoptotic waste as a source for autoantigens that induce and maintain autoimmune responses is discussed. METHODS: Apoptosis was detected by morphology and propidium iodide staining. In vitro phagocytosis of autologous apoptotic cells in cultured peripheral blood mononuclear cells was evaluated microscopically. Cross-feeding experiments were performed to investigate phagocytosis of heterologous apoptotic cells by in vitro-differentiated macrophages. Furthermore, the effect of annexin V on the phagocytosis of apoptotic cells was investigated. RESULTS: Reduced clearance of apoptotic cells in SLE patients was observed. The defective clearance appeared to reflect phagocyte dysfunction and not an abnormal execution of apoptosis. A similar picture was seen when in vitro-differentiated macrophages from control populations were treated with annexin V. CONCLUSION: Noninflammatory engulfment phagocytosis of apoptotic cells is decreased in SLE patients. Persistently circulating apoptotic waste may encounter inflammatory removal pathways and serve as immunogen for the induction of autoreactive lymphocytes and as antigen for immune complex formation.     

In situ evidence of neoplastic cell phagocytosis by macrophages in papillary thyroid cancer

We evaluated the occurrence, tissue distribution, and prognostic value of tumor-associated macrophages in 121 papillary thyroid carcinomas using immunohistochemical staining with anti-CD68 antibody in archival paraffin-embedded sections. Lymphocytic infiltration and dendritic cell presence were also evaluated. Three groups were identified according to the presence and characteristics of macrophages: 1) tumors without evidence of infiltrating macrophages: (n = 35); 2) tumors with infiltrating macrophages but no evidence of neoplastic cells phagocytosis (n = 68); and 3) tumors with infiltrating macrophages and in situ evidence of active neoplastic cell phagocytosis (n = 18). Neoplastic cell phagocytosis by macrophages was positively correlated with both lymphocytic infiltration and dendritic cells (P = 0.0000), whereas it was negatively correlated with vascular invasion (P = 0.0032). Distant metastases developed in none of the 18 tumors with neoplastic cell phagocytosis, but occurred in 15 of 103 of the remaining tumors (P = 0.0647) and were significantly and negatively associated with lymphocytic infiltration or dendritic cells. The present study indicates, therefore, that immune reaction, involving neoplastic cell phagocytosis by macrophages and lymphocytic infiltration, plays a role in the development of distant metastases in patients with papillary thyroid cancer.     

Age-related changes in oxygen and nutrient uptake by hindquarters in newborn pigs during cold-induced shivering

Newborn pigs rely essentially on shivering thermogenesis in the cold. In order to understand the rapid postnatal enhancement of thermogenic capacities in piglets, the oxygen and nutrient uptake of hindquarters was measured in vivo in 1- (n = 6) and 5-day-old (n = 6) animals at thermal neutrality and during cold exposure. The hindquarters were considered to represent a skeletal muscle compartment. Indirect calorimetry and arterio-venous techniques were used. The cold challenge (23 C at 1 day old and 15 C at 5 days old for 90 min) induced a similar increase (+90 %) in regulatory heat production at both ages. Hindquarters blood flow was higher at 5 days than 1 day old at thermal neutrality (26 +/- 3 vs. 17 +/- 1 ml min-1 (100 g hindquarters)-1) and its increase in the cold was much more marked (+65 % at 5 days old vs. +25 % at 1 day old). Oxygen extraction by the hindquarters rose from 30-35 % at thermal neutrality to 65-70 % in the cold at both ages. The calculated contribution of skeletal muscle to total oxygen consumption averaged 34-40 % at thermal neutrality and 50-64 % in the cold and skeletal muscle was the major contributor to regulatory thermogenesis. Based on hindquarters glucose uptake and lactate release, carbohydrate appeared to be an important fuel for shivering. However, net uptake of fatty acids increased progressively during cold exposure at 5 days old. The enhancement in muscular blood supply and fatty acid utilization during shivering is probably related to the postnatal improvement in the thermoregulatory response of the piglet.     

Morphological changes associated with long-term potentiation

Long-term potentiation (LTP) is a long-lasting form of synaptic plasticity induced by brief repetitive afferent stimulation that is thought to be associated with learning and memory. It is most commonly studied in the hippocampus where it may last for several weeks, and involves the synthesis of new proteins that might play a structural role. In this review we summarize the evidence in favor of modifications of neuronal architecture during LTP. We focus our attention on changes occurring at the level of single synapses, including components of postsynaptic dendrites (dendritic spines, the postsynaptic density, and synaptic curvature), of presynaptic terminals, and the formation of new synapses. We conclude that although many morphological changes at various sites have been observed during LTP, there is no definitive proof in favor of structural changes associated with LTP. However, morphological modifications remain a valid candidate for mechanisms of learning and memory.     

Membrane electrical properties associated with insulin receptor downregulation in human erythrocytes

We reviewed 29 consecutive patients after cemented femoral revision of cemented hip arthroplasties for osteolysis. After an average follow-up of 8.5 years, osteolysis had recurred in only two cases (6.9%) and 25 femoral components (86%) remained well fixed.     

Perioperative changes in complement associated with cardiopulmonary bypass

The total haemolytic complement (CH50), the complement components C3 and C4, the complement breakdown product C3d, alternative pathway activation and transferrin, were measured before, during and after cardiopulmonary bypass. As expected, CH50 decreased after heparinization, remained low during bypass and decreased further up to 8 h after bypass. C3 and C4 decreased significantly during bypass, continued to decrease for a further 8 h after bypass (by 35% and 40% respectively) and thereafter increased gradually up to 48 h. Although the depletions observed were suggestive of complement activation, there were no demonstrable increases in C3d, and in all patients the concentration of C3d remained within the normal range. Hence it was concluded that complement depletions of this magnitude were unlikely to result from complement activation. Non-specific changes in protein concentrations during bypass, as a result of dilution, redistribution or other unidentified factors, are more probable causes of the observed reductions. The acute phase response to surgery may be a factor in the subsequent increase in C3 and C4 which is seen 24 h after bypass. As transferrin concentrations in the plasma are known to decrease during this response the observed decrease in transferrin concentration would support this view.