Macrophages In Suspension Research Articles

Beneficial effects of activated macrophages on sulfur mustard-induced cutaneous burns, an in vivo experience

Objective: Macrophages are known to have key functions in almost every stage of wound healing and there is evidence for their beneficial effects in treating decubital ulcers and deep sternal wound infections in human. This study aimed to investigate the efficacy of a treatment with activated macrophages on ameliorating acute and long-term sulfur mustard (SM) induced skin injuries in the hairless guinea pig (HGP) model. Methods: HGP were exposed to SM vapor and treated with either a single or multiple intra-dermal injections of human activated macrophages in suspension (hAMS) into the wound bed. Clinical and histological evaluations were conducted up to 4 weeks post-exposure. Results: A single treatment with hAMS early after exposure (15 min and 6 h) resulted in a reduction in the number of damaged cells and vesications in the epidermis at 24 h. A substantial increase in cellular infiltration, mostly polymorphonuclears, was taking place in the hAMS-treated animals starting as early as 1 h after exposure. This flow of inflammatory cells continued, in the treated group, for at least 4 weeks, long after the injected macrophages were not detected. Repeated injections of hAMS (15 min, 48 h and 7 d post-exposure) decreased significantly the area of the wounds and improved the integrity of the barrier function as expressed by measuring trans-epidermal water loss up to 10 d. Conclusions: Our results indicate that the role of macrophages in wound healing is complex; their efficacy may depend on the timing of administration. Further investigation is required to determine whether they are required during the early phase of wound development and/or during the late phase of scar formation and remodeling.

Activation of adenosine A3 receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells

Adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been tested from the point of view of potentiating the effects of hematopoietic growth factors interleukin-3 (IL-3), stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) on the growth of hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in suspension of normal mouse bone marrow cells in vitro. IB-MECA alone induced no GM-CFC growth. Significant elevation of numbers of GM-CFC evoked by the combinations of IB-MECA with IL-3, SCF, or GM-CSF as compared with these growth factors alone has been noted. Combination of IB-MECA with G-CSF did not induce significantly higher numbers of GM-CFC in comparison with G-CSF alone. Joint action of three drugs, namely of IB-MECA + IL-3 + GM-CSF, produced significantly higher numbers of GM-CFC in comparison with the combinations of IB-MECA + IL-3, IB-MECA + GM-CSF, or IL-3 + GM-CSF. These results give evidence of a significant role of selective activation of adenosine A(3) receptors in stimulation of the growth of granulocyte/ macrophage hematopoietic progenitor cells.

Adhesion of human U937 macrophages to phosphorylcholine‐coated surfaces

A new type of amphiphilic phosphorylcholine (PC) polymer was used in this work to develop a cell culture surface that allows the attachment of U937 macrophages. The PC polymer was a random copolymer of N-isopropylacrylamide (45%), N-(phosphorylcholine)-N'-(ethylenedioxy-bis(ethyl)) acrylamide (41%), and the hydrophobic monomer N-(n-octadecyl) acrylamide (14%). Polypropylene (PP) films (1 cm2) were coated with the polymer solution by immersion. U937 macrophage suspensions were applied on PC polymer-coated surfaces and incubated for up to 72 h at 37 degrees C. While U937 cells did not adhere to PP, ammonia, nitrogen, or oxygen plasma-treated surfaces, they attached rapidly on PC-coated surfaces (< 1 h), proliferated, and stayed attached to the modified surface for at least 72 h, suggesting that unique features of the PC polymer, and the U937 macrophages, are responsible for the attachment of these cells. We compared the effect of Co2+ and Cr3+ ions on the expression of bone-resorbing cytokines (TNF-alpha, IL-6, IL-1beta) in U937 macrophages cultured on PC-coated surfaces to the response of U937 macrophages in suspension. Cytokine gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Addition of Co2+ and Cr3+ ions led to a significant increased expression of TNF-alpha in both cultured and suspension cells. On the other hand, Co2+ and Cr3+ ions had a weak stimulatory effect or no effect on IL-1beta and IL-6, respectively, in both cultured and suspension cells. In conclusion, the use of PC polymer-modified surfaces might offer promising new opportunities for the culture of human U937 cells and may also point to the mechanism by which macrophages interact with lipid bilayers of biological membranes.

Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages.

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.

Ratio imaging of enzyme activity using dual wavelength optical reporters.

The design of near-infrared fluorescent (NIRF) probes that are activated by specific proteases has, for the first time, allowed enzyme activity to be imaged in vivo. In the current study, we report on a method of imaging enzyme activity using two fluorescent probes that, together, provide improved quantitation of enzymatic activity. The method employs two chemically similar probes that differ in their degradability by cathepsin B. One probe consists of the NIRF dye Cy5.5 attached to a particulate carrier, a crosslinked iron oxide nanoparticle (CLIO), through cathepsin B cleavable L-arginyl peptides. A second probe consists of Cy3.5 attached to a CLIO through proteolytically resistant D-arginyl peptides. Using mixtures of the two probes, we have shown that the ratio of Cy5.5 to Cy3.5 fluorescence can be used to determine levels of cathepsin B in the environment of nanoparticles with macrophages in suspension. After intravenous injection, tissue fluorescence from the nondegradable Cy3.5-D-arginyl probe reflected nanoparticle accumulation, while fluorescence of the Cy5.5-L-arginyl probe was dependent on both accumulation and activation by cathepsin B. Dual wavelength ratio imaging can be used for the quantitative imaging of a variety of enzymes in clinically important settings, while the magnetic properties of the probes allow their detection by MR imaging.

Ratio Imaging of Enzyme Activity Using Dual Wavelength Optical Reporters

The design of near-infrared fluorescent (NIRF) probes that are activated by specific proteases has, for the first time, allowed enzyme activity to be imaged in vivo. In the current study, we report on a method of imaging enzyme activity using two fluorescent probes that, together, provide improved quantitation of enzymatic activity. The method employs two chemically similar probes that differ in their degradability by cathepsin B. One probe consists of the NIRF dye Cy5.5 attached to a particulate carrier, a crosslinked iron oxide nanoparticle (CLIO), through cathepsin B cleavable L-arginyl peptides. A second probe consists of Cy3.5 attached to a CLIO through proteolytically resistant D-arginyl peptides. Using mixtures of the two probes, we have shown that the ratio of Cy5.5 to Cy3.5 fluorescence can be used to determine levels of cathepsin B in the environment of nanoparticles with macrophages in suspension. After intravenous injection, tissue fluorescence from the nondegradable Cy3.5-D-arginyl probe reflected nanoparticle accumulation, while fluorescence of the Cy5.5-L-arginyl probe was dependent on both accumulation and activation by cathepsin B. Dual wavelength ratio imaging can be used for the quantitative imaging of a variety of enzymes in clinically important settings, while the magnetic properties of the probes allow their detection by MR imaging.

Metabolism of phosphatidylglycerol by alveolar macrophages in vitro.

In whole animal studies, it has been shown that turnover of surfactant dipalmitoylphosphatidylglycerol (DPPG) is faster than that of dipalmitoylphosphatidylcholine (DPPC). The goal of this investigation was to characterize the metabolism of DPPG by alveolar macrophages and to determine whether they contribute to the faster alveolar clearance of DPPG. Isolated rat alveolar macrophages were incubated with liposomes colabeled with [(3)H]DPPG and [(14)C]DPPC. Macrophages internalized both lipids in a time- and temperature-dependent manner. The uptake of both lipids was increased by surfactant protein (SP) A and by adherence of the macrophages to plastic slides. The isotope ratio of DPPC to DPPG internalized by macrophages in suspension in the absence of SP-A was significantly lower than the isotope ratio in liposomes, suggesting that macrophages preferentially internalize DPPG when SP-A is absent. Phospholipase activity in macrophage homogenate was higher toward sn-2-labeled DPPG than toward sn-2-labeled DPPC. These studies show that alveolar macrophages play an important role in catabolizing surfactant lipids and may be partially responsible for the relatively faster clearance of DPPG from the lung.

The porin OmpC ofSalmonella typhimuriummediates adherence to macrophages

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequence of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.

42] Enzyme-linked immunosorbent-based adhesion assays

Among the various adhesion assays that require some type of bacterial labeling to estimate the extent of bacterial adhesion to animal cells, the enzyme-linked immunosorbent assay (ELISA)-based system has been introduced as a convenient and sensitive means to enumerate adherent microorganisms. It is especially useful in systems employing animal cells immobilized on microtiter plates, such as tissue-culture cells and phagocytic cells, which tend to adhere naturally to plastic surfaces. Even cells in suspension can be immobilized on plastic surfaces either before or after performing the adhesion assay without affecting their ability to bind bacteria and at the same time retaining the same receptor specificity as that of the nonimmobilized cells. The chapter exemplifies a number of studies using ELISA-based systems to estimate adhesion of microorganisms to various substrata and describes an ELISA-based procedure for the enumeration of adhesion of type 1 fimbriated Klebsiella pneumoniae to mouse peritoneal macrophages in suspension.

In Vitro Dissolution of Uranium Oxide by Baboon Alveolar Macrophages

In vitro cellular dissolution tests for insoluble forms of uranium oxide are technically difficult with conventional methodology using adherent alveolar macrophages. The limited number of cells per flask and the slow dissolution rate in a large volume of nutritive medium are obvious restricting factors. Macrophages in suspension cannot be substituted because they represent different and poorly reproducible functional subtypes with regard to activation and enzyme secretion. Preliminary results on the dissolution of uranium oxide using immobilized alveolar macrophages are promising because large numbers of highly functional macrophages can be cultured in a limited volume. Cells were obtained by bronchoalveolar lavages performed on baboons (Papio papio) and then immobilized after the phagocytosis of uranium octoxide (U3O8) particles in alginate beads linked with Ca2+. The dissolution rate expressed as percentage of initial uranium content in cells was 0.039 +/- 0.016%/day for particles with a count median geometric diameter of 3.84 microns(sigma g = 1.84). A 2-fold increase in the dissolution rate was observed when the same number of particles was immobilized without macrophages. These results, obtained in vitro, suggest that the U3O8 preparation investigated should be assigned to inhalation class Y as recommended by the International Commission on Radiological Protection. Future experiments are intended to clarify this preliminary work and to examine the dissolution characteristics of other particles such as uranium dioxide. It is recommended that the dissolution rate should be measured over an interval of 3 weeks, which is compatible with the survival time of immobilized cells in culture and may reveal transformation states occurring with aging of the particles.

In vitro dissolution of uranium oxide by baboon alveolar macrophages.

In vitro cellular dissolution tests for insoluble forms of uranium oxide are technically difficult with conventional methodology using adherent alveolar macrophages. The limited number of cells per flask and the slow dissolution rate in a large volume of nutritive medium are obvious restricting factors. Macrophages in suspension cannot be substituted because they represent different and poorly reproducible functional subtypes with regard to activation and enzyme secretion. Preliminary results on the dissolution of uranium oxide using immobilized alveolar macrophages are promising because large numbers of highly functional macrophages can be cultured in a limited volume. Cells were obtained by bronchoalveolar lavages performed on baboons (Papio papio) and then immobilized after the phagocytosis of uranium octoxide (U3O8) particles in alginate beads linked with Ca2+. The dissolution rate expressed as percentage of initial uranium content in cells was 0.039 +/- 0.016%/day for particles with a count median geometric diameter of 3.84 microns(sigma g = 1.84). A 2-fold increase in the dissolution rate was observed when the same number of particles was immobilized without macrophages. These results, obtained in vitro, suggest that the U3O8 preparation investigated should be assigned to inhalation class Y as recommended by the International Commission on Radiological Protection. Future experiments are intended to clarify this preliminary work and to examine the dissolution characteristics of other particles such as uranium dioxide. It is recommended that the dissolution rate should be measured over an interval of 3 weeks, which is compatible with the survival time of immobilized cells in culture and may reveal transformation states occurring with aging of the particles.

Radioimmunoassay for platelet‐activating factor

A radioimmunoassay (RIA) for measurement of platelet-activating factor (PAF) was developed. At a final antiserum dilution of 1:640, the lowest detection limit of PAF was 0.1 pmol (50 pg). The standard curve obtained was suitable for measurement of PAF in amounts ranging from 0.1 pmol to 30 pmol. The antiserum showed high specificity. Cross-reaction for lysoPAF, lysophosphatidylcholine and long-chain phosphatidylcholines was very low (less than 0.025%). 1-Palmitoyl-2-acetyl-sn-glycero-3-phosphocholine cross-reacted slightly (6.25%). PAF exogenously added to macrophage suspensions was quantitatively determined by RIA after solvent extraction and high-performance liquid chromatographic separation. RIA was also used to estimate PAF formation after stimulation of rabbit alveolar macrophages in suspension with calcium ionophore A23187.

Neutral‐Red Uptake and Expression of Monocytic Antigens in Amniotic‐Fluid Mononuclear Phagocytes: Evaluation of a Novel Approach for Prenatal Diagnosis of Neural‐Tube Defects

In cases of fetal neural-tube defects macrophages are present in the amniotic fluid. We found that these viable phagocytic cells take up neutral-red and are easily identified as "red cells" by microscopic examination. This method is suitable for the rapid identification and counting of amniotic-fluid macrophages in suspension. We have studied 298 amniotic fluid samples. In the 226 normal cases studied, 0 to 1,200 macrophages per milliliter amniotic fluid have been found. In contrast, we found 1,250 to 99,000 macrophages per milliliter amniotic fluid in our 70 open neural tube defect (ONTD) cases. Statistical evaluation was performed to estimate the normal and pathologic ranges. Specificity and sensitivity of the neutral-red test and predictive value of positive and negative results have been calculated and presented in comparison with alpha-fetoprotein (AFP) determinations and ultrasonic methods. In 5 cases of anencephaly and 7 normal cases amniotic fluid cells were studied by immunocytochemistry: mononuclear cells present in the abnormal cases showed intense immunoreactivity for the Mo1 and Mo2 surface antigens of the phagocytic cell lineage.

Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels.

Cytosolic free Ca2+ ([Ca2+]i) homeostasis was investigated in mouse peritoneal macrophages and in the macrophage-like cell line J774. [Ca2+]i measurements were performed in both cells in suspension and cells in monolayers loaded with either quin2 or fura-2. Resting [Ca2+]i was 110-140 and 85-120 nM for cell suspensions and monolayers, respectively. There were no significant differences in [Ca2+]i between the two macrophage populations whether quin2 or fura-2 were used as Ca2+ indicators. Addition of heat-aggregated IgG, IgG-coated erythrocyte ghosts, or a rat monoclonal antibody (2.4G2) directed against mouse Fc receptor II induced a rise in [Ca2+]i. This [Ca2+]i increase was consistently observed in J774 and peritoneal macrophage suspensions and in J774 macrophage monolayers; in contrast it was observed inconsistently in peritoneal macrophages in monolayer cultures. The increase in [Ca2+]i induced by ligation of Fc receptors was inhibited totally in macrophages in suspension and by 80% in macrophages in monolayers by a short preincubation of macrophages with PMA; however, phagocytosis itself was unaffected. The effect of reducing cytosolic Ca2+ to very low concentrations on Fc receptor-mediated phagocytosis was also investigated. By incubating macrophages with high concentrations of quin2/AM in the absence of extracellular Ca2+, or by loading EGTA into the cytoplasm, the [Ca2+]i was buffered and clamped to 1-10 nM. Despite this, the phagocytosis of IgG-coated erythrocytes proceeded normally. These observations confirm the report of Young et al. (Young, J. D., S. S. Ko, and Z. A. Cohn. 1984. Proc. Natl. Acad. Sci. USA. 81:5430-5434) that ligation of Fc receptors causes Ca2+ mobilization in macrophages. However, these results confirm and extend the findings of McNeil et al. (McNeil, P. L., J. A. Swanson, S. D. Wright, S. C. Silverstein, and D. L. Taylor. 1986. J. Cell Biol. 102:1586-1592) that a rise in [Ca2+]i is not required for Fc receptor-mediated phagocytosis; and they provide direct evidence that Fc receptor-mediated phagocytosis occurs normally even at exceedingly low [Ca2+]i.

In vivo and in vitro expression of macrophage membrane interleukin 1 in response to soluble and particulate stimuli.

We examined the expression of a membrane form of interleukin 1 (IL 1) by macrophages. Murine peritoneal macrophages fixed immediately after harvesting, in suspension, did not show membrane IL 1. Membrane IL 1 was expressed after a 3-hr culture on plastic dishes. These findings allowed us to examine some conditions in vivo that may trigger the expression of this protein; that is, by fixing the macrophages in suspension we could determine if a given stimulus had an effect. We found that membrane IL 1 was expressed briefly after administration of live or dead Listeria monocytogenes or endotoxin. Serum proteins were ineffective. At the time of maximal activation of macrophages by live Listeria, membrane IL 1 was not expressed. Analysis of in vitro conditions indicated that expression of membrane IL 1 and Ia molecules could be dissociated. In culture, recombinant interferon-gamma induced Ia but no membrane IL 1. Uptake of dead Listeria organisms had no effect on Ia but triggered membrane IL 1. The stimulation of membrane IL 1 was not caused by phagocytosis per se: latex particles were ineffective. Opsonized red cells stimulated membrane IL 1 on macrophages that were activated in vivo by inflammatory stimuli.

The macrophage adherence phenomenon: Its relationship to prostaglandin E 2 and superoxide anion production and changes in transmembrane potential

Mononuclear phagocytes are undoubtedly the sine qua non of chronic inflammatory reactions. This is demonstrated by their unique ability to function as phagocytic, secretory, or effector cells during the course of an immune event. Although macrophages can perform a variety of immune tasks, their ability to function appropriately is dependent upon the mode of elicitation, the stimulus under investigation, the source of the macrophages (peritoneal, alveolar, etc.), and whether the macrophages are monolayers or in suspension. We have examined the relationship between adherent and non-adherent elicited peritoneal macrophages in terms of prostaglandin E 2 (PGE 2) and superoxide anion (O 2) production; in addition, we hae studied these elicited macrophages in suspension for their ability to undergo transmembrane potential changes in response to several stimuli. Non-adherent, elicited peritoneal macrophages demonstrated an increase in basal PGE 2 production, and were refractory to particulate stimulus. After monolayer formation, basal PGE 2 levels dropped and the cell could respond to both soluble and particulate stimuli. Only adherent macrophages could respond to a specific challenge and synthesize O 2. Both O 2 production and depolarization of the transmembrane potential were suppressed in cell in suspension. Furthermore, both exogenous PGE 2 and supernatant from macrophages in suspension could modulate O 2 production by PMA challenged macrophages monolayers. These studies indicate that PGE 2 may modulate macrophage function and dictate activity as macrophages go from the non-adherent to adherent state.

Rapid method for identification of macrophages in suspension by acid alpha-naphthyl acetate esterase activity.

A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.

Stimulated murine peritoneal macrophages in suspension and cell culture: enzyme determinations by biochemical and histochemical means.

Peritoneal exudate cells of mice were studied up to 4 days after i.p. injection of thioglycollate broth medium by means of conventional enzyme determinations and quantitative histochemical measurements of individual cells. Cells from the peritoneal cavity were either investigated immediately after harvesting or after culturing periods of up to six days for enzymic activities of aminopeptidase, esterase, lactate dehydrogenase and beta-galactosidase. A fairly good correlation exists between biochemical determinations of aminopeptidase and esterase activity and the mean of histochemical data. Following a sharp increase in the number of cells after stimulation, aminopeptidase, esterase and lactate dehydrogenase activities per cell were found to be increased. Moreover, the cells taken three or five days after simulation synthesized large amounts of aminopeptidase and esterase as shown by culturing experiments. This capacity of the cells was subsequently lost in cells harvested seven days after stimulation but beta-galactosidase increased and lactae dehydrogenase was more readily released into culture supernatants. The increase in aminopeptidase and esterase was dependent on protein synthesis since it was abolished by cycloheximide. Thioglycollate broth medium provokes immigration of cells into the peritoneal cavity where cells apparently differentiate by increasing their aminopeptidase and esterase concentrations, and by raising their intracellular catabolism rates, which leads eventually to the decay of the cells. The different enzymic phenotypes and the large heterogeneity at any time point after stimulation presumably also reflect different functional properties during the inflammatory process.

Different macrophage populations distinguished by means of fluorescent polysaccharides. Recognition and properties of marginal-zone macrophages.

Fluorescent conjugates of hydroxyethyl (OEt) starch of Ficoll are selectively ingested and retained in vivo by spleen marginal-zone (MZ) and lymph node marginal-sinus macrophages of mice, whereas similar conjugates of type 3 pneumococcal capsular polysaccharide (SIII) are generally retained by macrophages (Kupffer cells, histiocytes, macrophages of spleen, lymph nodes, bone marrow and peritoneal cavity). MZ and other macrophages are readily identifiable by fluorescence after injection in vivo of OEt starch and SIII labeled with tetraethylrhodamine isothiocyanate and fluorescein isothiocyanate. Collagenase digestion was required for recovery of intact MZ macrophages from spleen in single cell suspensions and for maximum yields of other macrophages. MZ macrophages are larger and morphologically distinct from other macrophages, but resemble them in respect of EA, EAC receptors and acid phosphates and nonspecific esterase content and are equally radio-resistant. The appear normal in CBA/N nude and in beige mice. Freshly isolated MZ macrophages in suspension have adherent lymphocytes, dispersible by EDTA treatment, with B but not T cell markers. It is suggested that selective adherence to MZ macrophages is a factor in determining B cell traffic. MZ macrophages did not have demonstrable surface I-A or I-EC antigens. Only 4--8% of other spleen macrophages freshly isolated by collagenase treatment expressed I-A in the same preparation, whereas 35% of other cells (lymphocytes and blasts) reacted with monoclonal anti-I-A and anti-I-EC. After adherence to glass or plastic, 40% or more red-pulp, but not MZ macrophages, became I-A-positive. When taken from mice recently restimulated with sheep erythrocytes, half the red-pulp macrophages expressed I-A even before adherence. The relation of MZ to other macrophages is not known. However, their properties are consistent with the demonstrated ability of thymus-independent antigens selectively taken up by these cells to elicit long-lasting IgM antibody responses by direct interaction with B cells. The unexpected observation that only a small proportion of spleen macrophages freshly isolated from unstimulated mice had detectable surface I-A, but that this proportion was much increased after attachment to plastic, is discussed in relation to the possibility that macrophages do not express surface Ia antigens unless they have been stimulated.

Ultrasonic effects on alveolar macrophages in suspension.

Rabbit alveolar macrophages in suspension were exposed to 5 or 10 min of continuous 2-MHz ultrasound with 5, 10, and 15 W/cm2 spatial average intensities. Viability as determined by dye exclusion decreased with increasing intensity. Pressure experiments indicated that this was a result of acoustic cavitation. Ultrasound induced clumping of cells and often reduced membrane ruffling. Some cells were disintegrated. Cells that appeared to be otherwise intact had swollen mitochondria with ruptured cristae.