Fluorescent Zymosan Research Articles

Characterising phagocytes and measuring phagocytosis from live Galleria mellonella larvae

ABSTRACT Over the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo mammalian replacement model organism for the study of human pathogens. Experimental readouts of response to infection are most often limited to observing the melanization cascade and quantifying larval death and, whilst transcriptomic and proteomic approaches, and methods to determine microbial load are also used, a more comprehensive toolkit of profiling infection over time could transform the applicability of this model. As an invertebrate, Galleria harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells – termed haemocytes. Although information on subtypes of haemocytes exists, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay Galleria haemocytes, but protocols include both centrifugation and fixation – physical methods which have the potential to affect haemocyte morphology prior to analysis. Here, we present a method for live haemocyte analysis by flow cytometry, revealing that Galleria haemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the Galleria haemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall β-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.

Direct modulation of microglial function by electrical field.

Non-invasive electric stimulation (ES) employing a low-intensity electric current presents a potential therapeutic modality that can be applied for treating retinal and brain neurodegenerative disorders. As neurons are known to respond directly to ES, the effects of ES on glia cells are poorly studied. A key question is if ES directly mediates microglial function or modulates their activity merely via neuron-glial signaling. Here, we demonstrated the direct effects of ES on microglia in the BV-2 cells—an immortalized murine microglial cell line. The low current ES in a biphasic ramp waveform, but not that of rectangular or sine waveforms, significantly suppressed the motility and migration of BV-2 microglia in culture without causing cytotoxicity. This was associated with diminished cytoskeleton reorganization and microvilli formation in BV-2 cultures, as demonstrated by immunostaining of cytoskeletal proteins, F-actin and β-tubulin, and scanning electron microscopy. Moreover, ES of a ramp waveform reduced microglial phagocytosis of fluorescent zymosan particles and suppressed lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression in BV-2 cells as shown by Proteome Profiler Mouse Cytokine Array. The results of quantitative PCR and immunostaining for cyclooxygenase-2, Interleukin 6, and Tumor Necrosis Factor-α corroborated the direct suppression of LPS-induced microglial responses by a ramp ES. Transcriptome profiling further demonstrated that ramp ES effectively suppressed nearly half of the LPS-induced genes, primarily relating to cellular motility, energy metabolism, and calcium signaling. Our results reveal a direct modulatory effect of ES on previously thought electrically “non-responsive” microglia and suggest a new avenue of employing ES for anti-inflammatory therapy.

Adipose-derived stem cells decolonize skin Staphylococcus aureus by enhancing phagocytic activity of peripheral blood mononuclear cells in the atopic rats.

Staphylococcus aureus (S. aureus) is known to induce apoptosis of host immune cells and impair phagocytic clearance, thereby being pivotal in the pathogenesis of atopic dermatitis (AD). Adipose-derived stem cells (ASCs) exert therapeutic effects against inflammatory and immune diseases. In the present study, we investigated whether systemic administration of ASCs restores the phagocytic activity of peripheral blood mononuclear cells (PBMCs) and decolonizes cutaneous S. aureus under AD conditions. AD was induced by injecting capsaicin into neonatal rat pups. ASCs were extracted from the subcutaneous adipose tissues of naïve rats and administered to AD rats once a week for a month. Systemic administration of ASCs ameliorated AD-like symptoms, such as dermatitis scores, serum IgE, IFN-γ+/IL-4+ cell ratio, and skin colonization by S. aureus in AD rats. Increased FasL mRNA and annexin V+/7-AAD+ cells in the PBMCs obtained from AD rats were drastically reversed when co-cultured with ASCs. In contrast, both PBMCs and CD163+ cells bearing fluorescent zymosan particles significantly increased in AD rats treated with ASCs. Additionally, the administration of ASCs led to an increase in the mRNA levels of antimicrobial peptides, such as cathelicidin and β-defensin, in the skin of AD rats. Our results demonstrate that systemic administration of ASCs led to decolonization of S. aureus by attenuating apoptosis of immune cells in addition to restoring phagocytic activity. This contributes to the improvement of skin conditions in AD rats. Therefore, administration of ASCs may be helpful in the treatment of patients with intractable AD.

Essential Oil from Eucalyptus globulus (Labill.) Activates Complement Receptor-Mediated Phagocytosis and Stimulates Podosome Formation in Human Monocyte-Derived Macrophages.

Eucalyptus essential oil and its major constituent eucalyptol are extensively employed in the cosmetic, food, and pharmaceutical industries and their clinical use has recently expanded worldwide as an adjuvant in the treatment of infective and inflammatory diseases. We previously demonstrated that essential oil from Eucalyptus globulus (Labill.) (EO) stimulates in vitro the phagocytic activity of human monocyte-derived macrophages and counteracts the myelotoxicity induced by the chemotherapeutic 5-fluorouracil in immunocompetent rats. Here we characterize some mechanistic aspects underlying the immunostimulatory ability exerted by EO on macrophages. The internalization of fluorescent beads, fluorescent zymosan BioParticles, or apoptotic cancer cells was evaluated by confocal microscopy. Pro-inflammatory cytokine and chemokine release was determined by flow cytometry using the BD cytometric bead array. Receptor involvement in EO-stimulated phagocytosis was assessed using complement- or IgG-opsonized zymosan particles. The localization and expression of podosome components was analyzed by confocal microscopy and western blot. The main results demonstrated that: EO-induced activation of a macrophage is ascribable to its major component eucalyptol, as recently demonstrated for other cells of innate immunity; EO implements pathogen internalization and clearance by stimulating the complement receptor-mediated phagocytosis; EO stimulates podosome formation and increases the expression of podosome components. These results confirm that EO extract is a potent activator of innate cell-mediated immunity and thereby increase the scientific evidence supporting an additional property of this plant extract besides the known antiseptic and anti-inflammatory properties.

Neuroprotective and Anti-Neuroinflammatory Effects of a Poisonous Plant Croton Tiglium Linn. Extract.

Neuroinflammation is involved in various neurological diseases. Activated microglia secrete many pro-inflammatory factors and induce neuronal cell death. Thus, the inhibition of excessive proinflammatory activity of microglia leads to a therapeutic effect that alleviates the progression of neuronal degeneration. In this study, we investigated the effect of Croton tiglium (C. tiglium) Linn. extract (CTE) on the production of pro- and anti-inflammatory mediators in microglia and astrocytes via RT-PCR, Western blot, and nitric oxide assay. Neurotoxicity was measured by cell viability assay and GFP image analysis. Phagocytosis of microglia was measured using fluorescent zymosan particles. CTE significantly inhibited the production of neurotoxic inflammatory factors, including nitric oxide and tumor necrosis factor-α. In addition, CTE increased the production of the neurotrophic factor, brain-derived neurotrophic factor, and the M2 phenotype of microglia. The culture medium retained after CTE treatment increased the survival of neurons, thereby indicating the neuroprotective effect of CTE. Our findings indicated that CTE inhibited pro-inflammatory response and increased the neuroprotective ability of microglia. In conclusion, although CTE is known to be a poisonous plant and listed on the FDA poisonous plant database, it can be used as a medicine if the amount is properly controlled. Our results suggested the potential benefits of CTE as a therapeutic agent for different neurodegenerative disorders involving neuroinflammation.

Plasmin Cleaves Complement iC3b, Preventing CR3/4 Mediated Phagocytosis By Macrophages

IntroductionThe plasmin(ogen) and complement systems are activated at sites of tissue injury and are involved in hemostasis, wound healing, inflammation and immune surveillance. Although the mechanisms are poorly understood, dysregulation of these systems underlie the pathogenesis and progression of inflammatory and vascular diseases. We aimed to characterize the relevant molecular interactions between the plasmin(ogen) and complement pathways.The three complement pathways converge with formation of C3-convertases that cleave C3 into C3a and C3b. C3a is liberated as an anaphylatoxin while C3b participates in further formation of the C3 and C5 convertases, thereby amplifying complement activation. To dampen the system, negative regulatory mechanisms exist. C3b is degraded to iC3b by the factor I (FI)/FH complex, which in turn is degraded to C3dg by the FI/complement receptor 1 (CR1) complex. iC3b and C3dg induce cellular responses by binding to complement receptors CR3 / CR4 / CR2, and CR2, respectively. Interactions of iC3b with CR3 or CR4 induce phagocytosis by macrophages, and binding of iC3b or C3dg to CR2 promotes B-cell responses.Recent studies show that plasmin proteolyses C3b and iC3b. We further characterized the plasmin cleavage sites in iC3b and evaluated the functional consequences in vitro. Methods and ResultsPlasmin cleavage of iC3b was examined over a range of concentrations and times. Plasmin (50 nM) generated a 40 kDa iC3b cleavage fragment (946TLD – PSR1303) which was notable for containing both C3dg (1002HLI – PSR1303) and the C3 thioester domain, necessary for opsonic binding to surfaces.We tested the relevance of this cleavage in phagocytosis assays using immunofluorescence and flow cytometry (Figure 1). C3b bound to the surface of fluorescent (Alexa 488) zymosan particles (C3b-zym), was treated with FI/FH to generate iC3b-zym, and subsequently incubated with FI/CR1 or plasmin to yield C3dg-zym or 946TLD – PSR1303-zym, respectively. Western blots confirmed that plasmin generated 946TLD – PSR1303 from iC3b-zym. The C3 fragment-zymosan species (C3b-zym, iC3b-zym, C3dg-zym and 946TLD – PSR1303-zym) were each incubated with macrophages (PMA-differentiated THP-1 cells) for 90 minutes. Cells were washed, stained and fixed for immunofluorescence, or suspended for flow cytometry. Figure 1, panel A shows macrophages stained with CellMask (red, cell membrane) and DAPI (blue, nucleus). Fluorescent zymosan is seen in green. No phagocytosis was detected with zymosan lacking C3 (zym alone), but there was a small amount with C3b-zym. In contrast, iC3b-zym was highly effective in inducing phagocytosis by most macrophages. This effect of iC3b-zym was abolished with FI/CR1 or plasmin, i.e. little phagocytosis was detected with C3dg-zym or 946TLD – PSR1303-zym. [Display omitted] Flow cytometry-based quantitative analyses confirmed the preceding findings (Figure 1, panel B), with a similar pattern of phagocytosis induced by the zymosan-bound fragments. No phagocytosis was detected with zymosan lacking C3. Phagocytosis of C3b-zym and iC3b-zym was 7±2% and 17±1% of cells, respectively. C3dg-zym and 946TLD – PSR1303-zym induced phagocytosis was <5%.We also evaluated the role of the complement receptors in mediating the effect of the C3b/iC3b fragments using CR3/4 and CR1 blocking antibodies. These confirmed that phagocytosis of iC3b-zym and C3b-zym is mediated by CR3/4 and CR1, respectively. ConclusionsPlasmin cleaves iC3b to form a redundant complement regulatory pathway with the FI/CR1 complex, but which notably does not require a cellular cofactor. Further studies will delineate the role of this and other plasmin-generated complement fragments in modulating innate immune and inflammatory responses. Disclosures:No relevant conflicts of interest to declare.

Functional Selection of Phagocytosis-Promoting Genes: Cell Sorting–Based Selection

Phagocytosis is a critical host defense mechanism that clears invading pathogens, apoptotic cells, and cell debris; it is an essential process for normal development, tissue remodeling, immune response, and inflammation. Here, a functional selection strategy was used to isolate novel phagocytosis-promoting genes. After the retroviral transfer of mouse brain cDNA library into NIH3T3 mouse fibroblast cells, cell sorting was used to select the cells that phagocytosed fluorescent zymosan particles. The cDNAs were retrieved from the selected cells and identified by DNA sequencing as eIF5A, Meg3, Tubb5, Sparcl-1, Uchl-1, Bsg (CD147), Ube2v1, and Pamr1. The phagocytosis-promoting activity for some of these cDNAs was confirmed by transient transfection in the independent phagocytosis assays. Thus, the unbiased selection procedure successfully identified multiple phagocytosis-promoting genes. The selection method can be applied to other cell-based assays where cells with a desired phenotype can be physically separated. Moreover, the new gene targets uncovered in this study could be relevant to biomolecule screening in search of phagocytosis-regulating agents. In a small-scale screen, a series of imidazopyridine compounds was tested to identify the small molecules that modulate eIF5A-mediated phagocytic activity. Several compounds that influenced the phagocytic activity can be further used as chemical-genetic tools to delineate the mechanisms of eIF5A action and be potential drug candidates that are capable of therapeutically modulating phagocytic activity.

Cystic Fibrosis Transmembrane Conductance Regulator-independent Phagosomal Acidification in Macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.

Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis

AbstractThe protein tyrosine kinase Syk plays a central role in Fcγ receptor–mediated phagocytosis in the adaptive immune system. We show here that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Macrophage-like differentiated HL60 cells and C3bi-opsonized zymosan comprised the pathogen-phagocyte system. C3bi-opsonized zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor 3. During this process, Syk became tyrosine phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into HL60 cells resulted in impaired phagocytosis. Quenching assays using fluorescent zymosan revealed that most of the attached zymosan particles were located inside parental HL60 cells, whereas few were ingested by the mutant cells. These data indicated that Syk is required for the engulfment of C3bi-opsonized zymosan. During C3bi-zymosan–induced phagocytosis, actin accumulation occurred around phagosomes and was followed by depolymerization, and further RhoA was activated together with tyrosine phosphorylation of Vav. These responses including the actin remodeling were suppressed in Syk-siRNA– or DN-Syk–expressing cells. Our results demonstrated that Syk plays an indispensable role in complement-mediated phagocytosis by regulating both actin dynamics and the RhoA activation pathway and that these functions of Syk lead to phagosome formation and pathogen engulfment.

A Role of the Protein Tyrosine Kinase, Syk in Complement-Mediated Phagocytosis: A Mechanism on the Engulfment of Pathogen.

A protein-tyrosine kinase, Syk plays a central role in Fc-gamma receptor-mediated phagocytosis in the adaptive immune system. Here we show that Syk also makes an essential role in compliment-mediated phagocytosis in innate immunity. For this purpose, HL60 cells induced to macrophage-like with vitamin D3 and TPA and C3bi-opsonized zymosan were used as the pathogen-phagocyte system. Time-lapse microscopic observation revealed that C3bi-opsonized zymosan particles were promptly attached to and sucked into the cells but non-opsonized zymosan was not, and therefore it was indicated that our phagocytosis system is mediated by the complement receptor 3 (CR3). Syk was tyrosine-phosphorylated by the treatment with C3bi-opsonized zymosan and marked accumulation of Syk was detected around the newly forming phagosomes. To further investigate the effect of Syk on complement-mediated phagocytosis, Syk-siRNA or dominant-negative Syk (DN-Syk) was introduced into HL60 cells. Quantitative analysis by flow cytometry showed that phagocytosis was severely impaired in DN-Syk or Syk-siRNA expressing HL60 cells. To determine whether the impaired capture of zymosan is due to reduced binding to CR3 or reduced engulfment after binding, the cells were treated with a quenching reagent which discriminates fluorescent zymosan particles between inside and outside the cells. In the parental HL60 cells most of the attached zymosan particles were inside the cells, while few particles were ingested in the case of DN-Syk or Syk-siRNA expressing HL60 cells. These data indicated that Syk is required for engulfment of C3bi-opsonized zymosan but not for attachment of zymosan to CR3. To clarify the molecular mechanism how Syk affects the phagosome formation and its engulfment, the behavior of F-actin was searched microscopically. At the early stage of complement-mediated phagocytosis, marked accumulation of F-actin occurred around each phagosome and was followed by rapid depolymerization in the parental HL60 cells. In contrast, phagosomes surrounded by the accumulated actin were significantly decreased in DN-Syk or Syk-siRNA expresssing cells. These results indicate that Syk promotes actin assembly around phagosomes prior to engulfment at the early stage of the phagocytosis. As for the analysis of the downstream pathway of Syk in our phagocytosis system, RhoA-signaling was investigated. Vav-RhoA pathway was activated in HL60 cells but transfer of DN-Syk or Syk-siRNA into HL60 cells reduced the activation of Vav-RhoA signaling. Therefore Syk may act as an activator of the RhoA-pathway in phagocytosis.Our results demonstrate that Syk is required for complement-mediated phagocytosis by regulating both actin accumulation around phagosomes and RhoA-signaling harmonically and finally leads to phagosome formation and its engulfment.

Stimulus-specific defect in the phagocytic pathways of annexin 1 null macrophages.

The role of the glucocorticoid-regulated protein annexin 1 during the process of phagocytosis has been studied using annexin 1 null peritoneal macrophages. Wild type and annexin 1 null macrophages were incubated with several distinct phagocytic targets. No differences were observed in rate or the maximal response with respect to IgG complexes or opsonised zymosan phagocytosis, as assessed by monitoring the production of reactive oxygen species. When annexin 1 null macrophages were incubated with non-opsonised zymosan particles, they exhibited impaired generation of reactive oxygen species, which was linked to a defect in binding of cells to the particles, as determined with fluorescent zymosan. This phenomenon was further confirmed by electron microscopy analysis, where annexin 1 null macrophages internalised fewer non-opsonised zymosan particles. Specific alterations in macrophage plasma membrane markers were observed in the annexin 1 null cells. Whereas no differences in dectin-1 and FcgammaR II/III expression were measured between the two genotypes, decreased membrane CD11b and F4/80 levels were measured selectively in macrophages lacking annexin 1. These cells also responded with an enhanced release of PGE(2) and COX-2 protein expression following addition of the soluble stimulants, LPS and heat-activated IgG. In conclusion, these results suggest that participation of endogenous annexin 1 during zymosan phagocytosis is critical and that this protein plays a tonic inhibitory role during macrophage activation.

Neutrophil elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo.

Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE(-/-)) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE(-/-) mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1beta and TNF-alpha and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE(-/-) mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1beta, KC, and macrophage inflammatory protein-1alpha in NE(-/-) mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.

Role of Oxidative Stress in Amiodarone-induced Toxicity.

BACKGROUND: The clinical usefulness of amiodarone for the treatment of cardiac arrhythmias is limited by multiorgan toxicity, especially pulmonary and hepatic. There are conflicting reports in the literature regarding the role of free radicals in the initiation of amiodarone-induced toxicity. We evaluated the possible oxidative stress in a chronic model that is known to manifest pulmonary toxicity. METHODS AND RESULTS: A group of 20 Fischer-344 rats were injected with 60 mg/kg/day of amiodarone for 21 days. A control group of 20 animals received only saline injections. The alveolar macrophages obtained by lung lavage were incubated with hydroethidine and opsonized green fluorescent zymosan particles to measure oxidative and phagocytic activities by flow cytometry. Malondialdehyde levels were measured to assess the extent of lipid peroxidation in lung, liver, spleen, kidney, and heart. Total phospholipid levels in all the collected tissues and distribution of phospholipid classes in the lung and the liver were measured. The levels of amiodarone and its metabolite desethylamiodarone in serum and all collected tissues were measured by high-performance liquid chromatography. The phagocytic activity of th emacrophages from treated animals was decreased by 18-22% (P <.03) compared to controls; however, the oxidative activities of control and treated groups were not significantly different. The tissue malondialdehyde levels were not significantly different except in the spleen where they increased after amiodarone treatment (18.2 +/- 1.1 vs 23.7 +/- 2.8 µM/g tissue, P <.0001). Malondyaldehyde levels were not significantly different when normalized to lipid phosphorous content. Lung, liver, and spleen showed significantly higher phospholipid levels in the treated group. The tissue amiodarone and desethylamiodarone levels in the treated group were highest in spleen followed by lung, liver, kidney, and heart. CONCLUSIONS: The results show that amiodarone-induced pulmonary and hepatic toxicity is not directly mediated by oxidative stress; however, increased lipid peroxidation in the spleen, although secondary to phospholipidosis, may be physiologically significant.

A synthetic hemoregulatory peptide (HP5B) inhibits human myelopoietic colony formation (CFU-GM) but not leukocyte phagocytosis in vitro.

A synthetic analog of a hemoregulatory peptide associated with mature human granulocyte (HP5B) has been investigated for inhibitory effects on human myelopoietic stem cells in vitro. In addition, it has been tested for effects on phagocytosis by human granulocytes and monocytes by use of an automatic flow cytometric method. A dose-dependent inhibition of colony formation was found after preincubation of bone marrow cells for 1 h at 37 degrees C in the range 10(7) -10(-11) mol/l. Above or below these concentrations, no inhibitory effects were seen. The degree of inhibition varied from experiment to experiment, indicating variable responsiveness of the donor cells. Maximal effect was of magnitude 90% inhibition, and the optimal dose was 10(7) mol/l. The peptide had no effect on the kinetics of phagocytosis by measurements of the uptake of fluorescent Zymosan particles or Staphylococcus aureus. This may indicate a selective effect on the precursor cells, with no effect on the functional state of their progeny, the granulocytes and monocytes.

In vitro studies of cryopreserved baboon granulocytes.

Granulocytes were isolated from the buffy coat of baboon blood by counterflow centrifugation. They were frozen in polypropylene tubes in 2.0 ml volumes containing 1 × 107 granulocytes. The medium consisted of 5% DMSO, 6% HES, 4% human serum albumin, and 6 mM glucose in Normosol‐R, pH 7.1. Granulocytes were cooled to 4 C for 30 minutes, then cooled at 4 C per minute to –80 C and stored for one to three weeks in liquid nitrogen at –197 C. Cooling rates of 1 C and 10 C per minute were less efficacious. Tubes were thawed manually with swirling for 130 seconds at 42 C in a water bath. The yield after thawing was 98 ± 14 per cent.Granulocytes isolated from fresh blood all fluoresced green when incubated with fluorescein diacetate (FDA). Less than one per cent of cells showed nuclear uptake of ethidium bromide (EB). After freezing and thawing, an average of 79 ± 8 per cent granulocytes fluoresced green with FDA and 21 ± 8 per cent fluoresced red with EB. About 98 ± 2 per cent of the granulocytes isolated from fresh blood exhibited a serum dependent capacity for ingestion of latex and yeast. Thawed granulocytes were diluted 1:4 dropwise with a medium containing 7% HES, 4% albumin, and 6 mM glucose in Normosol‐R, incubated with fluorescent latex or fluorescent zymosan at 37 C for 30 minutes and then washed twice. After this, 75 ± 5 per cent demonstrated serum‐dependent latex ingestion and 67 ± 8 per cent ingested zymosan.