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The present study evaluates electron spin resonance (ESR) and the spin trapper 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) for analysis of superoxide radical production by human neutrophils interacting with viable Staphylococcus aureus and Staphylococcus epidermidis bacteria. To avoid auto-activation due to interaction with glass surfaces, neutrophils were preincubated in plastic tubes until the peak response was reached, and then transferred to a quartz flat cell to record the ESR spectra. The time point for peak response was identified by parallel analysis of the bacteria-neutrophil interaction using luminol amplified chemiluminescence. We found detectable ESR spectra from neutrophils interacting with as few as five bacteria of the weak activating S. epidermidis per neutrophil. Addition of the NADPH oxidase inhibitor diphenylene iodonium totally abolished spectra. Catalase, DMSO or an iron chelator had no impact on the produced spectra and ionomycin, a selective activator of intracellular NADPH oxidase, gave significant ESR spectra. Taken together, our results indicate that DEPMPO is cell permeable and detects NADPH oxidase derived superoxide anions formed in phagosomes or released by human neutrophils phagocytosing viable S. aureus and S. epidermidis. The technique may be used as a sensitive tool to evaluate superoxide anion production in human neutrophils.

Cellular ageing is associated with accumulation of undegradable intralysosomal material, called lipofuscin. In order to accelerate the lipofuscin-accumulation, confluent, growth arrested human fibroblasts were cultured under hyperoxic conditions. To provide a better insight into the effects of lipofuscin on cellular functions, we compared lysosomal stability in control and lipofuscin-loaded human fibroblasts under conditions of lysosome-targeted stress induced by exposure to either the lysosomotropic detergent MSDH or the redox-cycling quinone naphthazarin. We show that lysosomal damage, assessed by acridine-orange relocation, translocation of cathepsin D to the cytosol, and alkalinization of lysosomes is more pronounced in control than in lipofuscin-loaded fibroblasts. Finding that lysosomal integrity was less affected or even preserved in case of lipofuscin-loaded cells enables us to suggest that lipofuscin exerts lysosome-stabilizing properties. Note: Because of an embargo period with Springer journals, the full text will become available at LiU E-Press, January 2009. Original publication: Yuri Stroikin, Hanna Mild, Uno Johansson, Karin Roberg and Karin Öllinger, Lysosome-targeted stress reveals increased stability of lipofuscin-containing lysosomes, 2008, AGE http://dx.doi.org/10.1007/S11357-007-9045-9. Copyright: The original publication is available at http://www.springerlink.comThe previous status of this article was Manuscript and the working title was Lipofuscin preserves lysosome integrity under conditions of organelle-targeted stress.

Summary Natrium aurothiomaleate (GSTM) is a useful disease-modifying anti-rheumatic drug, but causes a variety of immune-mediated adverse effects in many patients. A murine model was used to study further the interaction of GSTM with the immune system, including induction of systemic autoimmunity. Mice were given weekly intramuscular injections of GSTM and controls equimolar amounts of sodium thiomaleate. The effects of gold on lymphocyte subpopulations were determined by flow cytometry. Humoral autoimmunity was measured by indirect immunofluorescence and immunoblotting, and deposition of immunoglobulin and C3 used to assess immunopathology. Gold, in the form of GSTM, stimulated the murine immune system causing strain-dependent lymphoproliferation and autoimmunity, including a major histocompatibility complex (MHC)-restricted autoantibody response against the nucleolar protein fibrillarin. GSTM did not cause glomerular or vessel wall IgG deposits. However, it did elicit a strong B cell-stimulating effect, including both T helper 1 (Th1)- and Th2-dependent isotypes. All these effects on the immune system were dependent on the MHC genotype, emphasizing the clinical observations of a strong genetic linkage for the major adverse immune reactions seen with GSTM treatment.

Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-alpha, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 +/- 0.1 in healthy cells to 6.8 +/- 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 +/- 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F(0)F(1)-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 +/- 0.3, in the healthy population, to 4.8 +/- 0.3 and 5.5 +/- 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-alpha.

Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

Mercury and silver induce antinucleolar autoantibodies (ANoA) targeting the 34-kDa nucleolar protein fibrillarin in susceptible mouse strains. Mercury has the ability to cause a general activation of the immune system, but antigen-specific mechanisms following direct or indirect interaction between mercury and fibrillarin are now believed to play a crucial role in the autoimmune pathogenesis. Our previous studies showed that silver neither induced the systemic immune complex deposits nor the increase of serum immunoglobulins seen after mercury treatment. The main objective of this study was to examine the relation between activation of the immune system and the induction of ANoA.During 4 weeks of subcutaneous silver nitrate injections into mice of the susceptible A.SW and SJL strains and the resistant A.TL strain, the number of T and B cells as well as the expression of cell surface activation and proliferation markers were monitored by flow cytometry. The number of cytoplasmic Ig+ splenocytes was determined by direct immunofluorescence technique on slides, and serum Ig levels as well as anti-ssDNA anti anti-DNP antibodies were determined by ELISA. Serum ANoA were monitored by the indirect immunofluorescence technique.Silver caused in the susceptible strains a weaker and later activation and proliferation of T and B cells than mercury, and no significant polyclonal B cell activation. In contrast, the ANoA titer was not different from that seen in mercury-treated mice of the same strains. Silver-treated mice of the A.TL strain showed neither activation of the immune system nor ANoA.Despite being as effective as mercury inducing ANoA, silver caused only a slight activation of the immune system. This demonstrates that the massive activation of the immune system in mercury treatment is not necessary for the induction of ANoA, and indicates that (auto)antigen-specific mechanisms are likely to play a key role in mercury- and silver-induced murine autoimmunity.

Genetically mercury-susceptible (H-2 s ) mice in which the nude (athymic) mutation had been introduced, and euthymic (H-2 s ) mice treated with anti-CD4 monoclonal antibodies were used to determine the importance of T-helper (CD4 + ) cells for induction of autoimmunity by mercury, and to study the possibility of using anti-CD4 MAb for treatment of manifest autoimmunity. SJL/N and (A.SW × SJL- nu ) F1 × SJL- nu BC (H-2 s ) mice homozygous for the nude mutation ( nu/nu ) were treated with 10 mg HgCl 2 /litre drinking water for 6 weeks. These mice developed neither the antinucleolar antibodies (ANoA) nor the systemic immune-complex (IC) deposits seen in mercury-treated littermates heterozygous for the nude mutation ( nu /+). The nu/nu mice showed a significant and substantial reduction of splenocytes with pan-T-(CD3 + ), T-helper-(CD4 + ) and T-cytotoxic/suppressor (CD8 + ) markers, which was accompanied by a severe reduction of the proliferative response to Concanavalin A. Euthymic SJL/N mice given an initial intravenous (i.v.) injection of 100 μg anti-CD4 MAb (clone GK 1.5, rat IgG2b), followed by 6 weeks treatment with 100 μg anti-CD4 MAb intraperitoneally (i.p.) every third day in combination with 10 mg HgCl 2 /litre drinking water, did not develop ANoA or systemic IC-deposits. These features were seen in controls i.p. injected with rat IgG2b and given HgCl 2 in the drinking water. The anti-CD4 MAb-treated mice showed very few CD4 + splenocytes, but a significant increase of CD8 + cells and severely impaired T-cell function. The possibility of treating longstanding autoimmune conditions with anti-CD4 MAb was examined by giving euthymic SJL mice HgCl 2 for 3 months, followed by a mercury-free interval of 3 months and finally 7 weekly injections of 1 mg anti-CD4 MAb. This therapy caused a severe reduction of CD4 + cells, but there was no decline in the ANoA titre. In conclusion, induction of systemic autoimmunity by mercury was strictly dependent on T cells, specifically T-helper (CD4 + ) cells, and mercury-induced ANoA persisted for a long time after stopping mercury treatment. At this late stage, the autoimmune condition was no longer amenable for anti-CD4 MAb therapy.

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Bax activator Bid, and translocation of Bax to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin D-mediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid.

Dental amalgam fillings are the most important source of mercury exposure in the general population, but their potential to cause systemic health consequences is disputed. In this study, inbred mice genetically susceptible to mercury-induced immune aberrations were used to examine whether dental amalgam may interfere with the immune system and cause autoimmunity. Female SJL/N mice were implanted in the peritoneal cavity with 8-100 mg silver amalgam or silver alloy for 10 weeks or 6 months. Chronic hyperimmunoglobulinemia, serum IgG autoantibodies targeting the nucleolar protein fibrillarin, and systemic immune-complex deposits developed in a time- and dose-dependent manner after implantation of amalgam or alloy. Splenocytes from mice implanted with amalgam or alloy showed an increased expression of class II molecules. The functional capacity of splenic T and B cells was affected in a dose-dependent way: 10 weeks of low-dose and 6 months of high-dose amalgam implantation strongly increased mitogen-induced T and B cell proliferation, whereas 10 weeks of high-dose implantation decreased the proliferation. Not only mercury but also silver accumulated in the spleen and kidneys after amalgam implantation. In conclusion, dental amalgam implantation in a physiological body milieu causes chronic stimulation of the immune system with induction of systemic autoimmunity in genetically sensitive mice. Implantation of silver alloy not containing mercury also induced autoimmunity, suggesting that other elements, especially silver, have the potential to induce autoimmunity in genetically susceptible vertebrates. Accumulation of heavy metals, from dental amalgam and other sources, may lower the threshold of an individual metal to elicit immunological aberrations. We hypothesize that under appropriate conditions of genetic susceptibility and adequate body burden, heavy metal exposure from dental amalgam may contribute to immunological aberrations, which could lead to overt autoimmunity.

SPECIFIC AIMSThe overall objective of the present study was to examine lysosomal stability during apoptosis and the relationship between caspase activation and the lysosomal proteases cathepsins D and B with regard to their effects on apoptosis.PRINCIPAL FINDINGS1. Naphthazarin-induced apoptosis is dependent on the lysosomal protease cathepsin DThe caspase-3-like activity increased after 12 h of treatment with the redox cycling quinone naphthazarine (NZ). By comparison, the activity of cathepsin D was augmented after 4 h of NZ treatment and peaked at 16 h (Fig. 1A⤻ , B⤻ ). Moreover, increased levels of p53, a transcription factor for cathepsin D, were detected after exposure to NZ for 4 h (Fig. 1D⤻ ). The total protein level did not change in response to p53 induction and was estimated to 8.2 ± 3.5, 8.0 ± 2.1, and 7.1 ± 1.4 μg protein/10 000 cells after 0, 12, and 20 h respectively. We also found that NZ caused a rapid decrease in the activity of the lysosomal cysteine protease cathepsin B (Fig. 1C⤻ ). Fi...

We studied phagolysosomal pH in alveolar macrophages (AM) using fluorescein-labeled yeast (FYP) and silica particles (FSP) as probes. Fluorescence intensities from the ingested test particles were measured on populations of AM using fluorescence spectrometry and on individual phagolysosomes using fluorescence microscopy. Measurements were performed on rabbit AM, which had been incubated with FYP or FSP (in vitro procedure). We also instilled FYP or FSP via the trachea into rabbit lungs and after 1 day, 1 week, 1 month, and 3 months lavaged the lungs and measured the pH in AM (in vivo procedure). Phagolysosomal pH was independent of the number and size of the fluorescent particles. Measurements of populations of AM with fluorescence spectrometry and of individual phagolysosomes with fluorescence microscopy gave similar average pH. For the FYP, pH decreased during the first day after lavage both in the in vitro and the in vivo procedures. For the FSP, pH was unchanged during the same period. After 1 day pH was similar for both particles. Electron microscopy showed a larger number of lysosomes in contact with phagosomes and a higher percentage of vacuolated phagosomes for FYP than for FSP. In the in vivo procedure, pH was unchanged at least up to 1 month, and this pH was lower than that in the in vitro procedure. The difference was probably due to conditions at the time of phagocytosis. Particles retained in the lung parenchyma were within AM, and their location within the AM appeared unchanged from 1 week up to 3 months. Images FIGURE 2. A FIGURE 2. B

Fluorescein-labeled silica particles (FSP) were instilled into the tracheae of rabbits. Groups of four rabbits were killed after 24 hr, 1 week, 1 month, or 3 months and their lungs were lavaged. Phagolysosomal pH in the alveolar macrophages (AM) was measured using microscope fluorometry with FSP as a probe. Due to the marked decline of the fluorescence intensities from the FSP between 1 and 3 months after instillation, it was not possible to measure pH at 3 months, but the values from 24 hr, 1 week, and 1 month were quite similar, with group means of 4.8 and 4.9, respectively. Phagolysosomal pH in AM which phagocytized the FSP in vitro showed values about half a pH unit higher. AM from rabbits lavaged at 1 week had more lysosomes in contact with the FSP-containing phagolysosomes and a higher degree of vacuolization between the FSP and the phagolysosomal membrane than AM lavaged at 1 day. The location of the FSP in the AM appeared to be similar in rabbits lavaged after 1 week and 3 months. In histologic sections from the lungs the large majority of the FSP were within cells at all time points.

Alterations of cellular structures often found in ageing cells is mainly the result of production of reactive oxygen species and a consequence of aerobic life. Both oxidative stress and decreased degradative capacity of lysosomal system cause accumulation of intralysosomal age-related pigment called lipofuscin. To investigate the influence of lipofuscin on cell function, we compared survival of lipofuscin-loaded and control human fibroblasts following complete starvation induced by exposure to phosphate-buffered saline (PBS). Starving of control fibroblasts resulted in lysosomal alkalinisation, relocation of cathepsin D to the cytosol, caspase-3 activation and, finally, cell death, which became evident 72 h after the start of exposure to PBS. Increase of lysosomal pH was significantly less prominent in lipofuscin-loaded cells than in controls and was accompanied neither by leakage of cathepsin D nor by caspase-3 activation even 96 h after the initiation of starvation. Suppression of autophagy by 3-methyladenine (3-MA) accelerated cell death, while inhibition of cathepsin D delayed it, implying an important role of autophagy in cell survival during starvation and showing the involvement of lysosomes in starvation-induced cell death. Disturbed apoptotic response found in lipofuscin-loaded cells can be interpreted as an example of hormesis--an adaptation to low doses of otherwise harmful agents, in this case of lipofuscin, which has a protective effect at moderate amounts but becomes toxic at large quantities.

Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10 ng/ml TNF-alpha for 4 h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2+/-0.1 to 5.8+/-0.1 and the lysosomal increased from 4.3+/-0.4 to 5.2+/-0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

Mercury induces a systemic autoimmune condition characterized by auto-antibodies to the nucleolar protein fibrillarin (AFA) and systemic immune-complex (IC) deposits in genetically susceptible mouse strains. This study examines T cell activation and cytokine production following mercury exposure in genetically susceptible and resistant strains. Mercury injected s.c., according to the protocol for induction of autoimmunity, caused an early T cell activation, measured as an increase of IL-2-producing cells, and increased expression of the IL-2-receptor proteins CD25 and CD122 and of the proliferation marker CD71 on days 2-4 in the susceptible A.SW and A. TH strains. This was followed by a long-lasting increase in the number of T cells, dominated by CD4(+) cells. Mice of the susceptible A.SW strain showed a modest increase of TNF-alpha-, IFN-gamma-, and IL-4-producing cells after 4-6 days, and a very distinct increase of IL-4-producing cells on days 8-10. The susceptible SJL strain (H-2(s)), severely deficient in Th2-promoting CD4(+), NK1.1(+) T cells, showed no increase of IL-4(+) cells on days 8-10. Instead, the number of IFN-gamma-producing cells was increased. Susceptible mice developed an increase of Ig-producing cells, AFA, and systemic IC-deposits. Genetically mercury-resistant A.TL mice showed a minimal increase of T cells, but no increase in cytokine-producing cells. We conclude that autoimmunogenic doses of HgCl2 induce an activation and proliferation of T cells in genetically susceptible mouse strains, as well as a broad increase of cytokine-producing cells, followed by a late predominance of the Th2-associated IL-4. One strain, severely deficient in Th2-promoting CD4(+), NK1.1(+) T cells, lacked the increase in IL-4(+) cells, indicating that a predominantly Th2-response is not necessary for induction of autoimmunity by mercury. However, a Th2-dominated response led to a faster and stronger B cell activation.

Lipofuscin-specific autofluorescence of living, cultured rat neonatal myocardial myocytes was studied with respect to intensity and spectral characteristics. The autofluorescence emission spectrum changed over time indicating continuously ongoing intramolecular reorganisation of the pigment. Moreover, as compared with formaldehyde-fixed material native lipofuscin produced a considerable red-shifted autofluorescence, indicating formaldehyde-induced molecular modification or quenching of certain parts of the spectrum. The relationship between oxidative stress and lipofuscinogenesis was confirmed because the rate of lipofuscin accumulation was increased when cells were grown at increasing ambient oxygen concentrations.

In 1994 a poster entitled 'A technical survey of storage conditions in Swedish museums' was presented at the IIC Congress on Preventive Conservation in Ottawa, Canada. All information gathered during that survey has now been entered into a database called 'Gunnel'. Gunnel stores information about 27 museums with a total of 354 storerooms representing 99,000m of storage area. That is almost 50% of the total storage area of central, regional and local museums in Sweden. The general conclusion drawn from analysis of the database material is that 20% of the storage areas in central and regional museums and 40% in the local museums are unsuitable for the preservation of cultural property. It is also possible to study how different categories of materials and finds are stored. This is illustrated by a comparison between the storage conditions for archaeological iron and ethnological iron. Every year a lot of work and money are expended on the conservation of various categories of archaeological material in Sweden. One of the most sensitive materials is archaeological iron. Very specific climatic conditions are required before, during and after the conservation process. The optimal climate for storage of archaeological iron is an environment with a relative humidity (RH) below 18%. But how is the archaeological iron stored when the conservation is finished? The Gunnel database shows that, out of 22 storerooms (representing an area of 750m2), there is only one room with an RH of 30% or lower. There are four rooms with 35% RH, two with 40%, two with 45%, four with 50%, four with 55%, three with 60% and four with 65% or higher. These figures include the main museum for archaeology in Sweden plus some important regional and local museums with large archaeological collections. The study shows that less than 4% of the storerooms can be considered acceptable for storing archaeological iron. The recommended storage condition for ethnological iron is an environment with an RH not exceeding 50%. If we examine how this kind of iron is stored in Swedish museums we find that out of 58 storage rooms (representing 4000m) there are eight storage rooms with an RH of 30% or lower. There are nine with 350/0 RH, six with 40%, one with 45%, 10 with 50%, 11 with 55%, three with 60% and nine with 65% or higher [1]. It appears that 60% of this material is stored under acceptable climatic conditions. This study shows that a sensitive material like archaeological iron is stored under very poor climatic conditions in Sweden. There is hardly any acceptable storage of this material. As far as ethnological iron is concerned, the situation is better. There are at least 35 storerooms with an acceptable climate. If we compare the storage of archaeological iron with the grand total of storerooms surveyed in Sweden we find that there are about 50 storerooms with an RH of 30% or lower [1]. Clearly this is not where archaeological iron is stored! It is quite obvious that the time has come for a thorough reorganization of the storage of our collections. Lars-Uno Johansson works at the Institutefor Conservation of National Antiquities, Box 5405, 114 84 Stockholm, Sweden.

A method for measurement of phagolysosomal pH in individual alveolar macrophages based on a cytofluorometric technique with fluorescein-labeled silica particles (FSP) as a probe was developed. The size of the FSP, 3.0 or 5.0 microns, did not affect the result of the pH measurements. The average pH values, range 5.1-5.5, of individual particles in macrophages from three rabbits agreed well with the pH values obtained in a macrophage population using a fluorescence spectrometer and the FSP. The variation of pH in phagolysosomes in alveolar macrophages from five rabbits was investigated. Measurements were performed 3, 6, and 24 h after addition of the FSP in cells containing one particle as well as in cells containing two particles. The variation in pH was small, with a coefficient of variation less than 10% in all rabbits at all times. There was a significant correlation between values obtained from two phagolysosomes in the same cell, indicating a cell factor responsible for 10-30% of the total variance. The fact that the variation of pH is small in normal, untreated rabbit alveolar macrophages should be of importance when estimating alveolar clearance of inorganic particles due to dissolution in the acid phagolysosomal milieu.