Role of alveolar macrophages in precipitation of mineral elements inhaled as soluble aerosols.

The lysosomes of several varieties of cells such as the tubular proximal cell of the kidney and the alveolar macrophage have the ability to concentrate and precipitate several elements inhaled in water-soluble form, usually as phosphate. The mechanism involved is attributed to the high acid phosphatase activity of lysosomes and can be considered as an in vivo Gomori reaction. Among the elements studied, most of them are chemotoxic or radiotoxic (Cr; group IIIA: Al, Ga, In; rare earths: La, Ce, Tm; actinides: Th, U). In the lung macrophage, this mechanism of intralysosomal concentration and precipitation may prevent the diffusion of these toxic elements through the alveolar membrane. ImagesFIGURE 1.FIGURE 2.


Introduction
Previous investigations have shown that the phagolysosome of the lung macrophage has the capacity to dissolve solid particles of low aqueous solubility (1,2). We show in this paper that the inverse is also possible, namely, that the lysosome of the lung macrophage is also able to concentrate and precipitate several elements inhaled in water-soluble form. This particular lysosomal function may prevent the diffusion ofmany chemotoxic or radiotoxic elements to the bloodstream.
Submicron aerosols were generated from 1% solutions ofthese compounds and administered to rats in a facility that has been described previously (3). Six male Wistar rats, witha mean body weight of 250 g, were exposed for 5 hr per day for 5 days. The animals were killed on day 5, 3 hr after the termination of exposure. The particle number concentration measured in the chamber was 15 x 105/cm3, resulting in a mass concentration of 10-30 sAg/cm3. The mean diameter of the particles was about 0.1 tm.

Microanalytical Methods
The ultrastructure of the cells in lung sections was examined using a Philips EM 300 microscope. The intracellular localization of the elements was determined using an electron probe Camebax Cameca microanalyzer equipped with thallium acid phtalate (TAP), pentaerythritol (PET), and lithium fluoride (LiF) crystals. An electron microscope specially adapted to this apparatus allowed us to examine ultra-thin sections, select zones of interest, and focus the electron beam on the intracellular organelle to be analyzed.
The conditions ofanalysis were as follows: electron acceleration voltage, 45kV; probe diameter, 500 nm; probe current, 150 nA. Under these conditions, elements present under the microprobe ata concentration greaterthan 100 ppm couldbe detected.
The Kcx line ofphosphorus and aluminum were detected with the TAP crystal at sinO = 0.23969 and sinO = 0.3463, respectively. The Kca line ofchromium was detected with the PET crystal at sinO = 026072. The Ma line ofuranium was detected with the PET crystal at sinO = 0.44692 and the La line ofcerium at sinO = 0.29279.
We made counts on the line ofthe element analyzed and measured background noise on either side. Mean background noise was then subtracted from the counts obtained on the line. This operation was repeated three times for each inclusion analyzed.

Results
Electron microscopy and microanalysis permit the determination ofthe precise intracellular localization ofthe deposit and its ultrastructural appearance for each element.
Lung Sectionsfrom Rats Exrosedto UiunylNitre Aerosol. Exposure ofrats to U02(NO3)2.6H20 caused abnonnal deposits FIGURE 1. Dense deposits observed in a rat macrophage after inhalation of uranyl nitrate. The deposits consist of an accumulation of very thin needles. The lysosomal membrane is not visible in this unstained section. Electron probe X-ray analysis of the deposits shows that they contain uranium and phosphorus at a high concentration.
in macrophages and cells, localized in the lysosomes that consisted ofvery dense and thick deposits ofneedles about 30 nm in length. The needles were sometimes isolated but most were clustered. The Ka line ofphosphorus and the Ma line ofuranium were detected in all these deposits by microanalysis (Fig. 1). Lung Sections from Rats Exposed to Aluminum Chloride Aerosol. The deposits observed after exposure to AlCl3 in the lysosomes ofmacrophages had two different ultrastructures. The first type consisted ofdense spherules ofdifferent dimensions. Their contours are well defined, and they are composed entirely ofaggregates ofvery fine granules. The second type consisted of either very dense aggregates (about 3 nm) or of undulated filaments within the lysosomes.
Lung Sections from Rats Exposed to Chromium Chloride Aerosol. In the alveolar macrophages, the deposits observed in phagolysosomes of rats exposed to CrCl3 consisted of fine granules grouped in clusters. These were often concentrated in dense masses forming thick undulated filaments. In some lysosomes these filaments were abundant and formed parallel strata. Lung Sectionsfrom Rats Exposed to Cerium Chloride Aerosol. In the macrophages of rats exposed to CeCl3, we observed lysosomes containing dense deposits and other lysosomes containing myelinlike configurations. The deposits were localized in the dense lysosomes in the form of aggregates of either fine granules or of fine needles (30 nm in length). In the lysosomes containing myelinlike configurations, deposits were localized at the periphery in the form of granular aggregations. In the lyso-somes, the deposits were primarily in the form of fine needles. The Ka line of phosphorus and the La line of cerium were detected on all these deposits by microanalysis (Fig. 2).

Discussion
The role oflysosomes inthemechanismofconcentrationofcertain mineral elements has been studied previously in renal cells (4)(5)(6). WhentheelementsAl, U, Cr, and Ce are injectedin soluble form, they are concentrated inthe lysosomes ofrenal proximal tubule cells as insoluble phosphates. The intralysosomal concentration ofthese elements inphosphate form suggestsamechanism dependentontheenzymatic potential ofthe lysosome. It is known that this organelle contains numerous enzymes including acid phosphatases. These enzymes release free phosphate ions from different substrates. Thephosphate ions thus releasedcan precipitate elements such as Al, U, Cr, or Ce in an acid medium. This in vivo precipitation depends on a mechanism identical to that observed in vitro in the well-known cytochemical reaction of Gomori. The Gomori reaction is a chemical method that allows themicroscopic visalization inatissue sectionoforganelles with high phosphatase activity. After incubation of a tissue section with a solution containing a soluble lead salt, the lead is precipitated as an insoluble lead phosphate salt in organelles containing acid phosphatase, namely, in lysosomes. Other elements such as Al or Ce can also be precipitated as insoluble phosphate salts in lysosomes by the same cytochemical method. _l--_|_!|ll _ .~~~~~.-" M FIUR 2I Itayooadesdpsis in a ra marphg afe inaato ofacru hoiearsl h yooe oti ml n esrnltos Elctonprb X-a anayi show tha ths grnlain cotIn choimadpopou ,aihcnetain The similarity of the mechanism of in vivo selective concentration of mineral elements as insoluble phosphate salts in lysosomes to the in vitro Gomori reaction was first demonstrated for Al by showing that the deposits of AlPO4 observed in renal lysosomes after in vivo injection of soluble sats (7) are identical in their ultrastructure and chemical composition to those deposits obtained with in vitro Gomori cytochemical reaction using soluble aluminum salts instead oflead salts. In another study (6), the classical Gomori reaction was used to show that after Cr poisoning, the renal lysosomes that concentrated this element in phosphate form had a strong acid phosphatase activity. Moreover, the Cr injected in vio and the lead used in the Gomori reaction were found within the same lysosomes. Similar results were obtained in renal lysosomes after injection of soluble uranium and cerium salts.
In these experiments, quantitative studies using microanalysis have shown that for the elements Al, Ce, and Cr, the Al/P, Ce/P, and Cr/P atomic concentration ratios are close to 1 in the intralysosomal precipitates, suggesting that these are insoluble orthophosphates with formulas AIP04, CePO4, and CrPO4, respectively.
In the present study, the concentration phenomena observed in pulmonary cell lysosomes ofthe macrophages are identical to those we have observed previously in renal lysosomes. Reference deposits have the same ultrastructure and the same chemical composition with the same concentration ratios. These observations strongly suggest that the local precipitation mechanism in pulmonary cells is identical to that demonstrated for renal cells.