In vitro the expression of a sialic acid-binding receptor on murine macrophages, sialoadhesin, is regulated by exposure to an inducing agent present in serum. We have used immunocytochemistry to examine the macrophage populations of the nervous system in order to test whether this serum inducing agent (SIA) also regulates sialoadhesin expression in vivo and whether plasma proteins may influence the phenotype of macrophages of the nervous system. Microglia, the resident macrophages of the central nervous system, reside behind the blood-brain barrier and do not express sialoadhesin. Microglia and macrophage populations in the cicumven-tricular organs, choroid plexus and leptomeninges are exposed to plasma proteins and some macrophages express sialoadhesin at these sites. Injury to the CNS, which damages the blood-brain barrier, induces sialoadhesin expression on a proportion of macrophages and microglia within the parenchyma. The expression of sialoadhesin matches the temporal and spatial distribution of the plasma extravasation into the brain parenchyma.

These experiments show that exposure to SIA is necessary for sialoadhesin expression and lend further support to the idea that the phenotype of microglia is in part regulated by the presence of the blood-brain barrier.

The extracellular fluid around the cellular elements of the central nervous system (CNS) has a different composition from plasma. The difference in the composition of the brain fluids comes about by virtue of the blood-brain barrier, which prevents unrestricted transfer of plasma constituents across the CNS endothelium to the extracellular space and lies at the tight junctions between the CNS endothelium (Reese and Kamovsky, 1967). A major difference between the cerebrospinal fluid (CSF), which is in equilibrium with the fluids of the extracellular space, and plasma is that the CSF concentration of protein is about 200-fold less than that in plasma (Davson et al. 1987). This raises the question as to whether exposure to some plasma proteins might modulate properties of neuronal or nonneuronal components of the CNS in homeostasis, or following injuries that result in increased permeability of the blood-brain barrier.

An observation suggesting that plasma proteins might play a role in the modulation of macroglial cell development comes from studies on macroglial cell lineage in vitro (Raff et al. 1983). The authors showed that differentiation of the 0-2A progenitor cell, which gives rise to both oligodendrocytes and type 2 astrocytes, was influenced by the presence of serum. When 10% serum was present, mostly astrocytes were generated, while at very low serum concentrations the generation of oligodendrocytes predominated. Recent evidence showing that myelin is lacking from sites of the CNS which lack the blood-brain barrier suggests that differentiation of the O-2A cell may also be influenced in vivo (Perry and Lund, 1989). It has been shown that some populations of developing neurons have detectable levels of plasma proteins intracellularly, although the significance of this is not clear (Dziegielewska et al. 1981).

In the course of our studies on CNS macrophages we found that the expression of CD4 on rat CNS macrophages is in part modulated by exposure to plasma (Perry and Gordon, 1987). Microglia, the resident macrophages of the CNS (Perry and Gordon, 1988, 1991; Streit et al. 1988), are not normally exposed to plasma proteins and have barely detectable or undetectable levels of CD4, while the microglia of the circumventricular organs (CVOs) exposed to plasma proteins had readily detectable levels, as did the macrophages in the choroid plexus and the leptomen-inges (Perry and Gordon, 1987). Following injury to the CNS and damage to the blood-brain barrier, cells with the morphology of microglia were found to express CD4. These results suggest that some component of plasma may play a role in the regulation of the expression of CD4 on rat macrophages but the nature of any possible mediator is unknown.

Studies from this laboratory on resident bone marrow macrophages have described a plasma membrane lectin-like receptor with a specificity for sialylated components on erythrocytes (formerly designated SER, now sialoadhesin) and this receptor appears to be involved in interactions of haemopoietic cells with bone marrow and spleen stromal macrophages in situ (Crocker and Gordon, 1986; Crocker et al. 1988b). Further work has shown that the expression of this macrophage-specific receptor can be regulated in vitro by a species-restricted inducing activity (SIA) in serum (Crocker et al. 1988a). Monocytes or peritoneal macrophages that express low or undetectable levels of sialoadhesin can be induced to express high levels of receptor activity by incubation in plasma or serum for 35 days. Preliminary characterization suggests that this inducing activity is a constitutively expressed plasma protein, has an apparent molecular weight of about 65 kDa and is distinct from a variety of cytokines known to modulate macrophage function (Crocker et al. 1988a).

We were interested to learn whether we could use the expression of sialoadhesin as a probe for the role of plasma proteins in modulating the phenotype of macrophages associated with the CNS. We describe here the regulation of expression of sialoadhesin on macrophages in the nervous system, in normal mice and following local trauma to the CNS, which increases the permeability of the blood-brain barrier.

Mice of the Balb/c strain bred in the Sir William Dunn School of Pathology (Oxford) were used. For surgery, mice were anaesthetized with intraperitoneal (0.1 ml/5 g body weight) Avertin. Prior to perfusion mice were anaesthetized with a lethal dose of Avertin.

Surgical procedures

Mice were placed in a small-animal stereotaxic instrument and a small hole was made in the skull over the dorsal cerebrum with a dental drill. A small aspiration lesion was made in the cortical grey matter about 1-2 mm in diameter. A piece of gel-foam was placed in the lesion and the skin was sutured with fine silk. At least two animals were used for immunocytochemistry at 1, 3, 7, 10, 14 and 21 days after the operation.

Each of two animals at 3, 7, 14 and 21 days after the lesion was given an intravenous injection (i.v.) of 2 mg of horseradish peroxidase (HRP; Boehringer) in 0.25 ml 0.9% saline 10 min prior to perfusion.

Immunocytochemistry

After the appropriate survival time the animal was anaesthetized, exsanguinated with 0.9% physiological saline and perfused transcardially with periodate-lysine-paraformal-dehyde with a final concentration of 2% paraformaldehyde (McLean and Nakane, 1974). The brains were blocked, postfixed for 4–6 h at 4°C and then infiltrated with 30% sucrose in phosphate-buffered saline. The blocks were embedded in O.T.C. compound (Lamb) and rapidly frozen. Sections were cut at 10;un, picked up on gelatinized slides and stored at —30°C. Selected regions of the CNS from several normal adult, three-day-old, and ten-day-old mice were also prepared.

To detect the entire CNS macrophage population, including microglia, we used a monospecific rabbit antiserum directed against the mouse macrophage specific antigen F4/80 (Austyn and Gordon, 1981; Lawson et al. 1990). To detect sialoadhesin we used either a rat monoclonal antibody, SER-4 (Crocker et al. 1988b), or a rabbit antiserum prepared against purified receptor, details of which will be reported elsewhere. The distribution of staining with the monoclonal antibody and the antiserum did not differ significantly in mice except that the latter produced a greater degree of sensitivity allowing better morphological preservation.

The sections were incubated with the primary antibodies and binding was detected using the avidin-biotin complex immunoperoxidase method, with reagents purchased from Vector Laboratories. The horseradish peroxidase was visualized using diaminobenzidine as the chromogen. Control sections were incubated with omission of the primary antibodies or preimmune rabbit serum.

Histochemistry

After the appropriate survival time mice with cortical lesions that had been injected intravenously with horseradish peroxidase were anaesthetized, exsanguinated with 0.9% physiological saline and perfused transcardially with 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M phoshate buffer. The brain was blocked, infiltrated with 30% sucrose overnight and frozen sections were cut at 50 pm. The sections were reacted with a modified Hanker-Yates method to reveal the distribution of HRP (Perry and Linden, 1982).

Distribution of sialoadhesin in normal CNS

The major macrophage population in the CNS is the microglia (Perry et al. 1985). These highly ramified cells are ubiquitous, although they vary in density and morphology in different brain regions (Lawson et al. 1990). The F4/80 antiserum reveals the plasma membrane, cell bodies and a network of their processes; a typical field of the cortex is shown (Fig. 1A). In the choroid plexus and leptomeninges the F4/80 positive (F4/80+) cells have a typically stellate morphology (Fig. 1C) and resemble many other tissue macrophages outside the CNS such as Kupffer cells of the liver. In the circumventricular organs such as the subfornical organ and pituitary the microglia have a more compact morphology and are stained with F4/80 (Fig. 1E).

Fig. 1.

(A) F4/80+ microglia in the cortex of adult mouse. (B) Adult mouse cortex stained with an antiserum to sialoadhesin. Note there are no microglia stained. (C) Macrophages in the choroid plexus stained with F4/80 (arrows). (D) A few choroid plexus macrophages are stained by the antiserum to sialoadhesin (arrows). (E) Microglia and their processes in the subfornical organ are F4/80+ (arrows) and (F) express sialoadhesin (arrows). Scale bars, 50 μm.

Fig. 1.

(A) F4/80+ microglia in the cortex of adult mouse. (B) Adult mouse cortex stained with an antiserum to sialoadhesin. Note there are no microglia stained. (C) Macrophages in the choroid plexus stained with F4/80 (arrows). (D) A few choroid plexus macrophages are stained by the antiserum to sialoadhesin (arrows). (E) Microglia and their processes in the subfornical organ are F4/80+ (arrows) and (F) express sialoadhesin (arrows). Scale bars, 50 μm.

We have not seen sialoadhesin expression on microglia in the parenchyma of normal CNS where the bloodbrain barrier is intact (Fig. IB). The absence of staining on microglia is not a result of poor detection since we find staining for sialoadhesin on cells in the leptomeninges, choroid plexus and CVOs (Fig. 1D,F). The number of positively stained cells in these structures was not as great as the number of F4/80+ cells in adjacent sections. In order to give an estimate of the proportion of F4/80+ cells that were expressing siaload-hesin, we counted the numbers of F4/80+ and siaload-hesin-stained cells in the different structures in adjacent sections. The structure with the greatest proportion of sialoadhesin stained cells was the subfornical organ where as many as 70% of the cells may express this receptor. In the choroid plexus and posterior pituitary we estimate that about 20% and 40% respectively express sialoadhesin. Other sites in the CNS known to be permeable to plasma proteins, the median eminence and the olfactory fibre layer, have very few cells stained for sialoadhesin, although both sites contain many F4/80+ cells. Therefore the expression of sialoadhesin can be detected in sites normally exposed to plasma and thus to SIA, although the level of expression varies.

In the newborn mouse, macrophages associated with the leptomeninges and choroid plexus expressed sialoadhesin but at low levels. At none of the ages examined were macrophages and immature microglia within the parenchyma stained by sialoadhesin antiserum. The blood-brain barrier is intact by the time of birth (Risau et al. 1986).

Distribution of sialoadhesin in PNS

We examined one peripheral ganglion, the trigeminal ganglion, of the normal mouse to see whether resident macrophages of the peripheral nervous system respond to exposure to plasma proteins. The ganglion lacks a blood-brain barrier. The macrophages of the trigeminal ganglion are numerous and surround the cell bodies of the neurons; cells with a similar morphology and density are stained by antisera to F4/80 and sialoadhesin (Fig. 2A,B). Macrophages lying among the peripheral nerve fibres have a stellate morphology and many of these cells express sialoadhesin.

Fig. 2.

Macrophages (arrows) in the trigeminal ganglion are labelled by F4/8O (A) and express sialoadhesin (B). Bar, 50 μm.

Fig. 2.

Macrophages (arrows) in the trigeminal ganglion are labelled by F4/8O (A) and express sialoadhesin (B). Bar, 50 μm.

Injury induced sialoadhesin expression

A traumatic injury to the cortex results in destruction of tissue and the breakdown of the blood-brain barrier. Intravenous injection of HRP, followed by perfusion about 10 min later, 3 days after the cortical injury revealed that a small region of cortex up to 500 μm from the lesion boundaries was now exposed to plasma proteins (Fig. 3A). At the borders of the lesion many rounded cells or cells with short processes were loaded with HRP. At the limit of the spread of HRP into the parenchyma a few faintly stained neurons were visible and some resident microglia that had endocytosed the HRP were labelled (Fig. 3B). A similar picture was seen at seven days after the lesion although there were labelled cells with more ramified processes at the borders of the lesion, By 14 days after the injury the region over which the HRP had spread was more restricted and following injections 21 days after injury the HRP was excluded from the parenchyma.

Fig. 3.

(A) A small lesion in the cerebral cortex is filled with gel-foam (star). Following intravenous injection of HRP, 3 days after injury, the HRP has entered the surrounding tissue. (B) Macrophages/microglia in the tissue surrounding the lesion have endocytosed the HRP. Macrophages/microglia in the parenchyma around the lesion border express F4/80 (C and E) and sialoadhesin (D and F). Bars, A, 100 μm; B-F, 50 μm.

Fig. 3.

(A) A small lesion in the cerebral cortex is filled with gel-foam (star). Following intravenous injection of HRP, 3 days after injury, the HRP has entered the surrounding tissue. (B) Macrophages/microglia in the tissue surrounding the lesion have endocytosed the HRP. Macrophages/microglia in the parenchyma around the lesion border express F4/80 (C and E) and sialoadhesin (D and F). Bars, A, 100 μm; B-F, 50 μm.

One day after injury F4/80+ cells adjacent to the lesion appeared as distorted microglia and there were a few large round macrophages. There were no cells expressing sialoadhesin. Three days after injury the number of F4/80+ cells had dramatically increased up to a distance of 500 μm from the borders of the lesion. At the edge of the cavity there were many cells with large cell bodies, about 15 gm in diameter, and the majority of these cells had short processes emanating from them. Outside this region the F4/80+ cells were activated microglia with larger cell bodies and a greater number of processes than are seen on microglia in the normal brain. The expression of sialoadhesin was much more restricted and only a few cells close to the lesion border were stained; the majority were simple rounded cells but some had the morphology of activated microglia.

Seven days after injury there was a further increase in the density of F4/80+ cells around the lesion borders and these cells were rounded cells or had the appearance of activated microglia (Fig. 3C,E). There were now more cells stained by the sialoadhesin antiserum and these had a wide range of morphologies, but notably some of them were like activated microglia revealed by F4/80 (Fig. 3D,F). By ten days post injury the processes of F4/80+ cells formed a very dense network adjacent to the lesion but cells and processes expressing sialoadhesin did not reach this density.

To estimate the relative proportions of F4/80+ and sialoadhesin-stained cells around the lesion we counted the numbers of cells stained by the two different antisera in a strip of tissue, 180 (tm wide extending 450 μm from the lesion border, at about the middle of the cortical grey matter. Counts were made in each of two animals at 7 and 10 days post injury. The cells labelled for F4/80 or sialoadhesin were counted in adjacent sections. The proportion of sialoadhesin-stained cells ranged from 45 to 65% of the density of F4/80+ cells.

Sialoadhesin-expressing cells were still present 14 days after injury and the majority of the cells now had the form of resident microglia with a small number of fine long processes. These cells all lay within about 500 μm of the edge of the lesion. By 21 days after injury the only sialoadhesin-stained cells lay within the gel-foam at the lesion site; an occasional cell lay at the very edge of the lesion site.

A cortical aspiration lesion produces not only degenerating tissue at the borders of the lesion but also degeneration of processes at a distance from the lesion. For example, there will be degenerating processes in the cortical white matter, the corpus callosum and in the contralateral hemisphere. There were no cells revealed by the sialoadhesin antiserum in the callosum or the contralateral cortex, suggesting that degeneration products alone are not sufficient to induce sialoadhesin expression on the microglia at these locations.

The results are consistent with the hypothesis that expression of the macrophage-specific antigen sialoadhesin is regulated by exposure to plasma proteins. Where the blood-brain barrier is intact none of the macrophage-derived population, immature microglia or adult microglia express sialoadhesin, but in regions normally exposed to plasma proteins or following damage to the blood-brain barrier variable numbers of macrophages and microglia do so. The presence of degenerating neuronal processes was not of itself sufficient to produce expression of this antigen.

Previous in vitro experiments on the expression of sialoadhesin have shown that plasma contains a species-restricted inducing agent that is necessary for induction and maintenance of expression (Crocker et al. 1988a,b). The results presented here for macrophage populations associated with the nervous system confirm that in vivo a necessary component of sialoadhesin expression is the exposure to plasma. It is important to note that even among resident populations of cells exposed to plasma, and hence the inducing agent SIA, there is heterogeneity in the expression of the antigen. Not all the macrophages in the choroid plexus and circumventricular organs express sialoadhesin, and in other tissues such as liver the expression on Kupffer cells, the resident macrophages, can be relatively low despite their exposure to plasma.

The absence of sialoadhesin expression on microglia in the parenchyma of the CNS is consistent with results showing that SIA has an apparent molecular weight of 65 000 or more, which would not allow this agent to cross the blood-brain barrier (Crocker et al. 1988a). Following injury and breakdown of the blood-brain barrier a proportion of microglia are found to express sialoadhesin and although the origin of these cells, whether activated resident microglia or recruited cells, is not known, the important point is that sialoadhesin expression is compatible with microglia morphology but only when exposed to the inducing agent. The results of these experiments show that isolation of microglia from plasma component(s) modulates their phenotype and supports our previous experiments on the expression of CD4 on rat macrophages and microglia (Perry and Gordon, 1987).

Although possible functions of sialoadhesin in the CNS are not known, this macrophage-specific receptor has recently been shown to mediate sialic aciddependent attachment to certain gangliosides incorporated into erythrocyte plasma membranes (Crocker et al. 1991). In neural tissues, gangliosides comprise up to 10% of the total plasma membrane lipid and it has been suggested that they play important roles in neuronal functions such as modulation of synaptic transmission, ion permeability and fluxes, genesis of neurites and the development of myelin (Wiegandt, 1985; Chan, 1987; Facci et al. 1988). It could be argued, therefore, that the down-regulation of sialoadhesin in the CNS is important in preventing undesired interactions between the microglial plasma membrane and gangliosides expressed on neurons and macroglia.

At the present time it is not known how general this regulation by plasma proteins may be. The possibility that other aspects of macrophage function such as receptor expression, phagocytic and secretory activity may all partly be regulated by plasma proteins serves to highlight the potential importance of this component of the tissue macrophage microenvironment.

This work was supported by the Medical Research Council and The Wellcome Trust. VHP is a Wellcome Senior Research fellow, PRC was a Beit Memorial Fellow and Junior Research Fellow of Wolfson College, Oxford. We thank Mrs S. Cahusac for her assistance with the histology.

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