Marion Edmonds Smith

Macrophage and microglial responses to cytokines in vitro: Phagocytic activity, proteolytic enzyme release, and free radical production

Certain cytokines are believed to play a key role in the development of autoimmune demyelinating diseases. Little is known, however, about the effects of these cytokines in the regulation of the key event in myelin destruction, the phagocytosis of myelin by phagocytic cells. We investigated the effects of certain cytokines and growth factors on cultured peritoneal macrophages and microglia in respect to their various functions, phagocytosis, secreted proteolytic activity, and oxidative activity. Interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α), and lipopolysaccharide (LPS), all proinflammatory factors, actually decreased (IFN‐γ and LPS), or had no effect (TNF‐α) on myelin phagocytosis by macrophages, but substantially increased phagocytic activity by microglia. Surprisingly, interleukins 4 and 10 (IL‐4 and IL‐10), considered to be downregulating cytokines, increased phagocytic activity by macrophages, while with microglia, IL‐4 had no effect, but IL‐10 almost doubled myelin phagocytosis. Transforming growth factor‐β (TGF‐β) had no significant effect on either cell. These cytokines did not affect proteolytic secretion in microglia, while IFN‐γ and LPS induced a doubling of the secreted proteases. This proteolytic activity was almost completely suppressed by calpain inhibitors, although some gelatinase appeared to be present. Microglia exerted much more oxidative activity on the membranes than macrophages, and granulocyte‐macrophage colony stimulating factor (GM‐CSF) and interleukin 1β (IL‐1β) significantly increased microglial oxidative activity. The pattern of responses of macrophages and microglia to the cytokine types indicate that in cytokine‐driven autoimmune demyelinating disease, microglia may be the more aggressive cell in causing tissue injury by phagocytosis and oxidative injury, while infiltrating macrophages may produce most of the proteolytic activity thought to contribute to myelin destruction. J. Neurosci. Res. 54:68–78, 1998.

The turnover of myelin in the adult rat

Abstract The rate of loss of radioactivity of lipids of brain subcellular fractions was followed for 56 days after injection of adult rats with uniformly labeled [ 14 C]glucose. Myelin did not reach its maximum specific activity until 5 days after injection, then showed a slow rate of loss of radioactivity. When the turnover of the lipid components of purified myelin was measured, inositol phosphatide and lecithin were found to have a more active metabolism than the other myelin lipids which include serine phosphatide, sphingomyelin, ethanolamine phosphatide, cerebroside, cerebroside sulfate, and cholesterol. These results which agree with our previous findings indicate that the metabolism of myelin on the outer layers is identical to that laid down early in the life of the animal. Proteolipid protein showed a half-life of about 35 days, thus was one of the more active myelin components. The pattern of metabolism, though on a longer time scale in myelin, is similar to that of the most active brain membrane fraction. These results are discussed in relation to the possible structure of myelin membranes.

Immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis

Spinal cord sections from Lewis rats with acute experimental allergic encephalomyelitis (EAE) showed greatly increased staining of astrocytes when stained immunocytochemically for glial fibrillary acidic protein (GFAP). Fibrous processes in white matter were heavily stained early in the course of the disease when paralysis was first evident (10-12 days after injection of guinea pig spinal cord myelin), then protoplasmic astrocytes were stained in the gray matter and became more heavily stained at 20 days post-injection. The stained astrocytes were evenly distributed throughout the tissue, and did not correspond to the sites of the lesions. Spinal cord slices of control and EAE rats were incubated with [3H]amino acids, then cytoskeletal proteins were prepared in an enriched fraction, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands counted for radioactivity. In the EAE rat all cytoskeletal proteins, including the neurofilaments, vimentin, microtubules, GFAP and actin, showed increased uptake of radioactive amino acids. Immunoprecipitation of GFAP with specific antiserum showed increased radioactivity in the complex beginning at day 10 when cellular infiltration was beginning in the EAE animals. As the disease became acute, the radioactivity in the immunoprecipitated GFAP increased, in some cases to very high levels, then by day 18 when recovery was underway, the radioactivity had fallen to normal levels. Possible agents causing metabolic activation of protein synthesis in EAE animals include stimulating substances elaborated by infiltrating lymphoid cells, and the generalized edema accompanying the demyelinative condition. The activation of GFAP protein staining and metabolism in EAE might serve as a model for the activated growth of astrocyte processes which cause the severe gliosis seen in multiple sclerosis.

Metabolism of Myelin

By “metabolism of myelin,” we refer to the molecular events involved in the synthesis of myelin components, and the subsequent assembly, maintenance, and turnover of the myelin sheath. The deposition of myelin involves coordination of the synthesis of its various lipid and protein components, and the interaction of these components to give a stable membrane. Degradation of myelin components occurs as a reaction of the myelin sheath to injury, but may also be required for normal maintenance and remodeling of the membrane. The topographic features of the myelin sheath can be expected to limit partially the metabolism of myelin. The roles of membrane fluidity and cytoplasmic inclusions in the turnover of compact myelin lamellae are not well understood, but obviously metabolism in this system is integrally linked to both anatomical and membrane structure.

AN IN VITRO SYSTEM FOR THE STUDY OF MYELIN SYNTHESIS

Abstract— A system for the study of short‐term myelin synthesis in spinal cord tissue in vitro with [U‐14C]glucose as a lipid and protein precursor is described. The rates of lipid and protein incorporation into myelin are age‐dependent, but do not appear to behave similarly. Uptake of labelled carbon is most rapid in monophosphoinositide and lecithin, and slowest in cerebroside sulphate. Myelin protein seems to retain more metabolic activity than does the lipid. These findings are interpreted as support for the unit membrane hypothesis for myelin rather than the subunit model.

The metabolism of myelin lipids.

Publisher Summary This chapter discusses the metabolism of myelin lipids. The three myelin lipid constituents, namely, inositol phosphatide, lecithin, and serine phosphatide, have certain metabolic characteristics that differentiate them from another group of myelin lipids. The pattern of rates of turnover of lipids of myelin and other brain membrane fractions is similar; nevertheless, all myelin constituents have a relatively slow rate of metabolism compared with other brain fractions. Whether the differential behavior of the lipids is the result of enzymatic activity or a simple exchange is unknown. The rate of turnover may be related to the length of the fatty acid chains and also to interactions with other lipids. Calculations of the lipid compositions of myelin show the molar ratio of cholesterol to the stable lipids to be approximately one, which may be evidence for the postulated cholesterol–galactolipid or cholesterol–phospholipid complex. A more detailed knowledge of the behavior of the lipids related to data on the turnover of the several myelin proteins may provide insight into the molecular arrangement of myelin.

Experimental allergic neuritis in the Lewis rat.

Purified myelin from the peripheral nervous system of guinea pig, frog (Rana catesbeiana), rat, rabbit, beef, and human in Freunds adjuvant were injected into the Lewis rat. Groups of rats receiving injections of myelin from different species were examined for signs of dysfunction and lesions in the PNS and CNS. Injection of frog PNS myelin into the Lewis rat did not produce any clinical signs or lesions typical of experimental allergic neuritis (EAN) or experimental allergic encephalomyelitis (EAE). Injection of myelin from the PNS of rat, rabbit, beef, and human elicited clinical signs and lesions characteristic of EAN, while guinea pig myelin injection caused superimposed conditions of EAE and EAN. The myelin proteins from the various species were separated by polyacrylamide gel electrophoresis, the gels were scanned and the individual proteins measured. There did not appear to be a correlation between the amount of P2 protein contained in the different myelin species and the severity of the EAN symptoms and lesions produced. Although the Lewis rat is far more susceptible to EAE caused by guinea pig CNS myelin than by any other species, EAN can be easily induced in this animal by injection of PNS myelin from a number of species.

STUDIES ON THE MECHANISM OF DEMYELINATION: TRIETHYL TIN‐INDUCED DEMYELINATION

The metabolism of myelin undergoing breakdown as a result of edema induced by chronic administration of triethyl tin (TET) dissolved in the drinking water (10 mg/l.) was examined. The spinal cord showed more edema and loss of myelin than the brain. Uptake in vitro of [1‐14C]acetate into myelin lipids of slices of brain or spinal cord from TET‐treated rats was depressed until 4–5 weeks after the beginning of the regime, then rose to above normal levels. The uptake of [l‐14C]leucine into myelin protein rose within several weeks of TET treatment to levels averaging over 300 per cent of normal and remained high even after the TET was removed. The high levels of [l‐14C]leucine incorporation were inhibited by cycloheximide and were not explained by an increase in the size of the free amino acid pool. The three classes of myelin proteins, basic, proteolipid protein, and Wolfgram protein shared in the increased incorporation. Spinal cord myelin showed the greatest metabolic response, brain stem myelin less, and myelin from the forebrain was minimally affected by the TET treatment. Myelin prelabelled by intracisternal injection of [l‐14C]acetate and [l‐14C]leucine before the onset of TET administration showed faster turnover in myelin proteins in relation to the myelin lipids than the control in the most severely affected animals, but not in others less affected. A ‘floating fraction’ was observed floating on 10.5% (w/v) sucrose during the myelin purification. This fraction showed metabolic characteristics typical of myelin, and myelin‐labelling studies at various stages of the animals development showed it to be derived from recently synthesized myelin. The floating fraction from the brain contained less cerebroside and more lecithin than myelin, while the spinal cord floating fraction composition was much like that of myelin. The floating fractions contained less protein typical of myelin (basic and proteolipid protein) and more highmolecular‐weight protein which may have been derived from contaminating microsomes. The floating fraction was presumed to be partially deproteinated myelin. The use of TET‐treatment as model for demyelination as a result of edema and proceeding in the absence of macrophages is discussed.

Opsonization with Antimyelin Antibody Increases the Uptake and Intracellular Metabolism of Myelin in Inflammatory Macrophages

Abstract: In most demyelinating diseases, macrophages are believed to be active agents of myelin destruction. In experimental encephalomyelitis, these cells appear to strip off and ingest the myelin lamellae, and myelin debris has been observed within the cell body. We show here in vitro conditions in which rat peritoneal macrophages phagocytose and metabolize CNS myelin lipids. Purified rat myelin, prelabeled in vivo with [14C]acetate, was incubated with preimmune serum or rabbit antiserum to rat CNS myelin and added to macrophage monolayers. Myelin opsonized with antimyelin antibodies was more readily phagocytosed and metabolized by cultured macrophages than untreated myelin or that preincubated with preimmune serum. In the presence of macrophages, levels of myelin polar lipids and cholesterol decreased, whereas radioactive cholesterol ester and triglyceride accumulated. Up to five times as much radioactive cholesterol ester and about twice as much triglyceride accumulated in macrophage cultures containing antibody‐treated myelin as in cultures fed preimmune serum‐treated myelin or in those incubated with untreated myelin. Both the fatty acid and the cholesterol from cholesterol ester contained radioactive label: therefore, both were derived at least partly from the radioactive myelin lipid. Antiserum to myelin purified from peripheral nerve was almost as effective as that to CNS myelin in stimulating cholesterol metabolism, whereas antiserum to galactocerebroside was about 70% as active. Antiserum to basic protein had less effect, whereas antiserum to the myelin‐associated glycoprotein and proteolipid protein was inactive. Of the polar lipids, ethanolamine phosphatide was most degraded in both the antiserum‐ and preimmune serum‐treated myelin, with the diacyl form and plasmalogen form degraded about equally. These experiments indicate that myelin‐specific antibodies in inflammatory CNS lesions may participate in and stimulate macrophage‐mediated demyelination.

Phagocytosis of Myelin in Demyelinative Disease: A Review

In the cell-mediated demyelinating diseases such as experimental allergic encephalomyelitis and multiple sclerosis, as well as their peripheral nerve counterparts, the phagocytic cells are the agent of myelin destruction. Both resident microglia and peripheral macrophages invading the nervous system have been shown to phagocytize myelin, although microglia appear to be more active, especially at early stages of disease. Several different receptors on these cells have been implicated as myelin receptors, with the Fc- and complement receptors receiving the most attention. Other receptors, especially the macrophage scavenger receptor with its broad specificity deserves further exploration, especially in view of its affinity for phosphatidylserine, which becomes externalized with membrane disruption. Evidence is shown for cytokine regulation of phagocytic activity in both macrophages and microglia. Further investigation of the pathways of cytokine action on myelin phagocytosis through signal transduction molecules will be important for a further understanding of the events leading to myelin destruction in demyelinating diseases.