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β-interferon regulates the immunomodulatory activity of neonatal rodent microglia
Journal of Neuroimmunology 72 Ž1997. 11–19
b-interferon regulates the immunomodulatory activity of neonatal rodent microglia G.L. Hall a
a,b,)
, M.G. Wing c , D.A.S. Compston
a,b
, N.J. Scolding
a,b
UniÕersity of Cambridge, Department of Neurology, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK b MRC Cambridge Centre for Brain Repair, UniÕersity ForÕie site, Robinson Way, Cambridge CB2 2PY, UK c Molecular Immunopathology Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, UK Received 24 January 1996; revised 14 May 1996; accepted 19 June 1996
Abstract b-interferon Ž b-IFN. has both pro and anti-inflammatory properties, the balance of which leads to some suppression of disease activity in multiple sclerosis patients. Here, we examine the immunomodulation of neonatal rodent microglia, the principal CNS accessory cell, by b-IFN and consider the interaction of b-IFN and g-interferon Žg-IFN.. b-IFN and g-IFN inhibit microglial proliferation. b-IFN antagonises both g-IFN-induced upregulation of class II expression and the ability of g-IFN primed cells to mount a respiratory burst. In contrast, b-IFN upregulates microglial Fc receptor expression and augments tumour necrosis factor alpha secretion from suboptimally stimulated microglia. Keywords: Multiple sclerosis; Microglia; Beta interferon; Rodent
1. Introduction Multiple sclerosis ŽMS. is the commonest cause of acquired neurological disability in young adults. The precise mechanisms of immunologically mediated tissue damage are not fully understood, but there is a consensus that activated T lymphocytes cross the blood–brain barrier and initiate an inflammatory response within the central nervous system ŽCNS.. Soluble factors are important in myelin and oligodendrocyte injury w1x and infiltrating macrophages and activated resident microglia subsequently phagocytose myelin sheaths. Non-specific immunosuppressive and cytotoxic treatment has been relatively unsuccessful in the treatment of MS, but more recently, b-IFN has been assessed as a potential treatment. There is accumulating evidence that it may suppress some aspects of disease activity w1,2x and alter the natural history of MS. However, the exact mechanism of action of b-IFN in patients with MS has not yet been elucidated. Although first described on the basis of its antiviral properties, b-IFN has now been documented to have im-
) Corresponding author. Tel.: q44 Ž1223. 217842; fax: q44 Ž1223. 336941; e-mail: [email protected]
munomodulatory functions on a wide range of target cells. b-IFN has a direct antiproliferative action on some cells including macrophages and counteracts the mitogenic stimulus of certain cytokines w3,4x. It acts on large granular lymphocytes to increase killing, augments natural killer cell activity w5x and leads to increased generation of cytotoxic T lymphocytes w6x. B cell proliferation is augmented as is secretion of Ig M, G and A. The effects of b-IFN on accessory cells such as the peripheral macrophage are well characterised. b-IFN prevents proliferation and causes terminal differentiation and activation w7x. The resultant morphological changes include cell enlargement, spreading, pseudopod formation and vacuolation. Adherence is increased, as is the number and density of Fc receptors ŽFcR. w8x leading to increased phagocytosis. Destruction and detoxification of internalised pathogens is maximised by induction of lysosomal enzymes. Class I MHC expression is upregulated leading to increased class I restricted antigen presentation to CD8 cells. By contrast, in certain other accessory cells, namely the murine macrophage and the human astrocyte, not only does b-IFN not induce class II MHC, the expression of which is required for antigenspecific activation of CD4 q T cells via the T cell receptor ŽTCR., but it actually inhibits upregulation of class II surface expresson mediated by g-IFN w9–11x.
0165-5728r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 1 2 8 - 2
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In MS, T cells are thought to be activated within the periphery and enter the CNS where maintenance of activation occurs after encountering autoantigen. Immunoglobulin also contributes to the disease process; B lymphocytes are identified in the perivascular infiltrate w12x and oligoclonal bands in the cerebrospinal fluid and, furthermore, in the experimental allergic encephalomyelitis model, passive transfer of myelin oligodendrocyte glycoprotein-specific antibody results in a more severe disease with pathology resembling the primary demyelination seen in MS w13x. Central FcR expressing cells could directly phagocytose opsonised targets and immune complexes. Specific peptide within the immune complex would be processed and presented in the context of class II MHC, and hence activate CD4-positive T cells. In the CNS, microglia fulfill the criteria of immunologically active accessory cells; on activation in vitro they increase their expression of class II MHC w14,15x, and FcR w15x; they are phagocytic and can process and present antigen in an MHC restricted fashion to both resting and naive T cells w16–18x and can upregulate constitutive expression of the Th1 co-stimulatory molecule B7 w19x. In addition, microglia have important direct cytotoxic effector functions involving adherence to, and phagocytosis of, opsonised targets, the ability to mount a respiratory burst in response to appropriate stimulation w15x and the production of potentially harmful cytokines such as TNFa w20x. We have systematically studied the effects of recombinant rat b-IFN on a comprehensive range of immunological accessory and cytotoxic effector functions using cultured rodent microglia and have examined the interaction of b-IFN with g-IFN, which has been proposed as the main pro-inflammatory cytokine in the pathophysiology of MS. Neonatal rodent microglial morphology, class II MHC expression, proliferation, FcR expression, ability to mount a respiratory burst and TNFa secretion have each been examined. We identified both antagonistic and synergistic effects of these cytokines, re-emphasising the complexity of immune interactions between immunocompetent cells, cytokines and potentially beneficial immunotherapeutic agents.
2. Materials and methods 2.1. Cytokines Rat recombinant g-IFN and b-IFN were obtained from Biosource International ŽCamarillo, CA, USA.. g-IFN was used in all experiments at a concentration of 100 unitsrml. b- IFN was used at 200 unitsrml. 2.2. Tissue culture Microglia were isolated and cultured from 1–2-day-old Sprague– Dawley rats using the method of Giulian and
Baker w21x with modifications as described by Zajicek et al. w20x. This protocol was further modified such that, briefly, mixed brain cultures were shaken on day 8 for 2 h to remove microglia from the adherent cell population. The supernatant containing microglia was centrifuged at 900 rpm for 5 min, the cells were resuspended in Dulbecco’s modified Eagle’s medium ŽDMEM. with 10% foetal calf serum ŽFCS. and plated onto glass cover slips, for immunocytochemistry and respiratory burst experiments, and on to plastic in all other experiments, at the required density. After 30 min, adherent cells were washed twice with Hanks balanced salt solution ŽHBSS. to remove cell debris and any non-adherent cells. Microglia were then cultured at 378C in 5% CO 2 in DMEM with 10% FCS and used as described in the individual experiments. 2.3. Cell proliferation To measure cell proliferation, microglia were plated in triplicate onto flat-bottomed 96-well plates at a density of 1 = 10 5 cells per well. g-IFN, b-IFN, both interferons or neither were added and the microglia cultured for 48 h, after which, 7 m Cirml w3 Hxthymidine was added to each well and the microglia cultured for a further 4 h. The wells were then washed =3 with PBS to remove all unincorporated w3 Hx thymidine. The microglia were detached from the wells using trypsinrEDTA and cells collected using a cell harvester. The counts were read in a liquid scintillation b counter and the mean of triplicate wells was calculated and plotted. 2.4. Immunocytochemistry Class II MHC surface expression was identified by indirect immunocytochemistry using OX 17, a mouse IgG1 monoclonal antibody which recognises a monomorphic determinant on rat RT 1D, the rat homologue of mouse Ia-EŽSerotec., at 1:100 Žapproximately 34 m grml. as the primary antibody and a secondary FITC-labelled goat anti-mouse IgG ŽSigma. also at 1:100. Microglia were fixed with 2% paraformaldehyde subsequent to incubation with the secondary antibody, mounted in Vectashield and viewed under fluorescence microscopy using fluorescein optics. Appropriate controls were performed using an irrelevant mouse monoclonal IgG1 antibody or FITC-labelled goat anti-mouse IgG alone. 2.5. Expression of Fc receptors (FcR) 2.5.1. Preparation of opsonised sheep RBC Quantitative assessment of microglial FcR expression was performed using a rosette method modified from that of Stewart et al. w22x. Sheep red blood cells ŽRBC. in Alsever’s solution ŽTCS Microbiology, Bucks, UK. were washed Ž1,000 rpm. =3 in PBS with 10 mM sodium azide. RBC were then resuspended to a concentration of
G.L. Hall et al.r Journal of Neuroimmunology 72 (1997) 11–19
1 = 10 9 cellsrml. This is equivalent to a 4% solution of sheep RBC. Washed cells were incubated with an equal volume of a mouse IgG2b monoclonal antibody to sheep RBCs ŽSera-Lab. at a concentration of 10 m grml for 30 min at 378C. That concentration of antibody just below the agglutinating titre was determined in preliminary experiments. The opsonised sheep RBCs were washed =3 and stored at 48C for up to 4 days. 2.5.2. Detection of microglial FcR Microglia were plated in 12-well plates at 3.75 = 10 5 per well. After 30 min, cells were washed twice with HBSS and all washes were pooled. Any non-adherent cells were pelleted by centrifugation at 500 =g, resuspended and counted, allowing accurate estimation of the non-adherent cells and adherent cells remaining in the wells. Adherent microglia were cultured for 48 h either in the absence of cytokines or in the presence of b-IFN or g-IFN alone or together in DMEM with 10% FCS, then washed once with PBS prior to addition of the opsonised sRBCs. For this, opsonised sheep RBCs were washed =3 with PBS, resuspended to a concentration of 2.5 = 10 7 cellsrml in PBS with 10 mM sodium azide and 1 ml was added to each well. The plates were then incubated for 1 h on ice, then the wells were washed gently =3 and the sRBCs rosetted around microglia were then flash-lysed with 300 m l distilled water. The lysates were spun to remove any particulate matter and the haemoglobin content was assessed by the optical density reading at 412 nm in a spectrophotometer. A standard curve was constructed of the optical density readings of known numbers of RBCs lysed with 300 m l of distilled water. 2.6. Respiratory burst
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cytokines, with interferons alone or in combination, or with lipopolysaccharide ŽLPS. Ž1 m grml. as described. After 24 h in culture, supernatants were collected and spun at 3,000 rpm to remove non-adherent cells or cell debris. Supernatants were then assayed immediately or stored at y208C. Stored samples were thawed on only one occasion. Microglial production of soluble TNFa was assayed using the TNFa sensitive fibroblast cell line, L929, as previously described w23x. Briefly, 2 = 10 3 L929 fibroblasts in the log phase of their growth cycle were incubated in flat-bottomed 96-well plates in 50 m l of DMEM with 10% FCS for 72 h with 50 m l of microglial culture supernatant prepared as described below. At 72 h, the wells were pulsed with 7 m Cirml w3 Hxthymidine and incubated for a further 4 h. The wells were then washed =3 with PBS to remove all unincorporated w3 Hxthymidine. The fibroblasts were detached from the wells using trypsinrEDTA and cells harvested. Counts were read in a liquid scintillation b counter. A standard curve was constructed using recombinant rat TNFa .
3. Results 3.1. Microglial proliferation To study proliferation, microglia were cultured for 48 h, alone or in the presence of IFNs, and pulsed with w3 Hx thymidine; cells were harvested and radioactivity counted ŽFig. 1.. In the presence of b-IFN, there was approximately a 60% reduction in proliferation, whereas g-IFN alone, or in combination with b-IFN, completely inhibited proliferation.
Microglia were plated onto 19 mm glass cover slips at a density of 2 = 10 5 cells per cover slip, and were cultured for 48 h Žas described above. in the absence of IFNs or in the presence of g-IFN, b-IFN, or both IFNs. b-IFN was added 2 h prior to g-IFN, simultaneous with g-IFN or 2 h subsequent to g-IFN. At 48 h, coverslips were incubated for 45 min in 30% normal rat serum followed by incubation with affinity-purified FŽabX .2 fragment donkey anti-rat IgG ŽH q L, Jackson ImmunoResearch Laboratories, West Grove, PA. at 25 m grml and nitroblue tetrazolium ŽNBT. at 1 mgrml in PBS. Coverslips were then washed, fixed with 2% paraformaldehyde, mounted with Gurr ŽBDH Laboratory Supplies, Poole. and viewed under light microscopy. Respiratory burst products were identified by their ability to reduce NBT to insoluble formazan seen as a purple deposit. 2.7. Assay for tumour necrosis factor alpha (TNFa ) Microglia were cultured in flat-bottomed 96-well plates at a density of 2 = 10 5 microglia per well in the absence of
Fig. 1. Microglial proliferation in the presence of b-IFN and g-IFN. Microglia were cultured for 48 h either in the absence of added cytokines, or with b-IFN or g-IFN alone or together. At 48 h, microglial proliferation was assessed by 3 H incorporation following a 4-h pulse.
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3.2. Class II MHC induction Class II MHC expression was examined by indirect immunocytochemistry. Microglia were incubated for 48 h in the presence of 10% FCS without cytokines or with b-IFN, g-IFN, or both b-IFN and g-IFN ŽFig. 2.. g-IFNinduced marked upregulation of class II MHC expression ŽFig. 2c.. In the presence of b-IFN, microglia took on a stellate appearance ŽFig. 2b. easily distinguishable from the amoeboid forms induced by g-IFN ŽFig. 2c.. b-IFN did not affect basal class II expression ŽFig. 2b., but inhibited g-IFN-induced upregulation of class II ŽFig. 2d.. The granular staining seen in the absence of exogenous cytokines, or in the presence of either b-IFN alone or b-IFN and g-IFN in combination was also seen under rhodamine optics with equivalent intensity and, therefore, represents autofluorescence. 3.3. FcR expression To measure FcR expression, microglia were incubated with sheep RBCs opsonised with a mouse IgG2b monoclonal antibody to sheep RBCs antibody, an isotype which
would be predicted to bind to both low and high affinity microglial FcR w24x. An example of rosettes formed around microglia previously incubated with b-IFN is shown in Fig. 3a. To quantify the amount of rosetting, the sRBCs rosetting the microglia were lysed with water and the haemoglobin content measured using a spectrophotometer. By constructing a standard curve of the haemoglobin content of known numbers of sheep RBC and, given that the number of microglia per well was fixed Žand corrected for the number of non-adherent cells lost in the original washing step as described in Section 2, Materials and Methods., it was possible to plot the mean number of rosetted sheep RBCs per individual microglial cell ŽFig. 3b.. b-IFN induced a two-and-a-half-fold upregulation of microglial FcR expression ŽFig. 3b.. g-IFN upregulated FcR expression to a lesser Žbut nonetheless significant. extent. There appeared to be antagonism between b-IFN and g-IFN such that in the presence of both cytokines, receptor upregulation approximated more closely to that seen with g-IFN than with b-IFN alone. The experiment shown is representative of three experiments that all showed equivalent results.
Fig. 2. Detection of microglial MHC class II expression by immunocytochemistry. Microglia were incubated for 48 h Ža. in the absence of added cytokines, or with Žb. b-IFN, Žc. g-IFN and Žd. b-IFN and g-IFN. Surface class II expression was detected using the OX 17 antibody Žmagnification =400..
G.L. Hall et al.r Journal of Neuroimmunology 72 (1997) 11–19
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Fig. 3. Ža. Detection of microglial FcR expression. An example of rosettes formed by opsonised sheep RBC around microglia previously exposed to b-IFN for 48 h. Sheep RBCs were opsonised by a mouse monoclonal IgG2b antibody, the Fc portion of which is recognised by both low and high affinity Fc receptors Žmagnification =400.. Žb. Detection of microglial FcR expression; estimation of mean number of rosetted sRBC per microglia. With reference to a standard curve of haemoglobin content of lysates of predetermined numbers of sRBC plotted against the number of sRBC, the mean number of rosetted sRBC per microglia was calculated.
3.4. Respiratory burst experiments The effect of interferons on the ability of microglia to mount an oxidative burst, a critical step in microglial cytotoxicity, was studied. Microglia were cultured for 48 h in the absence of cytokines, and with b-IFN, or g-IFN ŽFig. 4a–c.. Microglia were then incubated in 30% normal rat serum followed by a FŽabX .2 fragment donkey anti-rat
IgG to cross-link FcR which should induce a respiratory burst in appropriately primed cells. As shown in Fig. 4c, g-IFN primed microglia to mount a respiratory burst. Some b-IFN primed cells were able to mount a respiratory burst but to a lesser extent seen as small, densely positive cells ŽFig. 4b.. No respiratory burst products were obtained if normal rat serum was omitted. In order to study the interactions of g-IFN and b-IFN
Fig. 4. Ability of b-IFN and g-IFN-primed microglia to mount a respiratory burst. After 48 h in culture in the absence of IFNs or in the presence of b-IFN or g-IFN either alone or in combination, microglia were examined for their ability to mount a respiratory burst following appropriate stimulation Žsee Section 2, Materials and methods.. Respiratory burst products were detected by reduction of NBT to insoluble formazan visualised as a purple deposit. The panels represent Ža. no cytokines, Žb. b-IFN, Žc. g-IFN and Žd–f. b-IFN added Žd. 2 h before Ž t y 2., Že. with Ž t 0 ., or Žf. 2 h after g-IFN Ž t q 2. Žmagnification of all micrographs =400..
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with respect to microglial oxidative burst, b-IFN was added 2 h before, at the same time as, or 2 h after g-IFN ŽFig. 4d–f.. The ability of g-IFN-primed microglia to produce a respiratory burst was significantly reduced in the presence of b-IFN and this phenomenon was dependent on the sequence in which the cytokines were added, being maximal when microglia were preincubated with b-IFN prior to the addition of g-IFN and abolished when b-IFN was added only 2 h after g-IFN ŽFig. 5.. A reciprocal effect was also seen: the small densely positive microglia observed after priming with b-IFN were rarely seen when g-IFN was also present. 3.5. Microglial TNFa secretion Preliminary experiments demonstrated that the antiproliferative effect of b-IFN on many cell types extends to the L929 fibroblast line used to detect TNFa secretion Ždata not shown.. To control for this effect, the inhibition of fibroblast proliferation induced by both b-IFN and g-IFN, alone or in combination, was subtracted from the overall effect of supernatant produced by microglia cultured in their presence. A standard curve was constructed using recombinant rat TNFa and, therefore, by comparison, the amount of TNFa secreted by microglia was calculated. As shown in Fig. 6, IFNs alone did not stimulate TNFa secretion but augmented that induced by submaximal doses of LPS. Both g-IFN and b-IFN increased LPS-induced TNFa secretion, although this effect was greater with g-IFN. No significant antagonism was seen between b-IFN and g-IFN on secretion of TNFa . The experiment shown is representative of a series of experiments that all showed the same trend. Interestingly, microglia in culture for more than 12 h prior
Fig. 5. Ability of b-IFN and g-IFN-primed microglia to mount a respiratory burst. The experiment of Fig. 4 has been quantitatively assessed and is represented graphically. Shown are the mean number of formazan positive cells Ž"S.E.M.. in each treatment category.
Fig. 6. Microglial TNFa secretion. The amount of TNFa secreted was assessed using the L929 fibroblast proliferation assay and by extrapolation from a standard curve plotted using rat recombinant TNFa .
to stimulation could not be induced to secrete TNFa Ždata not shown..
4. Discussion Our study shows that the effects of b-IFN and g-IFN on neonatal rodent microglia are both complex and interactive. The results do not produce a complete explanation for the clinical effects of b-IFN, but highlight the potential benefits and complications of cytokine treatment. Both b-IFN and g-IFN are antiproliferative for microglia alone or in combination and they augment TNFa secretion induced by sub-optimal concentrations of LPS. In contrast, b-IFN antagonises both g-IFN-induced upregulation of class II MHC surface expression and the ability of g-IFNprimed microglia to mount a respiratory burst. Microglial FcR expression is upregulated by b-IFN and to a lesser extent by g-IFN, but in addition, g-IFN antagonised the superior effect of b-IFN. The mechanism of this reciprocal antagonism is unlikely to occur through competition for cellular receptors as b-IFN and g-IFN bind to different ligand-specific receptors. In MS, the balance of pro- and anti-inflammatory properties of exogenously administered b-IFN appears to favour suppression of disease activity w1,2x. On peripheral macrophages, most actions of b-IFN are pro-inflammatory, the main exception being inhibition of g-IFN-induced upregulation of class II MHC expression w9,10x. This would suggest that, in isolation, b-IFN primes peripheral macrophages for direct cytotoxic tasks but specifically impairs initiation and maintenance of a class II restricted CD4 q T cell response. Here we show by immunocytochemistry that this inhibition of class II expression by b-IFN is also true for microglia, the resident CNS cell most capable of antigen presentation.
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b-IFN inhibits the proliferation of microglia as it does peripheral macrophages w3x, which may reflect the suppression of division which often precedes differentiation. One might, therefore, expect b-IFN to enhance cytotoxic properties by increasing FcR expression, respiratory burst and TNFa secretion, as was indeed shown. We have demonstrated that, with respect to FcR expression, microglia respond to b-IFN in a similar way to peripheral macrophages. In our experiments, b-IFN induced an almost three-fold increase in FcR expression which is in keeping with the findings of Vogel et al. Ž1983. who showed that b-IFN induces an equivalent upregulation of FcR expression on murine macrophages w25x. In addition, b-IFN induces upregulation of FcR expression to a level greater than that induced by g-IFN, a phenomenon also previously documented for peripheral macrophages w26x. Our results also show that in some respects there is reciprocal antagonism between b-IFN and g-IFN. In contrast to what we observe with respect to class II expression, g-IFN appears to antagonise b-IFN-induced upregulation so that in the presence of both cytokines, the level of FcR expression detected is less than that induced by b-IFN alone and more closely approximates to the level seen with g-IFN. Conversely, and in keeping with the effect on class II MHC antigen expression, b-IFN inhibits the ability of g-IFN-primed microglia to mount a respiratory burst. This is consistent with the work of Garotta et al. Ž1986., who described antagonism of type II IFN-induced oxygen radical production in human macrophages by type I IFN w27x. The fact that maximal inhibition is demonstrated with pre–exposure to b-IFN, and that this inhibitory effect of b-IFN is negligible if b-IFN is added only 2 h after g-IFN would be in keeping with transcriptional regulation. A precedent for this interpretation would be the finding that b-IFN-mediated inhibition of g-IFN-induced class II upregulation in murine macrophages and a human astrocytoma line occurred at the level of transcription w28,29x. However, despite these latter findings, we have demonstrated that, in neonatal rat microglia, b-IFN-mediated control of class II surface expression is controlled by a post-translational mechanism w30x. Further evidence of antagonism between type I and type II IFNs comes from recent work showing that using human peripheral macrophages, g-IFN antagonises the inhibitory effect of b-IFN on transferrin receptor expression w31x. However, we showed no antagonism between b-IFN and g-IFN on secretion of TNFa . Whilst neither induced TNFa secretion per se, both augmented the production induced by submaximal concentrations of LPS, demonstrating that in this context b-IFN is pro-inflammatory. This is of particular relevance in MS since TNFa has been detected in acute lesions and in vitro may be toxic to oligodendrocytes w32,33x, both of which implicate TNFa in the pathogenesis of MS. The principal pro-inflammatory cytokine in MS is g-
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IFN. It is secreted from activated T cells and is responsible for activation of accessory cells necessary for potentiation of the immune response w34x. Like TNFa , it is also detectable in active MS lesions w35x. A therapeutic trial of g-IFN was terminated early following increased frequency and severity of exacerbations amongst the treated group w36x. It seems reasonable to assume, therefore, that b-IFN is administered to patients with MS on a background of endogenous g-IFN production, and one can speculate that, whilst in the non-diseased state b-IFN might increase the cytotoxic functions of microglia, in active disease, FcR expression, the ability to mount a respiratory burst and class II MHC expression within lesions would each be reduced leading to impaired activation and potentiation of the Th cell arm of the immune response. This provides one hypothesis for how exogenous b-IFN might swing the balance in favour of disease suppression in MS. The excess of exacerbations noted during the first 3 months of b-IFN treatment w37x, may be explained by an initial increase in cytotoxicity mediated by elevated FcR expression and TNFa secretion prior to the balance being swung in favour of down-regulation of the Th cell mediated arm of the immune system. Other studies attempting to elucidate the mechanism of action of b-IFN in MS have concentrated on peripheral lymphocytes. These have shown an array of effects including decreased mitogen driven proliferation of both CD4 q and CD8 q T cells w38x and decreased secretion of the pro-inflammatory cytokines g-IFN, TNFa and lymphotoxin w39x. This is in contrast to the effect of b-IFN on TNFa secretion by microglia. Accessory cells such as macrophages Žand by extrapolation microglia., rather than T cells, provide the major source of TNFa . In addition, b-IFN augments suppressor cell function w40,41x and may increase secretion of suppressive cytokines TGF b-1 and IL-10 from activated T cells w42x. However, CD4 q T cells are dependent on class II positive antigen presenting cells for initiation and maintenance of activation. Given that myelin, the ultimate immunological target in MS, is exclusively confined to the CNS, it seems probable that maintenance and propagation of this specific immune response occurs centrally. The effects of b-IFN on peripheral T cells and on central microglia are likely to be equally important in mediating the therapeutic effects. With respect to neonatal rodent microglia, we have shown that the effect of b-IFN is immunomodulatory and if one can extrapolate to the human disease situation, the balance of the pro- and anti-inflammatory properties of b-IFN results in a degree of disease suppression. Acknowledgements This work was supported by the Wellcome Trust. We would like to thank Miss Sarah Stevens for expert technical support.
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expression on adult human microglia studied in vitro and in situ. Eur. J. Immunol. 24, 3031–3037. Zajicek, J.P., Wing, M.G., Scolding, N.J. and Compston, D.A.S. Ž1992. Interactions between oligodendrocytes and microglia. Brain 115, 1611–1631. Giulian, D. and Baker, T.J. Ž1986. Characterization of ameboid microglia isolated from developing mammalian brain. J. Neurosci. 6, 2163–2178. Stewart, J., Glass, E.J., Weir, D.M. and Daha, M.R. Ž1986. Macrophage membrane receptors. In: D.M. Weir ŽEd.., Handbook of Experimental Immunology: Cellular Immunology. Blackwell. Wing, M. G., Montgomery, A.M.P., Harley, C. and Lachmann, P.J. Ž1990. Cytostasis of different tumours by a murine PPD-reactive CD 4q T lymphocyte clone is mediated by interferon-g and tumour necrosis factor alone or synergistically. Clin. Exp. Immunol. 82, 208–213. Lu, H.-T., Riley, J.L., Babcock, G.T., Huston, M., Stark, G.R., Boss, J.M. and Ransohoff, R.M. Ž1995. Interferon ŽIFN. b acts downstream of IFN-g-induced class II transactivator messenger RNA accumulation to block major histocompatability complex class II gene expression and requires the 48-kD DNA-binding protein, ISGF3-g . J. Exp. Med. 182, 1517–1525. Vogel, S. N., Finbloom, D.S., English, K.E., Rosenstreich, D.L. and Langreth, S.G. Ž1983 . Interferon-induced enhancement of macrophage Fc receptor expression: b-interferon treatment of C3HrHeJ macrophages results in increased numbers and density of Fc receptors. J. Immunol. 130, 1210–1214. Durum, S. K. and Oppenheim, J.J. Ž1993. Proinflammatory cytokines and immunity. In: W.E. Paul ŽEd.., Fundamental Immunology. Raven Press, New York, pp. 801–883. Garotta, G., Talmadge, K.W., Richard, J., Pink, L., Dewald, B. and Baggiolini, M. Ž1986. Functional antagonism between type I and type II interferons on human macrophages. Biochem. Biophys. Res. Commun. 140, 948–954. Fertsch-Ruggio, D., Schoenberg, D.R. and Vogel, S.N. Ž1988. Induction of macrophage Ia antigen expression by rIFN-g and down regulation by IFN-a r b and dexamethasone are regulated transcriptionally. J. Immunol. 141, 1582–1589. Ransohoff, R. M., Devajyothi, C., Estes, M.L., Babcock, G., Rudick, R.A., Frohman, E.M. and Barna, B.P. Ž1991. Interferon-b specifically inhibits interferon-g-induced class II major histocompatability complex gene transcription in a human astrocytoma cell line. J. Neuroimmunol. 33, 103–112. Hall, G. L., J. Girdlestone, M. G. Wing, N. J. Scolding, and D. A. S. Compston. Ž1996.. Beta interferon inhibits gamma interferon-induced major histocompatability complex class II expression on neonatal rodent microglia through a post-translational block of class II intracellular transport. submitted. Testa, U., L. Conti, N. M. Sposi, B. Varano, E. Tritarelli, W. Malorni, P. Samoggia, G. Rainaldi, C. Pelschle, F. Belardelli, and S. Gessani. Ž1995.. IFN-b selectively down-regulates transferrin receptor expression in human peripheral blood macrophages by a posttranslational mechanism. J. Immunol. 115:427–435. Hofman, F. M., Hinton, D.R., Johnson, J. and Merill, J.E. Ž1990. Tumor necrosis factor identified in multiple sclerosis brain. J. Exp. Med. 170, 607–612. Selmaj, K. W. and Raine, C.S. Ž1988. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann. Neurol. 23, 339–346. Howard, M. C., Miyajimi, A. and Coffman, R. Ž1993. T-cell-derived cytokines and their receptors. In: P.W.E. ŽEd.., Fundamental Immunology, Raven Press, New York, pp. 763–800. Traugott, U. and Lebon, P. Ž1988. Demonstration of a , b , and g-interferon inactive chronic multiple sclerosis lesions. Ann. N.Y. Acad. Sci. 540, 309–311. Panitch, H. S., Hirsch, R.L., Schindler, J. and Johnson, K.P. Ž1987. Treatment of multiple sclerosis with g-inteferon: exacerbations asso-
G.L. Hall et al.r Journal of Neuroimmunology 72 (1997) 11–19 ciated with activation of the immune system. Neurology 37, 1097– 1102. w37x Rudge, P., Miller, D., Crimlisk, H. and Thorpe, J. Ž1995. Does interferon beta cause initial exacerbations of multiple sclerosis? The Lancet 345, 580. w38x Rudick, R. A., Carpenter, C.S., Cookfair, D.L., Tuohy, V.K. and Ransohoff, R.M. Ž1993. In vitro and in vivo inhibition of mitogendriven T-cell activation by recombinant interferon beta. Neurology 43, 2080–2087. w39x Noronha, A., Toscas, A. and Jensen, M.A. Ž1993. Interferon b decreases T cell activation and interferon g production in multiple sclerosis. J. Neuroimmunol. 46, 145–154.
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w40x Noronha, A., Toscas, A. and Jensen, M.A. Ž1990. Interferon beta augments suppressor cell function in multiple sclerosis. Annals of Neurology 27, 207–210. w41x Noronha, A., Toscas, A. and Jensen, M.A. Ž1992. Contrasting effects of alpha, beta, and gamma interferons on nonspecific suppressor cell functions in multiple sclerosis. Annals of Neurology 31, 103–106. w42x Shakir, S., Byskosh, P.V. and Reder, A.T. Ž1994. Interferon-b induces interleukin-10 mRNA in mononuclear cells ŽMNC.. Neurology 44, A211.
b-interferon regulates the immunomodulatory activity of neonatal rodent microglia G.L. Hall a
a,b,)
, M.G. Wing c , D.A.S. Compston
a,b
, N.J. Scolding
a,b
UniÕersity of Cambridge, Department of Neurology, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK b MRC Cambridge Centre for Brain Repair, UniÕersity ForÕie site, Robinson Way, Cambridge CB2 2PY, UK c Molecular Immunopathology Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, UK Received 24 January 1996; revised 14 May 1996; accepted 19 June 1996
Abstract b-interferon Ž b-IFN. has both pro and anti-inflammatory properties, the balance of which leads to some suppression of disease activity in multiple sclerosis patients. Here, we examine the immunomodulation of neonatal rodent microglia, the principal CNS accessory cell, by b-IFN and consider the interaction of b-IFN and g-interferon Žg-IFN.. b-IFN and g-IFN inhibit microglial proliferation. b-IFN antagonises both g-IFN-induced upregulation of class II expression and the ability of g-IFN primed cells to mount a respiratory burst. In contrast, b-IFN upregulates microglial Fc receptor expression and augments tumour necrosis factor alpha secretion from suboptimally stimulated microglia. Keywords: Multiple sclerosis; Microglia; Beta interferon; Rodent
1. Introduction Multiple sclerosis ŽMS. is the commonest cause of acquired neurological disability in young adults. The precise mechanisms of immunologically mediated tissue damage are not fully understood, but there is a consensus that activated T lymphocytes cross the blood–brain barrier and initiate an inflammatory response within the central nervous system ŽCNS.. Soluble factors are important in myelin and oligodendrocyte injury w1x and infiltrating macrophages and activated resident microglia subsequently phagocytose myelin sheaths. Non-specific immunosuppressive and cytotoxic treatment has been relatively unsuccessful in the treatment of MS, but more recently, b-IFN has been assessed as a potential treatment. There is accumulating evidence that it may suppress some aspects of disease activity w1,2x and alter the natural history of MS. However, the exact mechanism of action of b-IFN in patients with MS has not yet been elucidated. Although first described on the basis of its antiviral properties, b-IFN has now been documented to have im-
) Corresponding author. Tel.: q44 Ž1223. 217842; fax: q44 Ž1223. 336941; e-mail: [email protected]
munomodulatory functions on a wide range of target cells. b-IFN has a direct antiproliferative action on some cells including macrophages and counteracts the mitogenic stimulus of certain cytokines w3,4x. It acts on large granular lymphocytes to increase killing, augments natural killer cell activity w5x and leads to increased generation of cytotoxic T lymphocytes w6x. B cell proliferation is augmented as is secretion of Ig M, G and A. The effects of b-IFN on accessory cells such as the peripheral macrophage are well characterised. b-IFN prevents proliferation and causes terminal differentiation and activation w7x. The resultant morphological changes include cell enlargement, spreading, pseudopod formation and vacuolation. Adherence is increased, as is the number and density of Fc receptors ŽFcR. w8x leading to increased phagocytosis. Destruction and detoxification of internalised pathogens is maximised by induction of lysosomal enzymes. Class I MHC expression is upregulated leading to increased class I restricted antigen presentation to CD8 cells. By contrast, in certain other accessory cells, namely the murine macrophage and the human astrocyte, not only does b-IFN not induce class II MHC, the expression of which is required for antigenspecific activation of CD4 q T cells via the T cell receptor ŽTCR., but it actually inhibits upregulation of class II surface expresson mediated by g-IFN w9–11x.
0165-5728r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 1 2 8 - 2
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In MS, T cells are thought to be activated within the periphery and enter the CNS where maintenance of activation occurs after encountering autoantigen. Immunoglobulin also contributes to the disease process; B lymphocytes are identified in the perivascular infiltrate w12x and oligoclonal bands in the cerebrospinal fluid and, furthermore, in the experimental allergic encephalomyelitis model, passive transfer of myelin oligodendrocyte glycoprotein-specific antibody results in a more severe disease with pathology resembling the primary demyelination seen in MS w13x. Central FcR expressing cells could directly phagocytose opsonised targets and immune complexes. Specific peptide within the immune complex would be processed and presented in the context of class II MHC, and hence activate CD4-positive T cells. In the CNS, microglia fulfill the criteria of immunologically active accessory cells; on activation in vitro they increase their expression of class II MHC w14,15x, and FcR w15x; they are phagocytic and can process and present antigen in an MHC restricted fashion to both resting and naive T cells w16–18x and can upregulate constitutive expression of the Th1 co-stimulatory molecule B7 w19x. In addition, microglia have important direct cytotoxic effector functions involving adherence to, and phagocytosis of, opsonised targets, the ability to mount a respiratory burst in response to appropriate stimulation w15x and the production of potentially harmful cytokines such as TNFa w20x. We have systematically studied the effects of recombinant rat b-IFN on a comprehensive range of immunological accessory and cytotoxic effector functions using cultured rodent microglia and have examined the interaction of b-IFN with g-IFN, which has been proposed as the main pro-inflammatory cytokine in the pathophysiology of MS. Neonatal rodent microglial morphology, class II MHC expression, proliferation, FcR expression, ability to mount a respiratory burst and TNFa secretion have each been examined. We identified both antagonistic and synergistic effects of these cytokines, re-emphasising the complexity of immune interactions between immunocompetent cells, cytokines and potentially beneficial immunotherapeutic agents.
2. Materials and methods 2.1. Cytokines Rat recombinant g-IFN and b-IFN were obtained from Biosource International ŽCamarillo, CA, USA.. g-IFN was used in all experiments at a concentration of 100 unitsrml. b- IFN was used at 200 unitsrml. 2.2. Tissue culture Microglia were isolated and cultured from 1–2-day-old Sprague– Dawley rats using the method of Giulian and
Baker w21x with modifications as described by Zajicek et al. w20x. This protocol was further modified such that, briefly, mixed brain cultures were shaken on day 8 for 2 h to remove microglia from the adherent cell population. The supernatant containing microglia was centrifuged at 900 rpm for 5 min, the cells were resuspended in Dulbecco’s modified Eagle’s medium ŽDMEM. with 10% foetal calf serum ŽFCS. and plated onto glass cover slips, for immunocytochemistry and respiratory burst experiments, and on to plastic in all other experiments, at the required density. After 30 min, adherent cells were washed twice with Hanks balanced salt solution ŽHBSS. to remove cell debris and any non-adherent cells. Microglia were then cultured at 378C in 5% CO 2 in DMEM with 10% FCS and used as described in the individual experiments. 2.3. Cell proliferation To measure cell proliferation, microglia were plated in triplicate onto flat-bottomed 96-well plates at a density of 1 = 10 5 cells per well. g-IFN, b-IFN, both interferons or neither were added and the microglia cultured for 48 h, after which, 7 m Cirml w3 Hxthymidine was added to each well and the microglia cultured for a further 4 h. The wells were then washed =3 with PBS to remove all unincorporated w3 Hx thymidine. The microglia were detached from the wells using trypsinrEDTA and cells collected using a cell harvester. The counts were read in a liquid scintillation b counter and the mean of triplicate wells was calculated and plotted. 2.4. Immunocytochemistry Class II MHC surface expression was identified by indirect immunocytochemistry using OX 17, a mouse IgG1 monoclonal antibody which recognises a monomorphic determinant on rat RT 1D, the rat homologue of mouse Ia-EŽSerotec., at 1:100 Žapproximately 34 m grml. as the primary antibody and a secondary FITC-labelled goat anti-mouse IgG ŽSigma. also at 1:100. Microglia were fixed with 2% paraformaldehyde subsequent to incubation with the secondary antibody, mounted in Vectashield and viewed under fluorescence microscopy using fluorescein optics. Appropriate controls were performed using an irrelevant mouse monoclonal IgG1 antibody or FITC-labelled goat anti-mouse IgG alone. 2.5. Expression of Fc receptors (FcR) 2.5.1. Preparation of opsonised sheep RBC Quantitative assessment of microglial FcR expression was performed using a rosette method modified from that of Stewart et al. w22x. Sheep red blood cells ŽRBC. in Alsever’s solution ŽTCS Microbiology, Bucks, UK. were washed Ž1,000 rpm. =3 in PBS with 10 mM sodium azide. RBC were then resuspended to a concentration of
G.L. Hall et al.r Journal of Neuroimmunology 72 (1997) 11–19
1 = 10 9 cellsrml. This is equivalent to a 4% solution of sheep RBC. Washed cells were incubated with an equal volume of a mouse IgG2b monoclonal antibody to sheep RBCs ŽSera-Lab. at a concentration of 10 m grml for 30 min at 378C. That concentration of antibody just below the agglutinating titre was determined in preliminary experiments. The opsonised sheep RBCs were washed =3 and stored at 48C for up to 4 days. 2.5.2. Detection of microglial FcR Microglia were plated in 12-well plates at 3.75 = 10 5 per well. After 30 min, cells were washed twice with HBSS and all washes were pooled. Any non-adherent cells were pelleted by centrifugation at 500 =g, resuspended and counted, allowing accurate estimation of the non-adherent cells and adherent cells remaining in the wells. Adherent microglia were cultured for 48 h either in the absence of cytokines or in the presence of b-IFN or g-IFN alone or together in DMEM with 10% FCS, then washed once with PBS prior to addition of the opsonised sRBCs. For this, opsonised sheep RBCs were washed =3 with PBS, resuspended to a concentration of 2.5 = 10 7 cellsrml in PBS with 10 mM sodium azide and 1 ml was added to each well. The plates were then incubated for 1 h on ice, then the wells were washed gently =3 and the sRBCs rosetted around microglia were then flash-lysed with 300 m l distilled water. The lysates were spun to remove any particulate matter and the haemoglobin content was assessed by the optical density reading at 412 nm in a spectrophotometer. A standard curve was constructed of the optical density readings of known numbers of RBCs lysed with 300 m l of distilled water. 2.6. Respiratory burst
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cytokines, with interferons alone or in combination, or with lipopolysaccharide ŽLPS. Ž1 m grml. as described. After 24 h in culture, supernatants were collected and spun at 3,000 rpm to remove non-adherent cells or cell debris. Supernatants were then assayed immediately or stored at y208C. Stored samples were thawed on only one occasion. Microglial production of soluble TNFa was assayed using the TNFa sensitive fibroblast cell line, L929, as previously described w23x. Briefly, 2 = 10 3 L929 fibroblasts in the log phase of their growth cycle were incubated in flat-bottomed 96-well plates in 50 m l of DMEM with 10% FCS for 72 h with 50 m l of microglial culture supernatant prepared as described below. At 72 h, the wells were pulsed with 7 m Cirml w3 Hxthymidine and incubated for a further 4 h. The wells were then washed =3 with PBS to remove all unincorporated w3 Hxthymidine. The fibroblasts were detached from the wells using trypsinrEDTA and cells harvested. Counts were read in a liquid scintillation b counter. A standard curve was constructed using recombinant rat TNFa .
3. Results 3.1. Microglial proliferation To study proliferation, microglia were cultured for 48 h, alone or in the presence of IFNs, and pulsed with w3 Hx thymidine; cells were harvested and radioactivity counted ŽFig. 1.. In the presence of b-IFN, there was approximately a 60% reduction in proliferation, whereas g-IFN alone, or in combination with b-IFN, completely inhibited proliferation.
Microglia were plated onto 19 mm glass cover slips at a density of 2 = 10 5 cells per cover slip, and were cultured for 48 h Žas described above. in the absence of IFNs or in the presence of g-IFN, b-IFN, or both IFNs. b-IFN was added 2 h prior to g-IFN, simultaneous with g-IFN or 2 h subsequent to g-IFN. At 48 h, coverslips were incubated for 45 min in 30% normal rat serum followed by incubation with affinity-purified FŽabX .2 fragment donkey anti-rat IgG ŽH q L, Jackson ImmunoResearch Laboratories, West Grove, PA. at 25 m grml and nitroblue tetrazolium ŽNBT. at 1 mgrml in PBS. Coverslips were then washed, fixed with 2% paraformaldehyde, mounted with Gurr ŽBDH Laboratory Supplies, Poole. and viewed under light microscopy. Respiratory burst products were identified by their ability to reduce NBT to insoluble formazan seen as a purple deposit. 2.7. Assay for tumour necrosis factor alpha (TNFa ) Microglia were cultured in flat-bottomed 96-well plates at a density of 2 = 10 5 microglia per well in the absence of
Fig. 1. Microglial proliferation in the presence of b-IFN and g-IFN. Microglia were cultured for 48 h either in the absence of added cytokines, or with b-IFN or g-IFN alone or together. At 48 h, microglial proliferation was assessed by 3 H incorporation following a 4-h pulse.
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3.2. Class II MHC induction Class II MHC expression was examined by indirect immunocytochemistry. Microglia were incubated for 48 h in the presence of 10% FCS without cytokines or with b-IFN, g-IFN, or both b-IFN and g-IFN ŽFig. 2.. g-IFNinduced marked upregulation of class II MHC expression ŽFig. 2c.. In the presence of b-IFN, microglia took on a stellate appearance ŽFig. 2b. easily distinguishable from the amoeboid forms induced by g-IFN ŽFig. 2c.. b-IFN did not affect basal class II expression ŽFig. 2b., but inhibited g-IFN-induced upregulation of class II ŽFig. 2d.. The granular staining seen in the absence of exogenous cytokines, or in the presence of either b-IFN alone or b-IFN and g-IFN in combination was also seen under rhodamine optics with equivalent intensity and, therefore, represents autofluorescence. 3.3. FcR expression To measure FcR expression, microglia were incubated with sheep RBCs opsonised with a mouse IgG2b monoclonal antibody to sheep RBCs antibody, an isotype which
would be predicted to bind to both low and high affinity microglial FcR w24x. An example of rosettes formed around microglia previously incubated with b-IFN is shown in Fig. 3a. To quantify the amount of rosetting, the sRBCs rosetting the microglia were lysed with water and the haemoglobin content measured using a spectrophotometer. By constructing a standard curve of the haemoglobin content of known numbers of sheep RBC and, given that the number of microglia per well was fixed Žand corrected for the number of non-adherent cells lost in the original washing step as described in Section 2, Materials and Methods., it was possible to plot the mean number of rosetted sheep RBCs per individual microglial cell ŽFig. 3b.. b-IFN induced a two-and-a-half-fold upregulation of microglial FcR expression ŽFig. 3b.. g-IFN upregulated FcR expression to a lesser Žbut nonetheless significant. extent. There appeared to be antagonism between b-IFN and g-IFN such that in the presence of both cytokines, receptor upregulation approximated more closely to that seen with g-IFN than with b-IFN alone. The experiment shown is representative of three experiments that all showed equivalent results.
Fig. 2. Detection of microglial MHC class II expression by immunocytochemistry. Microglia were incubated for 48 h Ža. in the absence of added cytokines, or with Žb. b-IFN, Žc. g-IFN and Žd. b-IFN and g-IFN. Surface class II expression was detected using the OX 17 antibody Žmagnification =400..
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Fig. 3. Ža. Detection of microglial FcR expression. An example of rosettes formed by opsonised sheep RBC around microglia previously exposed to b-IFN for 48 h. Sheep RBCs were opsonised by a mouse monoclonal IgG2b antibody, the Fc portion of which is recognised by both low and high affinity Fc receptors Žmagnification =400.. Žb. Detection of microglial FcR expression; estimation of mean number of rosetted sRBC per microglia. With reference to a standard curve of haemoglobin content of lysates of predetermined numbers of sRBC plotted against the number of sRBC, the mean number of rosetted sRBC per microglia was calculated.
3.4. Respiratory burst experiments The effect of interferons on the ability of microglia to mount an oxidative burst, a critical step in microglial cytotoxicity, was studied. Microglia were cultured for 48 h in the absence of cytokines, and with b-IFN, or g-IFN ŽFig. 4a–c.. Microglia were then incubated in 30% normal rat serum followed by a FŽabX .2 fragment donkey anti-rat
IgG to cross-link FcR which should induce a respiratory burst in appropriately primed cells. As shown in Fig. 4c, g-IFN primed microglia to mount a respiratory burst. Some b-IFN primed cells were able to mount a respiratory burst but to a lesser extent seen as small, densely positive cells ŽFig. 4b.. No respiratory burst products were obtained if normal rat serum was omitted. In order to study the interactions of g-IFN and b-IFN
Fig. 4. Ability of b-IFN and g-IFN-primed microglia to mount a respiratory burst. After 48 h in culture in the absence of IFNs or in the presence of b-IFN or g-IFN either alone or in combination, microglia were examined for their ability to mount a respiratory burst following appropriate stimulation Žsee Section 2, Materials and methods.. Respiratory burst products were detected by reduction of NBT to insoluble formazan visualised as a purple deposit. The panels represent Ža. no cytokines, Žb. b-IFN, Žc. g-IFN and Žd–f. b-IFN added Žd. 2 h before Ž t y 2., Že. with Ž t 0 ., or Žf. 2 h after g-IFN Ž t q 2. Žmagnification of all micrographs =400..
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with respect to microglial oxidative burst, b-IFN was added 2 h before, at the same time as, or 2 h after g-IFN ŽFig. 4d–f.. The ability of g-IFN-primed microglia to produce a respiratory burst was significantly reduced in the presence of b-IFN and this phenomenon was dependent on the sequence in which the cytokines were added, being maximal when microglia were preincubated with b-IFN prior to the addition of g-IFN and abolished when b-IFN was added only 2 h after g-IFN ŽFig. 5.. A reciprocal effect was also seen: the small densely positive microglia observed after priming with b-IFN were rarely seen when g-IFN was also present. 3.5. Microglial TNFa secretion Preliminary experiments demonstrated that the antiproliferative effect of b-IFN on many cell types extends to the L929 fibroblast line used to detect TNFa secretion Ždata not shown.. To control for this effect, the inhibition of fibroblast proliferation induced by both b-IFN and g-IFN, alone or in combination, was subtracted from the overall effect of supernatant produced by microglia cultured in their presence. A standard curve was constructed using recombinant rat TNFa and, therefore, by comparison, the amount of TNFa secreted by microglia was calculated. As shown in Fig. 6, IFNs alone did not stimulate TNFa secretion but augmented that induced by submaximal doses of LPS. Both g-IFN and b-IFN increased LPS-induced TNFa secretion, although this effect was greater with g-IFN. No significant antagonism was seen between b-IFN and g-IFN on secretion of TNFa . The experiment shown is representative of a series of experiments that all showed the same trend. Interestingly, microglia in culture for more than 12 h prior
Fig. 5. Ability of b-IFN and g-IFN-primed microglia to mount a respiratory burst. The experiment of Fig. 4 has been quantitatively assessed and is represented graphically. Shown are the mean number of formazan positive cells Ž"S.E.M.. in each treatment category.
Fig. 6. Microglial TNFa secretion. The amount of TNFa secreted was assessed using the L929 fibroblast proliferation assay and by extrapolation from a standard curve plotted using rat recombinant TNFa .
to stimulation could not be induced to secrete TNFa Ždata not shown..
4. Discussion Our study shows that the effects of b-IFN and g-IFN on neonatal rodent microglia are both complex and interactive. The results do not produce a complete explanation for the clinical effects of b-IFN, but highlight the potential benefits and complications of cytokine treatment. Both b-IFN and g-IFN are antiproliferative for microglia alone or in combination and they augment TNFa secretion induced by sub-optimal concentrations of LPS. In contrast, b-IFN antagonises both g-IFN-induced upregulation of class II MHC surface expression and the ability of g-IFNprimed microglia to mount a respiratory burst. Microglial FcR expression is upregulated by b-IFN and to a lesser extent by g-IFN, but in addition, g-IFN antagonised the superior effect of b-IFN. The mechanism of this reciprocal antagonism is unlikely to occur through competition for cellular receptors as b-IFN and g-IFN bind to different ligand-specific receptors. In MS, the balance of pro- and anti-inflammatory properties of exogenously administered b-IFN appears to favour suppression of disease activity w1,2x. On peripheral macrophages, most actions of b-IFN are pro-inflammatory, the main exception being inhibition of g-IFN-induced upregulation of class II MHC expression w9,10x. This would suggest that, in isolation, b-IFN primes peripheral macrophages for direct cytotoxic tasks but specifically impairs initiation and maintenance of a class II restricted CD4 q T cell response. Here we show by immunocytochemistry that this inhibition of class II expression by b-IFN is also true for microglia, the resident CNS cell most capable of antigen presentation.
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b-IFN inhibits the proliferation of microglia as it does peripheral macrophages w3x, which may reflect the suppression of division which often precedes differentiation. One might, therefore, expect b-IFN to enhance cytotoxic properties by increasing FcR expression, respiratory burst and TNFa secretion, as was indeed shown. We have demonstrated that, with respect to FcR expression, microglia respond to b-IFN in a similar way to peripheral macrophages. In our experiments, b-IFN induced an almost three-fold increase in FcR expression which is in keeping with the findings of Vogel et al. Ž1983. who showed that b-IFN induces an equivalent upregulation of FcR expression on murine macrophages w25x. In addition, b-IFN induces upregulation of FcR expression to a level greater than that induced by g-IFN, a phenomenon also previously documented for peripheral macrophages w26x. Our results also show that in some respects there is reciprocal antagonism between b-IFN and g-IFN. In contrast to what we observe with respect to class II expression, g-IFN appears to antagonise b-IFN-induced upregulation so that in the presence of both cytokines, the level of FcR expression detected is less than that induced by b-IFN alone and more closely approximates to the level seen with g-IFN. Conversely, and in keeping with the effect on class II MHC antigen expression, b-IFN inhibits the ability of g-IFN-primed microglia to mount a respiratory burst. This is consistent with the work of Garotta et al. Ž1986., who described antagonism of type II IFN-induced oxygen radical production in human macrophages by type I IFN w27x. The fact that maximal inhibition is demonstrated with pre–exposure to b-IFN, and that this inhibitory effect of b-IFN is negligible if b-IFN is added only 2 h after g-IFN would be in keeping with transcriptional regulation. A precedent for this interpretation would be the finding that b-IFN-mediated inhibition of g-IFN-induced class II upregulation in murine macrophages and a human astrocytoma line occurred at the level of transcription w28,29x. However, despite these latter findings, we have demonstrated that, in neonatal rat microglia, b-IFN-mediated control of class II surface expression is controlled by a post-translational mechanism w30x. Further evidence of antagonism between type I and type II IFNs comes from recent work showing that using human peripheral macrophages, g-IFN antagonises the inhibitory effect of b-IFN on transferrin receptor expression w31x. However, we showed no antagonism between b-IFN and g-IFN on secretion of TNFa . Whilst neither induced TNFa secretion per se, both augmented the production induced by submaximal concentrations of LPS, demonstrating that in this context b-IFN is pro-inflammatory. This is of particular relevance in MS since TNFa has been detected in acute lesions and in vitro may be toxic to oligodendrocytes w32,33x, both of which implicate TNFa in the pathogenesis of MS. The principal pro-inflammatory cytokine in MS is g-
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IFN. It is secreted from activated T cells and is responsible for activation of accessory cells necessary for potentiation of the immune response w34x. Like TNFa , it is also detectable in active MS lesions w35x. A therapeutic trial of g-IFN was terminated early following increased frequency and severity of exacerbations amongst the treated group w36x. It seems reasonable to assume, therefore, that b-IFN is administered to patients with MS on a background of endogenous g-IFN production, and one can speculate that, whilst in the non-diseased state b-IFN might increase the cytotoxic functions of microglia, in active disease, FcR expression, the ability to mount a respiratory burst and class II MHC expression within lesions would each be reduced leading to impaired activation and potentiation of the Th cell arm of the immune response. This provides one hypothesis for how exogenous b-IFN might swing the balance in favour of disease suppression in MS. The excess of exacerbations noted during the first 3 months of b-IFN treatment w37x, may be explained by an initial increase in cytotoxicity mediated by elevated FcR expression and TNFa secretion prior to the balance being swung in favour of down-regulation of the Th cell mediated arm of the immune system. Other studies attempting to elucidate the mechanism of action of b-IFN in MS have concentrated on peripheral lymphocytes. These have shown an array of effects including decreased mitogen driven proliferation of both CD4 q and CD8 q T cells w38x and decreased secretion of the pro-inflammatory cytokines g-IFN, TNFa and lymphotoxin w39x. This is in contrast to the effect of b-IFN on TNFa secretion by microglia. Accessory cells such as macrophages Žand by extrapolation microglia., rather than T cells, provide the major source of TNFa . In addition, b-IFN augments suppressor cell function w40,41x and may increase secretion of suppressive cytokines TGF b-1 and IL-10 from activated T cells w42x. However, CD4 q T cells are dependent on class II positive antigen presenting cells for initiation and maintenance of activation. Given that myelin, the ultimate immunological target in MS, is exclusively confined to the CNS, it seems probable that maintenance and propagation of this specific immune response occurs centrally. The effects of b-IFN on peripheral T cells and on central microglia are likely to be equally important in mediating the therapeutic effects. With respect to neonatal rodent microglia, we have shown that the effect of b-IFN is immunomodulatory and if one can extrapolate to the human disease situation, the balance of the pro- and anti-inflammatory properties of b-IFN results in a degree of disease suppression. Acknowledgements This work was supported by the Wellcome Trust. We would like to thank Miss Sarah Stevens for expert technical support.
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