Abnormal production of interleukin-1 by microglia from trisomy 16 mice

Neuro,wience Lcuer,~, 132 ~[991 ) 27(I ?,74 ~'~ 1991 Elsevier Scientific Publishers Ireland Ltd. I)304-3940/91/5 03.50 ,4DO NIS 0304394091006668

270

NSL 08184

Abnormal production of interleukin-1 by microglia from trisomy 16 mice C.A. Colton 1'2, J. Yao 1'2, R.E. Taffs 3, J.E. Keri xa and M.L. Oster-Granitea 1Department of Physiology and Biophysics, Georgetown University Medical School, Washington, DC (U.S.A.), 2The Laboratory of Biophysics. NINDS, NIH, Washington, DC (U.S.A.), aNIH, NIAID, Laboratory of Immunology, Biochemistry and lmmunopharmacology Unit, Bethesda, MD (U.S.A.) and 4Developmental Genetics Laboratory, Department of Physiology and Departments of Neuroscience and of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD (U.S.A.) (Received 5 June 1991; Revised version received 13 August 1991; Accepted 13 August 1991)

Key words: Microglia; Trisomy 16 mouse; Down syndrome; Interleukin-1; Interferon The production of interleukin-1 (IL-1) was examined in cultured CNS microglia obtained from trisomy 16 (Tsl6) fetal mouse brain, a model system for studies relevant to Down syndrome (DS). When compared to microgtia from their normal littermates, Tsl6 microglia produced significantly higher levels of IL-1 activity both before and following stimulation with lipopolysaccharide (LPS). IL-1 release was stimulated by c~/flinterferon (IFN) in the normal but not Tsl6 microglial cultures. The overall level of IL-I production in normal littermates, however, was still less than that seen in Tsl6. Thus, microglia from Tsl6 mice may function in an inappropriate manner and, if this abnormality occurs in vivo, may have wide ranging effects on a developing nervous system.

Since its discovery as an endogenous pyrogen in the 1940s, interleukin-I (IL-1) has been established as an important regulatory agent for a variety of cell types, particularly those cells involved in the immune response and inflammation [9, 34]. IL-1 is now known to promote secretion of the acute phase proteins, prostaglandins E and proteases as well as to induce cellular differentiation and proliferation [8, 9, 34]. Its role in the body's defense against stress and injury has been well described. IL-1 is also involved in response of the central nervous system (CNS) to injury and has been shown by Giulian and Lachman [17] to increase in levels around stab wounds. Furthermore, IL-1 can modulate the 'stress' reaction by affecting the production of hypothalamicpituitary hormones such as adrenocorticotropin, growth hormone and somatostatin [4, 27, 38]. As in other tissues, IL-1 in the CNS not only enhances proliferation of cells at the wound site, with resultant gliosis [17], but also induces the secretion of other growth promoting factors such as nerve growth factor [5]. IL-t may also regulate neuronal growth during development [5]. IL-1 has been detected in at least 3 types of CNS cells; astrocytes, anterior pituitary cells and the CNS macrophages, the microglia [5, 13, 18, 27]. Among these cell populations, microglia are clearly a major source of IL-1 found in the CNS. Giulian and Lachman [17] and others Correspondence." C.A. Colton, Department of Physiology and Biophysics, Georgetown University Medical School, Washington, DC, U.S.A.

[18, 19, 23] have shown that cultured neonatal rat microglia release IL-1 when stimulated with lipopolysaccharide (LPS). The same is probably true in situ since injection of LPS in combination with ~ interferon (IFN) induces the expression of I L - I t mRNA in rat brain [24]. Control of IL-1 levels in the brain, however, is notwell understood. For example, abnormal production of IL- 1 has been recently associated with neurodegenerative diseases such as Alzheimer's disease (AD) and Down syndrome (DS). In both these conditions, deposition of amyloid arising from amyloid precursor protein (APP) and the formation of senile (neuritic) plaques are major neuropathological characteristics. IL-I may play a role in the pathogenesis of both processes, since IL-I treatment enhances the expression of APP mRNA in human endothelial cells [20], and increased IL-1 immunoreactivity in microgtia has been observed in the senile plaques of the brains of AD and DS individuals [21]. In order to further explore the relationship between IL-I and the neuropathology associated with DS, we have examined the secretion of IL-t by CNS microglia cultured from trisomy 16 (Tsl6) mice. Mouse chromosome 16 (MMU 16) and the distal portion of human chromosome 21 (HSA 21) demonstrate homology for many genes, including SOD1, IFNRC, APP and others [11, 12, 33]. When present in triplicate, the genes on the distal region of human chromosome 21 are associated with a DS phenotype. Thus, because of the genetic similarities, the Tsl6 mouse has become a model for studies

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relevant to DS which cannot be performed in DS individuals. Generation qf trisomy 16 mice. Trisomy 16 mice are generated via a breeding scheme as described in detail by Gropp et al. [22] and Gearhart et al. [14]. Briefly, mice which are homozygous for a particular Robertsonian (Rb) translocation chromosome, i.e., Rb(16,17), are bred to mice homozygous for a different Rb translocation chromosome, i.e., Rb(9,16). The progeny are doubly heterozygous and each contain chromosome 16 within a metacentric chromosome. Male mice from this stock are then mated to C57BL/6J females, containing a normal set of acrocentric chromosomes. The offspring of this mating contains approximately 34% trisomy 16 conceptuses. Monozygous offspring die at or around the time of implantation. Tsl6 fetuses were tentatively identified by their characteristic edematous phenotype and their identity was confirmed by karyotypic analysis using minced liver [ 10]. Microglial culture. Primary cultures from Tsl6 and their normal littermate mice were prepared by removing the cerebral hemispheres from conceptuses at day 15 of gestation. Hemispheres were placed into Leibovitz's medium (L-15) containing gentamycin and the meninges and associated blood vessels were carefully removed using a binocular microscope. The cerebral hemispheres were then rinsed in fresh L-15 to remove any adherent blood cells, transferred to D M E M (at 1 brain/ml of D M E M ) and gently triturated to disperse the cells. The suspended cells were then plated directly into a 96 well culture dish (50/d/well) and grown at 37°C in a humidified, 5% CO 2, 95% air atmosphere. Tsl6 microglia and normal, littermate microglia were grown separately in D M E M supplemented with 10% horse serum and 25/tg/ ml gentamycin. This media differs from that previously used [6] in that horse serum replaced fetal calf serum. We have found these conditions to promote proliferation of murine microglia while being unfavorable for other cell types. To determine the number of microglia in each well for both Tsl6 and normal littermate cultures, cells were stained and then counted using a Nikon binocular microscope. Microglia were identified by the binding of the lectin, GS- 1 coupled to peroxidase, while astrocytes were identified by standard immunocytochemical techniques using a TRITC-labelled antibody against glial fibrillary acidic protein (GFAP) [7, 35]. The cultures under our growth conditions were found to be essentially only microglia, containing greater than 95% microglia. The average number of microglia/mm2for Tsl6 cultures was 87 cells/ram 2 ( n = 2 4 wells counted) compared to 261 cells/ mm 2 in the normal littermate cultures ( n = 2 9 wells counted). These values represented 8400 cells/well for

the normal littermate and 2800 cells/well for the Tsl6 cultures. The difference in cell number between normal and Tsl6 cultures probably reflects the inherent lower number of total cells seen in Ts 16 brain [31 ]. Interleukin-1 bioassav. IL-1 levels in the supernatants from Tsl6 and normal littermate microglia were measured using the DI0.G4.1 cell proliferation bioassay as described by Kaye et al. [26]. Briefly, at day 12 in culture the microglial cells in the 96 well plates were washed, placed into serum-free media (HL-I, Ventrex) and then stimulated for 24 hours to produce IL-I by treatment with LPS or ~/fl IFN or were left untreated (unstimulated controls) for the same duration. A sample of the supernatants from each well was then diluted (1 part supernatant and 9 parts serum-free medium) and added to a 96-well plate containing D10.G4.1 (DI0) cells plus 2.5/Lg/ml Concanavalin A. Following a 48 h incubation, 0.5 /iCi [3H]thymidine was added to each well and the cells were incubated for an additional 12 h. The D10 cells were then harvested onto glass fiber filters using an automatic cell harvester. Radioactivity was determined using liquid scintillation counting and the average counts per minute (cpm) for 3 separate wells per experimental condition for a minimum of 3 different litter groups was obtained. A litter group was defined as the microglia obtained from the pooled Tsl 6 cerebral hemispheres and the pooled normal littermate cerebral hemispheres from a single, pregnant female mouse. Units of IL-I were determined from a standard curve using recombinant mouse IL-I (Genzyme) where one unit of IL-I was defined as that concentration required to produce a halt'maximal incorporation of [aH]thymidine by 20× 1(}~ D 10 cells. Because of the difference in microglial number in each well between the Tsl6 and normal littermate cultures, IL-1 activity was normalized to 1000 cells. Statistical analysis utilized a one way ANOVA. In some experiments, unstimulated IL-I activity was also measured at days 4, 7 and 11 of culture by sampling the growth media from the microglial cultures. These samples were then assayed as described above. Endogenous endotoxin level was measured in the culture supernatants using the Limulus amoebocyte lysate (LAL) assay (Cape Cod Associates). Endotoxin was 0.25 EU/ml or less. IL-2 activity was tested using IL-2 dependent longterm cultured murine cells (CTLL) incubated for 24 h in 96 well plates in the presence of a graded dilution of the experimental supernatants [16]. Proliferation of the CTLL cells was assayed by incorporation of [~H]thymidine (1/~Ci/ml) for 4 h. Both unstimulated and stimulated production of lL-1 is higher in Tsl6 microglial cultures than in those oF their normal littermates. In unstimulated microglia for a 24 h

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period, IL-1 activity of the Tsl6 cultures was 2.4 fold higher than in the cultures of normal littermate controls. This higher unstimulated level was detected as early as day 4 in culture and was seen throughout the sampling period (Table I). 1 The activity was attributed to IL-I~ by an antibody neutralization assay using a polyclonal anti-IL-1 ~ antibody (Genzyme) (data not shown). To eliminate the possibility that endogenous endotoxin contributed to the higher activity in the Tsl6 supernatants, endotoxin levels were measured using the Limulus amoebocyte lysate assay. Endotoxin levels were the same for both culture groups and were 0.25 EU/ml or less. Since IL-2 in the supernatants could also influence proliferation of the D10 cells, IL-2 activity was measured using a separate, sensitive bioassay for IL-2. No IL-2 activity was detected in any of the supernatants. Treatment with LPS produced a significant increase (p< 0.001) in IL-1 activity compared to the untreated condition in both Tsl6 and normal littermate microglia (Fig. 1). Tsl6 microglia tended to have a larger percent increase from the unstimulated condition (360%) compared to the normal littermate microglia (330%). Furthermore, the overall level of IL-I was significantly higher (P<0.001) in LPS-stimulated Tsl6 microglia compared to LPS-stimulated normal littermate microglia. In normal microglia, the [3H]thymidine uptake of D10 cells induced by 1 pg/ml LPS was 18.7_+ 1.1 cpm ( x 10 -3) per 1000 microglia (n= 17 wells assayed from a minimum of 6 different litter groups) while in Tsl6 microglia the level was 55.6+3.5 cpm ( x 10 -3) per 1000 microglia (n = 15 wells assayed from a minimum of 5 different litter groups). Treatment with a higher concentration of LPS (10/tg/ml) demonstrated the same phenomenon, that is IL-1 activity in the supernatants from Tsl6

TABLE I IL-1 ACTIVITY OF SUPERNATANTS FROM Tsl6 AND NORMAL LITTERMATE MICROGLIAL CULTURES Data represents the average [3H]thymidine uptake by D 10 cells in cpm (× 10-3)+S.E.M. incubated in the presence of diluted supernatant samples taken from the media of normal littermate and Ts 16 microglial cultures at day 4, 7, and 11 of culture. Number of wells assayed =9 for each time point. Day

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Fig. 1. LPS-stimulated production of IL-1 by cultured Tsl6 microglia and their normal littermates. Bar heights represent the average values of [3H]thymidine uptake in counts per minute (× 10-3)_+S.E.M. by DI0 cells in the presence of diluted supernatants from Tsl6 and normal littermate microglia. Microglia were either untreated or treated for 24 h with I/zg/ml and 10/lg/ml LPS (026:B6, E. coli). At least 17 wells from 6 litter groups for normal microglia and 15 wells from 5 litter groups for Ts 16 microglia were assayed for IL-I per experimental condition. Data were normalized to 1000 microglia. *Significant difference (P<0.001) between untreated and LPS-treated. **Significant difference (P < 0.001) between Tsl 6 and normal,

microglia was higher than that seen in the normal littermate microglia. However, the increased dose of LPS did not significantly increase IL-1 activity from that observed after treatment with 1 pg/ml LPS. To further investigate IL-I production by Tst6 microglia, we examined the effect of e/fl IFN treatment on IL-1 activity. Following treatment of microglial cultures for 24 h with murine o~/fl IFN, levels of IL- 1 activity were again higher in the Tsl6 microglia compared to their normal littermates (Fig. 2). The response of Ts16 microglia to e/fl IFN, however, was different from that observed in normal littermates, c~/fl IFN did not significantly change IL-1 activity from the unstimulated condition at either 100 or 1000 U/ml of ot/fl IFN. In the cultured normal littermate microglia, IL-I activity was significantly increased (P< 0,05) at the 1000 U/ml dose of c~/fl IFN but was not changed at 100 U/ml. While it is not surprizing that microglia cultured from Tsl6 mouse cerebral hemispheres produce IL-I [t3, 17-19], the significantly higher IL-I activity in supernatants from Tsl6 microglia compared to supernatants from normal, littermate microglia is surprizing. Both unstimulated and stimulated IL-1 activity was higher. For unstimulated Tsl6 microglia, this difference was evident from day 4 of culture and remained throughout the culture period. When stimulated by LPS, both Tsl6 and normal littermate microglia increased IL-1 production.

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Fig. 2. Effect of~/fl IFN on IL-1 production by Tsl6 and normal littermate microglia. Bar heights represent the average [3H]thymidine uptake in cpm (x 10-3)+S.E.M. of DI0 cells in the presence of diluted supernatants from IFN-treated Tsl6 and normal littermate microglia. Microglia were treated with 100 or 1000 U/ml of murine ~,'fl IFN for 24 h. The number of wells assayed varied from 6 to 33, using a minimum of 3 culture groups. Data was normalized to I000 microglia. *Significant difference (P<0.05) between untreated and IFNtreated. **Significantdifference (P < 0.001 ) between normal and Ts 16.

However, IL-1 activity was again higher in T s l 6 mice. Other agents such as :qlfl I F N are k n o w n to induce ILl p r o d u c t i o n by m o n o c y t e s a n d tissue m a c r o p h a g e s [1, 14, 30, 39]. Since C N S microglia are of m o n o c y t i c origin a n d share m o r p h o l o g i c a l a n d f u n c t i o n a l characteristics with other tissue m a c r o p h a g e s [6, 13, 19, 32, 36, 41], a response to c~/fl I F N would be expected. T r e a t m e n t with ~/fl I F N increased the release of IL-I above u n s t i m u lated levels in n o r m a l littermate microglia. However, the same c~/fl I F N c o n c e n t r a t i o n range did n o t increase IL-1 levels in T s l 6 microglia. The lack of a response is interesting, especially since the cell surface receptor for ~/fl I F N is triplicated in both H S A 21 a n d M M U 16 [11, 37]. In fact, the cellular response to ~/fl I F N in cultured DS cells a n d T s l 6 fibroblasts is 7 times higher rather than the expected 1.5 fold increase p r o d u c e d by the gene dosage effect of triplication of the :Ufl I F N receptor [12, 29]. O u r data m a y indicate that the d o s ~ r e s p o n s e curve in T s l 6 has shifted b e y o n d the range of~/fl I F N concentrations studied. The elevated p r o d u c t i o n of IL-1 in the T s l 6 microglia is indicative of a 'fully activated' m a c r o p h a g e similar to that seen at sites of injury in the C N S [1, 19, 39]. T s l 6 microglia also d e m o n s t r a t e an e n h a n c e d p r o d u c t i o n o f superoxide a n i o n , further suggesting that these m a c r o phages f u n c t i o n in an a b n o r m a l m a n n e r [3, 6]. The consequence of such an i n a p p r o p r i a t e l y increased level of IL-I in the C N S of T s l 6 mice or in the b r a i n s of DS individuals is not clear. At the least, a d i s r u p t i o n in the nor-

a n d Melissa H u n t e r for their excellent technical help. The work was s u p p o r t e d in part by individual research g r a n t ( H D 19932 to M . L . O . G . ) a n d a n i m a l costs were supported in part by the following p r o g r a m grants ( H D 19920, H D M.L.O.G.)

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