A Second Generation Transgenic Mouse Model Expressing Both Hemoglobin S (HbS) and HbS-Antilles Results in Increased Phenotypic Severity By M.E. Fabry, A. Sengupta, S.M. Suzuka, F. Costantini, E.M. Rubin, J. Hofrichter, G. Christoph, E. Manci, D. Culberson. S.M. Factor, and R.L. Nagel We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human (I and PS chains ((1~/3'[p'"~I) with a line expressing human a and fiSAdmlh' (B'"). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype becausethe reduced oxygenaffinity and solubility of the might enhance the rate and extent of polymer formation. We obtained mice that expressed both @ and /3kAnti'lr. The doubly transgenic mice that are heterozygous for deletion of mouse (Brn) occurred with reduced frequency andthose that are homozygous for deletion of mouse /3"jD' (BMDD) occurred at a much reduced frequency and suffered early mortality. Human (I was 58% of all (I globin for all animals, whereas and pS"'" were 34% and 28% of all j3 globins for mice and 4 2% and 36% for pMWmice. Hematocrit, Hb, and mean corpuscular Hbwere normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus ( 1 ~ / 3 ' [ / 3 ~ ~mice 1 older than 30 days (10.0% f 1.0% v 4.3% r 0.4%; P < .001, mean -c SE, n = 20 and n = 10, respectively) andcontrol mice (3.9% k 0.4%). Reticulocytosis was more severe in mice less than 30 days old (>20% for (~~g'p~~@ mice). " ] The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of ( 1 ~ / 3 " ~ ~mice " ~ 1with a variable number of very densecells.Delay times for polymerization of Hb in red blood cells from ( 1 ~ / 3 ~ / 3 ~ ~ [ f f ' " Imice were shorter than those of ( 1 " / 3 ~ [ / 3 ~mice, ~ ~ 1 and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating a b i l i in control mice under ambient conditions is 2,846 f 294 mOsm and was reduced 30% t o 1,958 k 240 mOsm, P c4 x in all miceexpressing both transgenes.We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease. Q 7 9 9 5 by The American Society of Hematology. A mouse pMaJo' (Hbb'"" '), which we symbolize as p"" or p""" to increase expression of the p globin transgenes. The SAD mouse4.' incorporates all three mutations (the sickle mutation plus the Antilles and D-Punjab) in one p chain and is the product of site-directed mutagenesis grafted onto a S-Antilles /3 gene. Mice that are also heterozygous for the mouse pMajor deletion express this trimutated chain at moderate levels (29% p"") and are characterized by anemia in the neonatal period, but notin the adult mice. In addition, they die after short exposure to hypoxia' and suffer from high intrauterine mortality. The efforts reported here had two goals: (1) to develop a transgenic mouse model that is intermediate between the mild-to-moderate phenotypes and the severe SAD mouse and (2) to test the hypothesis that the pathology observed /3"'" / 3 ' N ANIMAL MODEL for sickle cell disease has been a long sought goal that has recently been realized in several laboratories by the generation of transgenic mouse lines expressing hemoglobin S (HbS) or derivative forms of this mutant Hb."6 The animals generated have a range of pathophysiology from severe phenotypes that resulted in increased perinatal mortality to lines that exhibited moderate or mild pathology under ambient conditions. Two naturally occurring mutations increase the severity of HbS-related disease. One of them is HbS-Antilles, which contains, in addition to the HbS mutation at p6 (Glu-rVal), a second mutation in the same chain at p23 (Val+Ile). HbSAntilles has low oxygen affinity and low solubility under deoxy conditions. In contrast with sickle trait individuals, the heterozygous individuals with HbS-Antilles have significant pathology. The other mutation is HbD-Punjab, which occurs at p123 in a gene that does not contain the sickle mutation. This trans mutation results in pathology when the patient is a double heterozygote for both PS and ,L?D-PunJab because of assembly of tetramers that contain one p chain from each of the two mutated genes. Both of these mutations have beenused to create transgenic mice. Transgenic mice using the S-Antilles mutation were generated by Rubin et a13 and were found to have a slightly reduced hematocrit and a small number of irreversibly sickled cells (ISCs) that increased when the animals were subjected to hypoxia (a hypobaric chamber at 0.42 atm, which is equivalent to 8.4% 02). The animals studied, which were homozygous for mouse PMaJor deletion, expressed about 5 1% of their &globin as flS-Antil'es; however, only 10% of the a chains were human a (aH), and therefore, most of the flS-Antilles was found in dimers that contained mouse a-globin. As shown by Rhoda et al,' mouse a chains interfere with polymerization nearly as effectively as do human y chains. All of the mice described in this report make use of animals either heterozygous or homozygous for the deletion of the Blood, Vol 86, No 6 (September 15). 1995 pp 2419-2428 From theDepartments of Medicine and Department of Pathology, Albert Einstein College of Medicine, Bronx, NY; the Department of Genetics and Development, Columbia University, New York, NY; the Cell and Molecular Biology Division, Lawrence Berkeley Laboratory, Berkeley, CA: Laboratory of Chemical Physics, NlH, Bethesda, MD: and the Centralized Pathology Unit for Sickle Cell Disease, University of South Alabama, Mobile, AL. Submitted November 2, 1994; accepted April 13, 1995. Supported in part by National Institutes of Health Sickle Cell Center Grant No. HL-38655 and a Grant-in-Aidfrom the New York Branch of the American Heart Association. Address reprint requests to M.E. Fabry, PhD, Albert Einstein College of Medicine, Department of Medicine, Vllmann 921, 1300 Morris Park Ave, Bronx, NY 10461. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. Q 1995 by The American Society of Hematology. 0006-4971/95/8606-0143$3.00/0 2419 2420 FABRY ET AL under hypoxic conditions in the aHpS[pMDD] mouse is caused by polymerization of HbS. To achieve this end we have bred the transgenic mouse line aHPS[PMDD], which has a phenotype with moderate pathology under ambient conditions and more severe pathology under hypoxic condition^,'.^ to the aH/3S-Anti11es line. The rationale is that the combination of the low oxygen affinity, low solubility HbS-Antilles, and the high level of human (Y and PS expression of the aHpS[pMDD] line might result in an enhanced rate and extent of polymerization and a more severe phenotype than either of their parents. MATERIALS AND METHODS Hematologic Parameters Mice were bled from the tail using protocols approved by the animal studies committee and blood samples were analyzed for reticulocytes using the Sysmex R-1000 system (Sysmex, Toa, Japan) or by staining with thiazole orange and evaluating by fluorescenceactivated cell sorting (FACS) (Lysys 11; Becton Dickinson, Mountain View, CA). Serum samples were analyzed using the Chem-l system (Technicon, Tarrytown, NY). High-Performance Liquid Chromatography (HPLC) The globin composition was determined by HPLC using a denaturing solvfnt that separates the globin chains and a Vydac largepore (300 A) C, column (Separations Group, Hesperia, CA), 4.6 X 250 mm with a modified acetonitrilekl,O/trifluoroacetic acid gradient similar to that used by Schroeder et all0 for separating human chains. Two buffers were used, A (0.18% trifluoroacetic acid in 36% acetonitrile) and B (0.18% TFA in 46% acetonitrile). Starting with 38% B, the percentage of B was increased by 0.583% per minute until all of the globin chains were eluted. Slow Deoxygenation Blood samples were collected directly from the mouse tail into heparinized capillary tubes. The samples were carefully mixed in the tubes and then immediately sealed on both ends with tubesealing compound. They were allowed to sit at room temperature for approximately 24 hours. The hematocrit tubes were then placed in a nitrogen-filled glove bag where the ends of the tubes were cut off. By using a piece of small bore plastic tubing attached to the end of a needle and syringe, the cells were pushed out into vials containing degassed 2%glutaraldehyde in phosphate-buffered saline, pH 7.4. CSAT Blood from several mice with identical HPLC chromatograms was collected and washed into saline. The cells were hemolyzed by the addition of distilled water and by rapidly freezing and thawing several times. They were spun down in a microfuge and the clear red supernatant removed. The resulting hemoglobin was dialyzed and concentrated versus 0.1 m o m potassium phosphate buffer, pH 7.35 at 25°C. Samples were deoxygenated on ice by adding enough sodium dithionite solution to give a final concentration equal to three times the heme concentration. The samples were transferred anaerobically to CsATtubes filled with paraffin oil. They were incubated at 25°C overnight in a nitrogen atmosphere. The mouse Hb samples were centrifuged for 2 hours at 35,000 rpm at 25°C with purifiedHbSused as a control. The supernatants were removed anaerobically and concentrations and deoxy pHs were determined. Density Gradient The density gradient screening ofredblood cells (RBCs) was performed by Percoll/Larex gradients." Percoll (colloidal silica coated with polyvinylpyrrolidone; Pharmacia, Upsalla, Sweden) and Larex (arabino galactan polysaccharide; Larex International, Duluth, MN) gradients were formed from a mixture of Percoll and Larex (density 1.207 g/mL, determined from refractive index), water, and 10-fold balanced salts in a ratio of3.5:3.0:2.8:0.7. The pH and osmolarity are adjusted to 7.35 and 330 mOsm, respectively. Batches are prepared and frozen in 50-mL aliquots. A 0.1-mL aliquot of a well-mixed sample of whole blood with the hematocrit adjusted to 50 was added to 5.9 mL of the gradient mix for analytical determinations of RBC density distribution. The tubes are well mixed, and spun in a rotor (model 5-21; Beckman Instruments, Irvine, CA) for 25 minutes at 35,000g at 37°C. The resulting gradient is continuous but nonlinear. Each group of eight tubes included a sample from a control mouse and a tube with Pharmacia density-marker beads to ensure constant conditions. Scrupulous care in resuspending blood samples at all stages is critical in these measurements because the densest cells selectively adhere to the bottom of the tube. Delay Time Delay times were measured for three aHps/?s-A"t[pMD] mice and mouse under conditions that are physiologic one aHpsps~A"t[OMDD] for mice (pH 7.35 and330 mOsm). The plasma osmolarity characteristic for these mice was determined by 16 separate measurements on control C57BLJ6J mice in which we found an average plasma osmolarity of 331 2 16 mOsm. A similar value was found in the literature." Mice were bled approximately 1 hour before the delay time measurement was made; the cells were washed into a physiologic buffer (330 mOsm for mice") and diluted to Hct 0.1. The cells were then equilibrated with a humidified mixture of CO and COz at 37°C until pH 7.35 was reached (by adjusting the percent CO,) and transferred into a closed Dvorak-Stotter cell. The RBCs were allowed to settle in the cell while maintaining a temperature of 37°C for 20 minutes until most of the cells were adherent to the polylysine-coated glass surface. After this, the cell was inverted over a microscope. and the CO was flashed off the Hb by exposure to a focused laser beam at 514.5 nm. Onset of polymer formation was measured by continuously recording light scattering as previously described.l3.I4Although the actual measurements were more difficult because of the small size of the mouse RBCs, the polymerization curves obtained from mouse RBCs by this protocol were similar in shape and magnitude to those obtained from human RBCs. Urine-Concentrating Ability Because of the greater fragility of pS/lps-""' mice, they were deand four prived of water for 7 daytime hours. Six aHps/?S-A"t[pMD] a"~s~s~A"'[/?MDD] mice were examined. This less rigorous protocol (usually the mice are deprived of water overnight) allowed us to observe the animals and rescue any animals showing signs of lethargy or listlessness. At the end of this period urine was collected onto Parafilm and the osmolarity was measured after a 1:lO dilution with distilled water. Pathology A total of 7 aHpspS-""'[lpMDD] mice were examined; 4 of the animals (ages 87, 88, 151, and 157 days) were killed during the course of experimentation, 2 died spontaneously, and 1 was killed because it appeared tobe terminal (30, 91, and 103 days). Tissues were preserved in buffered formalin. Slides were stained with hematoxy- 2421 A TRANSGENIC MOUSE WITH HbSPLUSHbS-ANTILLES Table 1. Hematologic Properties of Transgenic Mice Reticulocytes % (age > 30 dl Mouse C57BU6J (n = 6 ) U MCV MCH MCHC IfL) (pg/cell) lg/dL) 3.9 f 0.4 45.4 f 0.9 14.5 2 1.0 33.7 ? 0.4 4.3 f 0.4 43.0 ? 1.4 14.1 2 0.7 35.7 2 1.7 14.2 34.6 2 1.5 W LLY a H p S l p D 1 (n = 10) (IHpS.Ant[ pMDD] (n = 5) ,HpSpS.Am[ 5.1 t- 0.7 43.6 f 0.3 I 0.9 pMD] (n = 9.5 4) f 1.2 42.2 ? 1.8 14.4 2 0.2 36.2 2 1.1 ,HpSpS-Am[pMDD] (n = 22) 10.0 2 1.0 43.5 f 1.5 14.9 I 0.5 36.2 2 0.7 Abbreviation: MCV, mean corpuscular volume. lin-eosin or trichrome. The reported results arefor the animals killed during the course of experimentation. RESULTS Hematology and Hb Composition Hematocrits, Hb, and mean corpuscular hemoglobin (MCH) were normal for all adult mice. Mice bearing both transgenes expressed both 8' and p'"'nlilles as well as human a;however, the percent of PS and fl'-Anlillcr expressed as the percent of all p chains for both DMDand OMDDmice was less than that expressed by either parent that was homozygous for the pMaJordeletion (pMDD) (Table 1). Expression of human a was 58% ? 1% of all a globin for all animals, whereas the expression of p' and pS"'"i''es was 34% 2 2% and 28% 5 1% of all p globins for PM"mice and 42% f 3% and 36% ? 2% for DOMDD mice (Table 2). aHpSpS-Ant [PMDD] Fig 1. Percoll-Larexcontinuousdensity gradient with density mouse, an marker beads, a control C57BLK.J mouse, an uHpS[pMDDl a"pspsAmIpMDI mouse, and an ( ~ ~ p ~ p mouse. ~ - ~Notethe ~ [ p retic~ ~ ulocytes (low-density cells at the top of the tube) in both of the the high-density cells in the #f3*p" doublytransgenicmiceand [pMDD] mouse. ~ ] Sickling Cells from aHflSfl"A"'[PMDD] mice were allowed to sickle slowly and were subjected to scanning electron microscopy Density gradients (Fig 1) were completed for control (Fig 2). More than 95% of the cells sickled and many exhib(C57BW6J). aHfl'[fiMDD], aHPSPS-Ant[pMD], and c~~fl~fl'-~"' ited a profusion of spicules. [pMDD] mice. As previously reported, the aHfl'[pMDD] mice had a mean corpuscular hemoglobin concentration (MCHC) CS*7 that is about 3 g/dL higher than that of the control mice. Both ,Hpqp5-Anl[pMD] and a H p S p S A n l [pMDD] mice also had The concentration of the Hb in equilibrium with the deoxy denser cells than control mice; in addition, they hada populagel phase was determined for aHfl'[pMDD], aHpsps-Anl[fiMD]. and aHpSpS-Anl [pMDD] tion of low-density cells that were predominantly reticulotransgenic miceandwascompared cytes, and aHflSfls"'nl[pMDD] mice had a population of higher with mixtures of HbS with HbAor HbF (Table 3). We found density cells. The aHflSfl'"'"l[PMDD] mice had the largest perthat the CSATfor aHpps-A"l[flMDD] mice was nearly 3 g/dL cent of high-density cells seen in any of the mice that we less than that of the aHpS[[BMDD] mice. Althoughthese values have examined to date. are only slightly less than those for a sickle trait patient, the tendency to form polymer in vivo will be higher because of the lower p50 of S-Antilles and the higher MCHC of mouse RBCs. Table 2. Hemoglobin Expression of Transgenic Mouse Lines Density Gradient Mouse 0' + /3SAm/All0 Os/All 0 2 2.4 74.7 f 2.4 74.7 2 2.4 - 11.1 2 2.7 50.9 2 2.3 - 50.9 2 2.3 2 1.4 62.6 I 2.1 34.4 f 2.0 28.3 2 .8 ? 2.3 42.2 I 2.8 35.9 2 1.7 aHIAll a Os.A"'lAll0 Delay Times Uy?S[ (n = 20) 55.9 ,HpS.Am[pMDD] (n = 4) ,HpSpS-Am[ pMD] (n = 8) 57.8 ,HpSpS.Am[ In = 10) pMDD] 58.2 2 78.2 .9 The time between deoxygenation and the onset of polymer formation was measured using laser photolysis of CO-Hb to produce deoxy-Hb followed by detection of polymer formation by light scattering. Three mice with aHps~"-"'[pMD] and one mouse with aHflsp'-Ant[pM"D] were tested and compared with three mice with the aHp'[flMD"] genotype (Fig 3). We found that the major difference was in the number of cells FABRY ET AL 2422 Fig 2. Scanningelectronmicrograph RBCs of from a ,HpSpS.Ant[pMDD] mouse that have been deoxygenated for 24 hours. Note the multispiculated forms, which are not common in aHpS[pMDDi mice. requiring 100 seconds or more to formpolymer. In mice with the a"~Sps"'n'[~MD"] phenotype, more than 95% of the cellssickled in lessthan 50 seconds,whereas in al'flSflS"'"' [pM"]and aHps[[pMD"1 mice, 16.5% and 39%. respectively, requiredmorethan 100 seconds to sickle. Weknowfrom previous experiments that more than 95% of the cells will sickle if maintained under deoxy conditions for long enough, thus even though these cells required more than 300 seconds to form polymer, they are able to sickle. Less obvious in the cumulative plots used are the cells withveryshort delay times (between 1% and 5% at .01, .OS, and . l seconds) of the aHp?p?-l\rU [pMD] and the aHp"pS"'"'[pM""l mice. hand-counting methods, but that the results were much more reproducible, in part because of the much larger number of cells used for each determination. Adult ~ r " p ' p ~ " ' " ' [ ~ ~ " ] micehad increased levels of reticulocytes (Table 1 ) and adult ~ r " p ~ ~ ~ " ' " 'had [ p "levels " ~ ] of reticulocytes higher than either parentline. Reticulocyte levels werehigherforthe doubly transgenic mice versus aHp'[pM""] mice older than 30 days (10.0% t 1.0% 11 4.3% t 0.4%, P < .001, mean t SE, n = 20 and n = 10. respectively) and control mice (3.9% t 0.4%). This effect was even more marked in mice under 30 days of age in which the ~ r ~ ~ ~ ~ ~ "mice ' " were ' [ ~ ~ " " ] found to have 20% reticulocytes (Fig 4). Reticulocytes Urine-Concentrating Ability Reticulocytes were measured usingthe automated Sysmex system and thiazole orange with FACS analysis. We found that there was a consistently lower percentage of reticulocytes recorded with theSysmex system when compared with Urine-concentrating ability after a 7-hour-long deprivation of water was reduced under ambient conditions in the PS/ fls"'"' mice that were either hetero- or homozygous for the mouse P""J'"deletion when compared with control mice (Fig 5). The values obtained were more varied because of the shorter period of water deprivation, butthemore fragile nature of the pslps"'"' mice dictated this protocol. Reduced urine-concentrating ability is a characteristic of sickle cell disease and urine osmolarity was reduced 30% to 1,958 Z 240 mOsm under ambient conditions in all mice expressing both transgenes ( P < 4 X IO-'; 2,846 t 294 mOsm in control mice). Table 3. CsAT for SamplesContaining p" Sample C s ~ gldL r Purified HbS HbS/HbA 40:60 HbSlmouse 50:50 HbS/HbF 50:50 17 2 1 27 2 2 31 2 1 30 2 1 28.9 t 1 34+ 27.2 2 1 25.9 2 1 a n p 1p D D I aHflS-Ant[ p D D ] aHljSpS.Anl nHpflS.Am[ [P D I p D D ] 4(mix - HbS/HbA 4060) g/dL -10 0 4 3 1.5 J+ 0 -1.1 Serum Enzymes Two serum enzymes associated with liver damage were analyzed: aspartate amino transferase and alanine amino 2423 A TRANSGENIC MOUSE WITH HbS PLUS HbS-ANTILLES transferase. For aspartate amino transferase, the aHPSPS-Ant [pMDD] mice had an average value of 167 -C 23 U/L versus the control C57BV6J mice, which had 52 2 4 U L (mean rC_ SE, P < .002, n = 15 and n = 8, respectively). For alanine amino transferase, the aHpSpS-An'[/3MDD3 mice had an average value of 166 32 U/L versus the control C57BV6J mice, which had 27 2 1 U/L (mean 2 SE, P < .005, n = 15 and n = 8, respectively). Mice heterozygous for the pmajor deletion aHPSPS-Ant[pMD] had intermediate values (Fig 6). I 25t: 0 Pathology A total of seven aHps~s~A"'[pMDD] mice were examined using hematoxylin-eosin and trichrome stains. The results reported are, unless otherwise indicated, for the four animals that were killed during experimentation, but the other three animals had very similar and, in some cases, more severe pathology. Brain. The two younger animals had congestion, occasional red neurons and rare pyknotic neurons. Red neurons were also seen in three other aHpSp"-""'[pMDD] mice. The red neurons may be indicative of hypoxic episodes secondary to focal vasoocclusion; however, they would not be regarded as significant in the absence of the more severe findings in the two older animals. The two older animals had neuronal dropout, pyknotic neurons and supporting cells, and pyknotic Purkinje cells in the cerebellum (Fig 7A). Lung. The lungs of the two older animals manifested 0 10 20 30 40 c. c 50 a"! 60 70 80 90 100 l l .01 .05 l l .l .5 l l ' 5 l l 10 l t 50 100 - L c300 Delay time in seconds Fig 3. Cumulative delay times for three &""~"DD1 mice, three a"/3s/3sA~"Dl mice, and an aHf3'/3sAR[BMwI mouse. Note that the aH/3'/3"~/3""l mouse (0)has more than 95% of cells with delay times (the time between complete deoxygenation and the onset of polymer formation) less than 50 seconds. In theC Y " ~ ' / ~ ~ ~ " mice ~~"I (01 and &"'" [p-] mice (A). 16.5% and 39%. respectively, of the cells required more than 100 seconds to sickle. In addition, note that both the aH/3'~"""1/3"l and the a"/P/3sM1~01 mice have more cells with short (less than 0.1 second) delay times, although this is hard to see in cumulative plots. 01 0 I I l I I 50 100 150 200 250 I I I 300 350 400 450 Mouse age in days Fig 4. Percent reticulocytes versus mouse age in days: aH/3s@MDD1 (0) and a"/3s/3BM"vMDDI (0).Note thevery high reticulocyte count of a"p'ps~A"[f3MDD1 mice at 30 days of age. septal thickening (Fig 7B) and interstitial fibrosis with some platelet thrombi. Severe congestion and intra-alveolar hemorrhage were noted focally in all four animals. Liver. In all cases examined, we found multifocal ischemic infarcts of ages varying from recent (1 to 2 days old) to remote (several days to several weeks old) with dilatation of central veins and a preserved rim of viable cells around the central vein, suggesting recent andor prolonged ischemic events (Fig 7C). One of the older mice showed evidence of past infarcts, but did not have evidence of recent infarcts. Areas of recent infarct featured focal coagulative necrosis with degenerating hepatocytes showing nuclear chromalysis and smudged cytoplasmic details with ghost-like outlines of hepatic plates and sinusoids. The areas of remote ischemic injury featured complete coagulative necrosis with loss of nuclei and amorphous eosinophilic outlines of hepatocytes and sinusoids. This is a new feature that has not been reported in other sickle transgenic mice. Occasional Kupffer cells could be visualized in the trichrome stained slides with RBC fragments (not shown). There was no appreciable iron deposition in the liver, but scarring and fibrosis was seen in older animals. Figure 7C illustrates old and new lesions in the liver. In the two older animals, fibroticand collapsed areas were seen. The liver was enlarged in aHPSPS-Ant[PMDD] mice versus C57BW6J of the same age (6.3% t 0.7% of body weight v 4.8% t 0.8%, n = 8 and n = 5, respectively; P < ,009; Table 4). Spleen. Spleen to body weight was calculated for eight animals and was enlarged in aH/3sps-Ant[pMDD] mice versus C57BL/6J of the same age (0.68% 5 0.27% of body weight v 0.26% f 0.04%,n = 8 and n = 5 respectively; P < .W6 Table 4) with significant fibrosis (visualized with trichrome stain, not shown), marked congestion with an expansion of 2424 FABRY ET AL red pulp, but without excessive iron accumulation. Focal 350 fibrosis was noted in the two older animals (15 1 and 157 days). Kidney. The kidneyhad congested glomeruli andthe afferent vessels were dilated. There was focal fibrosis in the medulla and the cortical-medullary junction, but only one 6 250 U) animal had fibrosis in the cortex. Glomerular and peritubular e S! vessels were markedly congested as were vessels in the paU) 200 pilla. The kidney was enlarged in aH,@@S-Ant[pMDD] mice l versus C57BL/6J of the same age (0.85% -C 0.12% of body weight v 0.57% ? 0.03%, n = 8 and n = 5 respectively; P 'z 150 U < .0005; Table 4). 0 AspAmTx 0 5 e 8 +-. Percent ps/ps~*"' Mice and Length of Survival ; 100 a Fewer aH~SPS-Ant[PMDD] mice survived to 10 days than were predicted. Mating aH/3s[[pM"D] micewith aHPS-Anl 50 [pMD"] mice wouldbe expected to produce 25%of four different genotypes: aHpS[pMDD], aH/3S-An'[pMDD], (-)[pMD"] 01 l I I I (homozygous for pmaJo' deletion, but no transgene), and aHPSPAnt [p"""], but 28.6%, 34.9%, 27%, and 9.5% were found instead (P < .03, n = 63). Not all aHpsps-An'[pM"D] mice were produced from this type of mating. In contrast, Fig 6. Aspartate amino transferase ( 0 )and alanine amino transmating two aHpS[pMDD] mice produces 73% aHps[[p"""] ferase (0) in U/L for control (C57BI).n"ps [pmD], a"pSB6"*tpMDl, and c ~ " p ~ p " ~ "mice. l All animals were maintained under ambient mice and 27% (-)[pMDD] mice; indicating that the conditions. Ages range from48 to 271 days. aHpS[pMDD] transgenic phenotype is more robust than the homozygous /?"'J"'deletion. Of 22 aH/3sps-Ant[pMDD] mice 3200 3000 2800 2600 2400 2200 2000 1800 1600 Fig 5. Urine-concentrating ability for control mice (males, e; females, 0)and p/p"* mice after a 7-hour deprivation of water. Female mice ara slightly offset to the right. Theps/psh mouse group contains animals that are both homozygous(0.all males) and heterozygous (males, females, 0)for the mouse p""'" deletion. +; surviving to 10 days, 5 died of natural causes between days 20 and 138, 1 additional mouse died accidentally during the course of experimentation, 8 were killed during the course of experimentation, and the 2 oldest surviving mice are 1 year old. Thus, more than 20% of these mice died before 5 months of age, which is in contrast with the 30 months that is normal for aHpS[pMDD] mice and C57BU6J mice. DISCUSSION We have previously reported on transgenic mice that express high levels of human a and PS (aHpS[pMDD]).5,6 We found that these mice had pathology under ambient conditions that could be made more severe when the mice were subjected to hypoxia'; therefore, we concluded that the enhanced HbS polymer formation induced by hypoxia resulted in increased vasoocciusion. If this interpretation is correct, mechanisms that increase polymer formation should result in increased pathology. To test this hypothesis and create a mouse with a more severe phenotype, we have introduced /3S-Anti"es into . the aHpS[pMDD] line, examined indicators of polymer formation, and compared these with hematology, urine-concentrating ability, and histopathology. The rate of polymer formation is extremely sensitive to the concentration of deoxy Hb, with a concentration dependence exceeding the 30th power,'',16 whereas the extent of polymer formation at equilibrium is directly proportional to the deoxy-Hb concentration. If polymer formation occurs slowly (with a long delay time), cells may be reoxygenated in the lung before polymer formation occurs. Delay times that are long with respect to the mean time of circulation would be expected to protect the animal under most circumstances; however, if adhesion or stasis occurs or the transit time is NSGENIC A 2425 Fig 7. Hematoxylin-eosin-stained sections of brain (A), lung (B),and liver (Cl. In (A), cerebellum from a 151-day-old ~~"&3""rp"l mouse showing pyknotic (filled arrow) and normal (open arrow) Purkinje cells. The pyknotic cells are hyperchromaticwith shrunken nuclei and are adjacent to cells with normal nuclei, consistentwith focal vasoocclusion. Each Purkinjecell is served by a single capillary (curvedarrow) and, hence, focal loss of them cells is an expected result of sickle cell vasoocclusion of single capillaries. One such capillary that ispacked with cells is indicated by the curved arrow (original magnification x 160). In (B),lung parenchyma with alveolar capillary congestion and focally thickened alveolar septa(arrows)from a 157-day-oldaHpsfis.h[fiMDDl mouse (original magnification x 160). In (C), a low-power view (original magnification x 6 5 ) of the liier is seen on the right showing multiple infarcts; the boxed areas areshown at higher magnification. The top of (C) (a) shows a recent ischemic injury with partial nuclear chromalysis; the bottom of (C) (b) shows a more remote ischemic injury with coagulative necrosis of hepatocytes(original magnification x 280). low solubility of HbS-Antilles leads to a lower CsATand very long, the delay time will be less important than the shorter delay times for polymerformation in RBCs in ability of the cell to form polymer. Intracellular polymer these transgenic mice. All of these properties interact to formation results in rigid, nondeformable cells with inproduce enhanced vasoocclusion, RBC destruction, and creased viscosity and potential for vasoocclusion,'7 and pathology, increased spontaneous death and, in the case therefore, both intrinsic and extrinsic factors that affect of the aH/3S@S-Ant[/3MDD] mice, a birthrate that is half of polymer formation are expected to affect pathology. that expected. We havegenerated and studiedtransgenicmicethat The amount of Hb per RBC is constant for all mice regardexpress both PS and @S-Antilles on a background which is less of whether they are C57BV6J control mice or express either heterozygous forthe pMaJor deletion ((.u"@~@~one or two transgenes. Consequently, although we do not A"'[pm])or homozygous for thedeletion ([(.u"@~@~know if this down regulation occurs at the level of transcripht[@MDD]). We find that these mice have a more severe tion, translation, or polypeptide chain assembly, the expresphenotype than either of the two parent lines. Features of sion of more than one transgene will result in reduced expresthese mice that will lead to an increased concentration of sion of each of the individual transgenes. deoxy PSand pS-Aatilles are (1) higher percent of human a A major difference between human sickle cell disease and and the sum of the mutated @-globin chains (Ps plus p" all transgenic mouse lines described to date is the absence Anti'les); (2) the decreased oxygen affinity of @S-Ao'i11es,which of adult anemia. Both human and animal studies indicate results in the presenceof more deoxy @S-Antil1es; and (3) the that the low hematocrit characteristic of sickle cell disease greater degree of dehydration of the RBCs of the doubly is protective against vasoocclusive events, and that increased transgenic mice, which increases the intracellular Hb conhematocrit results in greater pathology because of increased centration. The combination of these properties with the 2426 FABRY ET AL Table 4. Organ Weights for Control and Transgenic Mice cytes were elevated compared with the parent lines. This effect was particularly intense in mice younger than 30 days of age. In the neonatal mouse, RBCs are larger than those C57B1/6J 5 135 2.6 2 0.4 48.0 t- 1.6 5.7 2 0.3 of the adult and the level of 2,3-DPG expression is less than aH@I p D D 1 9 147 5.7 t 3.1 55.6 2 1.7 7.4 t 1.1 aHpSpS-Ant that of adult mice2’; in control mice, adult values for both [pMDD] 8 131 6.8 2 63.3 2.7 2 8.5 6.6 2 1.2 of these properties are attained by the end of the first 4 weeks Pcontrol v pSpS-Ant of life.” One possibility is thatone of these two effects (large <.006 <.009 1.0005 size or reduced 2,3-DPG) is responsible for the enhanced * Mouse age in days. rate of RBC destruction. In the aHflSPS-Ant[pMDD] mouse, the t Percent of body weight x 10. greater number of dense cells (Fig 2) may contribute to the increased reticulocyte count. Density gradients of human patients homozygous for HbS bulk blood viscosity. Thus, a low hematocrit in a transgenic (SS) have a large and variable population of dense RBCs animal model wouldresult in less sickling-related pathology. that is composed of both dense discocytes and ISCs.” The Nevertheless, the probable origin of the absence of anemia mechanism by which this population is formed is still not needstobe considered. In transgenic mice, a mechanism fully resolved, but it is generally agreed that it is the result contributing to the absence of anemia is the relative right of repeated cyclesinof vivo which results in shift of the oxygen equilibrium in mouse RBCs (because the potassium loss and dehydration. Density gradients of host Hb has a higher ~ 5 0 ~Another ). major component of aHps@s^nt[pMDD] mice indicate the presence of both a large the level of hemolysis in human sickle cell disease that is reticulocyte population and a much larger population of high absent in the mouse is the formation of very dense cells with density RBCs than i s seen for either of the parent lines. a life span of only 2 to 4 days. We have reported the presence Romero et all8 have shown that deoxygenation of transgenic of a deoxy potassium efflux, a unique characteristic of human mouse RBCs results in a deoxy potassium effluxthat is sickle cells, in RBCs of the aHPs[[PMD”] mouse that would analogous to that uniquely observed in human SS patients. be expected to contribute to formation of dense cells. Howde Francheschi et a124have recently reported a Ca2+-stimuever, the K:CI cotransport detectable inthe mouse lacks lated K+ channel in the SAD mouse analogous to that obmany of the characteristics found in human RBCs, and the served by Romero et a]’* in theaHpS[pMDD] mouse that probhigh activity of calmodulin-activated Ca2’-ATPase (which ably is responsible for the deoxy K+ efflux in both cases. is correlated with Ca2+-pumpactivity) in mouse RBCs may An increased population of high-density cells is seen in partially protect PS transgenic mouse RBCs from dehydraaHps[[pMDD] mice that have been exposed to hypoxia5;in this tion even inthe presence of a deoxy potassium efflux.” This case, the increased population was attributed to increased in may account for the small proportion of ISCs found in all vivo sickling. Similarly, we speculate that the increased RBC transgenic mouse models19and will also contribute to the density seenin the aHpsps~Ant[pMDD] mouse is caused by absence of adult anemia. nesum of pS and pS-Antilles In. aHpsps-Ant[/?MDD] mice is increased in vivo sickling under ambient conditions. The time between complete deoxygenation and the onset close to 80% compared with 72% PS in our previous line, of polymer formation (delay time) was measured in individaHpS[pMD”]. Because the human aHexpression is higher in ual RBCs for aHpspS-“”‘[PM”] and aHPSPS-Ant[pMDD] mice these animals (close to 60%) compared with the S-Antilles and compared with the parent aHps[pMDD] mouse Iine (Fig mice and the sum of PS plus pS-Antilles is higher than the 3). We found that both the aHPSPS-Ant~MD] and the aH,BSPS-An‘ expression of PS in the aHpS[,BMDD] mouse line, we antici[pMDD] mice had more cells with delay times less than 100 pated and found that the concentration of deoxygenated Hb msec and fewer cells with delay times greater than 100 secin equilibrium with the polymer (CSAT) for the aH@SpS-Ant onds than the aHpS[pM””] mice; however, in aHfls@s-An‘ [pMDD] mouse is less than that for either of the parents and [P”””] mice, 95% of the cells sickled in 50 seconds or less. less than that for a mixture of HbS and HbA similar to that It is not known whether the deleterious effects of a small found in sickle trait RBCs. The physiologic impact of this percent of cells with very short delay times is moreimportant CsAT must be interpreted in the context of other RBC properthan the protective effect of a larger percent of cells with ties; relevant here is the increased MCHC of transgenic long delay times. Although the aHpS,f?S-Ant[,BMD] mice had a mouse RBCs under physiologic conditions, resulting from smaller percent of polymerizable Hb plus pS-Antille.i) dense cell formation and the high normal plasma osmolarity than the aHps[[pMDD] mice, they had shorter delay times that in mice (330 mOsm). Therefore, the same CsAr will result appear to be a contradiction. The explanation for this disin a shorter delay time in transgenic mouse RBCs than it crepancy canbe found in the properties of HbS-Antilles. will in human cells. Furthermore, CsAT and delay times are HbS-Antilles differs from HbS in both its decreased oxygen measured under fully deoxygenated conditions and will not affinity (9 mm Hg v 5.5 mm Hg, respectively”) and in its reflect the important in vivo effect of the reduced oxygen decreased solubility in the deoxy state (1 1 g/dL versus 18.4 affinity of cells containing PS-Antilles. g/dL, respectively”). In vivo, we expect oxygen affinity to In adult mice, increased reticulocyte counts are an indicaplay a strong role, because RBCs are usually only partially tion of increased erythropoiesis and when they are combined deoxygenated, and the low p50 of HbS-Antilles will increase with an analysis of spleen histopathology, reticulocytosis can the concentration of deoxy Hb present and hence shorten be attributed to an increased rate of RBC destruction. In delay times. However, under the experimental conditions adult aHpsps-An‘[pMD] and aH@5PS-Ant[PMDD] mice, reticuloMouse Kidneyt LivertSpleent n Age* vs A TRANSGENIC MOUSE WITH HbS PLUS HbS-ANTILLES 2421 occlusion of individual capillaries, one would expect to find of cell-by-cell delay times, complete deoxygenation occurs normal Purkinje cells next to those that have become pykbefore data collection begins at 2 milliseconds and hence notic as is seen in Fig 6A. These findings are compatible the oxygen affinity of the Hb will not affect the delay time. with those found in some patients with sickle cell anemia On the other hand, the solubility of the Hb will play a major and related syndromes. role because Eaton et all5 have shown that the delay time Final manifestations of increased pathology are the reis proportional to the fractional saturation. The increased duced birthrate and high level of spontaneous death of douexpression of human a chains will also contribute to enbly transgenic mice that are homozygous for the mouse pMajor hanced polymer formation because the mouse a chain plays deletion. The cause of early death was not clearly estabthe same inhibitory role toward polymerization in the lished, but it appears to have multiple causes including contransgenic mouse as the human y chain in sickle cell anemia gestion and repeated hepatic infarcts. patients.' Hence, the short delay times can be attributed to In a separate set of experiments on the exposed cremaster the lower solubility of HbS-Antilles, which manifests itself of living aHpsps-An'[pMDD] mice, Kaul et a13' have reported in the lower CsATdiscussed above and the higher expression observing occasional sickled cells, RBC adhesion, and reof human a. duced RBC velocity. This is the first time that in vivo RBC Loss of urine-concentrating ability is a characteristic of adhesion has been reported. The reduced RBC velocity when human sickle cell patients that is found even in sickle trait compared with control mice is compatible with increased patients. The kidney is expected to be particularly sensitive viscosity caused by in vivo polymer formation. to sickle cell vasoocclusion because of the high osmolarity, We conclude that the pS/pS-A"' mice have a more severe low pH, and relatively low oxygen tensions that are characphenotype than either parent line and that this increased teristic of that organ. A urine-concentrating defect is not severity is correlated with more extensive polymer formafound in aHpS[pMDD] mice unless they are subjected to sevtion. Many of the pathologic features seen in the pS/pS-Ant eral days of h y p ~ x i a We . ~ found that urine-concentrating mice under ambient conditions can only be elicited from the ability was spontaneously decreased in both aH/3s/3S-A"'[pMD] parent lines by placing them under hypoxic conditions, and crHpSpS-"'[pMDD] under ambient conditions. Because the which also results in more extensive polymer formation. percentage of PS and PS"""'of the aHPS/3S-Ant[pMD] mice is Because the pathology observed in the pS/pS-Ant mice is similess than the percentage of PSin the parent crHps[/3MDD] mice, lar to that observed in the parent strain under hypoxic condithe reduced oxygen affinity of the S-Antilles Hb probably tions, this is consistent with our earlier conclusion that the plays a major role in the loss of urine-concentrating ability. increased Rl3C density and renal concentrating defect seen On the other hand, the loss of urine-concentrating ability cannot be attributed to hypoxic renal damage in these aniin the aHpS[pMDD] mice under hypoxic conditions was the mals. result of increased polymer formation and vasoocclusion Organ damageto four of the doubly transgenic animals that caused by nondeformable RBCs. Comparing these mice to werehomozygous for themouse pMaJor deletion (aHflsps-h' the parent lines mayallow us to dissect out the features of sickle cell disease that are most important in creating [pMDD] and were killed for experimentation was determined histologically. Congestion was characteristic of all tissues. pathology. The liver showed numerous areas of both old and new focal necrosis suggestive of episodic occurrence of ischemic NOTEADDED IN PROOF events. The observation that aspartate amino transferase and At 10 days of age, aHpsps-Ant[fiMDD] mice have a hematoalanine amino transferase levels were elevated in pSpS-Antcrit of 37 t 2 versus adult mice, which have a hematocrit mice indicates that the process of liver infarction is an ongoof 16 5 2.5 (mean t SE, n = 3 and 16, respectively; P < ing event in living animals. Focal necrosis of the liver has .00002). Therefore, neonatal anemia is also a characteristic been reported in both autopsy and biopsy specimens of sickle of these mice. cell patientsz6-" and has been attributed to vasoocclusive events by some auth0rs.2~ The spleen was enlarged more than REFERENCES 2.5-fold when compared with age-matched control mice, and 1 . GreavesDR,FraserP,Vidal MA, Hedges MA, Ropers D, older animals had areas of focal fibrosis that are compatible Luzzatto L, Grosveld F Atransgenic mouse model of sickle cell with ischemic events. In the lungs, focal intra-alveolar hemdisorder.Nature 343:183, 1990 orrhage and platelet thrombi were noted in all animals and 2. Ryan T M , Townes TM, Reilly MP, Asakura T, Palmiter RP, septal thickening was characteristic of older aHflSpS-AntBehringerRR:Human sickle hemoglobin intransgenic mice. Sci[PMDD] mice. The kidneys were enlarged and showed modest ence 247566, 1990 3. Rubin EM, Witkowska HE, Spangler E, Curtin P, Lubin BH: fibrosis in the medulla and cortico-medullary junction and Hypoxia-induced in vivo sickling of transgenic mouse red cells. J congested vessels in the glomeruli and papilla. In young Clin Invest 87:639, 1991 (less than 60 days) animals, occasional red neurons and rare 4. Trudel M, Saadane N, Garel M, Bardakdjuan-Michau J, Bloupyknotic neurons were found in the brain that are suggestive quit Y, Guerquin-Kern 3, Rouyer-Fessard P, Vidaud D, Pachniss A, of ischemic events. In older animals, franksigns of ischemia Romeo P, Beuzard Y, Costantini F M : Towards a transgenic mouse such as neuronal dropout, pyknotic neurons and supporting model of sickle cell disease: Hemoglobin S A D . EMBO J 10:3157, cells, and pyknotic Purkinje cells in the cerebellum appeared. 1991 The Purkinje cells are particularly interesting because each 5. FabryME,CostantiniF,Pachnis A, Suzuka SM, Bank N, cell is served by a single capillary. If sickled cells result in Aynedjian HS, Factor SM, Nagel RL:High expression of human beta 2428 S- and alpha-globins in transgenic mice: Erythrocyte abnormalities, organ damage, and the effect of hypoxia. Proc Natl Acad Sci USA 89:12155, 1992 6. FabryME,Nagel RL, Pachnis A, Suzuka SM, Costantini F: High expression of human beta S- and alpba-globins in transgenic mice: Hemoglobin composition and hematological consequences. Proc Natl Acad Sci USA 89:12150, 1992 7. Rhoda MD, Domenget C, Vidaud M, Bardakdjian Micbau J, Rouyer Fessard P, Rosa J, Beuzard Y: Mouse alpha chains inhibit polymerization of hemoglobin induced by human beta S or beta S Antilles chains. Biochim Biophys Acta 952:208, 1988 8. Skow LC, Burkhart BA, Johnson FM, Popp RA, Popp DM, Goldberg S Z , Anderson W, Barnett LB, Lewis SE: A mouse model for beta-thalassemia. Cell 34:1043, 1983 9. Trudel M, De Paepe ME, Chretien N, Saadane N. Jacmain J, Sorette M, Hoang T, Beuzard Y: Sickle cell disease of transgenic SAD mice. Blood 84:3 189, 1994 10. Schroeder WA, Shelton JB, Shelton JR, Huynh V, Teplow DB: High performance liquid chromatographic separation ofthe globin chains of non-human hemoglobins. Hemoglobin 9:461, 1985 1 I , Fabry ME, Mears JG, Patel P, Schaefer Rego K, Carmichael LD, Martinez G, Nagel RL: Dense cells in sickle cell anemia: The effects of gene interaction. Blood 64:1042, 1984 12. Crispins CG: Handbook on the Laboratory Mouse. Springfield, IL, Thomas, 1975 13. Mozzarelli A, Hofrichter J, Eaton WA: Delay time of hemoglobin S polymerization prevents most cells from sickling in vivo. Science 237:500, 1987 14. Coletta M, Hofrichter J, Ferrone FA, Eaton WA: Kinetics of sickle haemoglobin polymerization in single red cells. Nature 300: 194, 1982 15. Eaton WA, Hofrichter J: Hemoglobin S gelation and sickle cell disease. Blood 70:1245, 1987 16. Eaton WA, Hofrichter J: Sickle cell hemoglobin polymerization. Adv Protein Chem 40:63, 1990 17. Kaul DK, Nagel RL: Sickle cell vasoocclusion: Many issues and some answers. Experientia 495, 1993 (review) 18. Romero J, Schwartz RS, Fabry ME, Nagel RL, Canessa M: Deoxygenation ofred cells from transgenic mice with high HbS FABRY ET AL stimulates a charybdotoxin-sensitive K' efflux andforms dense cells. Clin Res 41:307A, 1993 (abstr) 19. Fabry ME: Transgenic animal models of sickle cell disease. Experientia 49:28, 1993 (review) 20. Scott AF, Bunn HF, Brush AH: The phylogenetic distribution of red cell 2,3 diphosphoglycerate and its interaction with mammalian hemoglobins. J Exp Zoo1 201:269, 1977 21. Petschow R, Petschow D, Bartels R, Bauman R, Bartels H: Regulation of oxygen affinity in blood of fetal, newborn, and adult mouse. Respir Physiol 35:271, 1978 22. Brugnara C: Membrane transport of Na and K and cell dehydration in sickle erythrocytes. Experientia 49:100, 1993 (review) 23. Joiner CH: Cation transport and volume regulation in sickle red blood cells. Am J Physiol 264:C251, 1993 (review) 24. de Franceschi L, Saadane N, Trudel M, Alper SL, Brugnara C, Beuzard Y: Treatment withoral clotrimazole blocks Ca(2+)activated K+ transport and reverses erythrocyte dehydration in transgenic SAD mice. A model for therapy of sickle cell disease. J Clin Invest 93:1670, 1994 25. Monplaisir N, Merault G, Poyart C, Rhoda MD, Craescu C, Vidaud M, Galacteros F, Blouquit Y, Rosa J: Hemoglobin S Antilles: A variant with lower solubility than hemoglobin S and producing sickle cell disease in heterozygotes. ProcNatlAcadSciUSA 83:9363, 1986 26. Mills LR, Mwakyusa D, Milner PF: Histopathologic features of liver biopsy specimens in sickle cell disease. Arch Pathol Lab Med 112:290, 1988 27. Schubert 'IT: Hepatobiliary system in sickle cell disease. Gastroenterology 90:2013, 1986 (review) 28. Bauer TW, Moore GW, Hutchins GM: The liver in sickle cell disease. A clinicopathologic study of 70 patients. Am J Med 69:833, 1980 29. Charlotte F, Bachir D,NenertM, Mavier P, Galacteros F, Dhumeaux D, Zafrani ES: Vascular lesion of the liver in sickle cell disease. Arch Pathol Lab Med 119:46, 1995 30. Kaul DK, Fabry ME, Costantini F, Rubin EM, Nagel RL: In vivo red cell-endothelial interaction and intravascular sickling in a transgenic mouse line expressing sickle hemoglobin. Blood 84:867a, 1994 (abstr)
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