ARGUS-10 (Hamamatsu Photonics, Hamamatsu, Ja- pan). Length of cancellous ... Co., St. Louis, MO) as substrate and fast blue RR salt. (Sigma Chemical Co.) ...
American Journal of Pathology, Vol. 147, No. 6, December 1995 Copyngjht C) American Society for Investigative Pathology
Bone Marrow Neutrophilia and Suppressed Bone Turnover in Human Interleukin-6 Transgenic Mice A Cellular Relationship among Hematopoietic Cells, Osteoblasts, and Osteoclasts Mediated by Stromal Cells in Bone Marrow
Hidetomo Kitamura, Hiromitsu Kawata, Fumiaki Takahashi, Yoshinobu Higuchi, Tatsuya Furuichi, and Hiroyuki Ohkawa From the Fuji-Gotemba Research Laboratory, Chugai Pharmaceutical Co., Komakado, Gotemba City, Shizuoka, Japan
To elucidate the effect of interleukin-6 (IL-6) on bone and bone marrow (BM), human IL-6 transgenic mice (hIL-6 tgm) were produced Their bone and BM were examined histologically, radiologicaly, histomorphometrically, and hematologically on a temporal basis. hIL-6 tgm showed histologicaly evident neutrophilia in BM. Increase in precursors of granulocytes and monocytes in hIL-6 tgm was demonstrated by an assay for colony forming unit in culture (CFU-C) of BM cells. Decrease in osteoblasts and osteoid and suppression of primary spongiosa formation were predominantly observed in hIL-6 tgm at 14 weeks old, the terminal stage of lifefor hIL-6tgm. An assay for colony forming unit in fibroblastic (CFU-F) of BM cells revealed a decrease in osteoblast precursor (with regard to alkaline phosphatase-positive colonies) in hIL-6 tgm at 15 weeks old Histomorphometry demonstrated a decrease of both osteoclast number and bone resorption in hIL-6 tgm. These results suggested that enhanced granulocytic hematopoiesis, suppressed bone turnover, and alteration of cellular population in stromal cells in BM occurred in hIL-6 tgrn Thus we provide new findings that facilitate understanding of celular interrelationships among hematopoietic cells, osteoblasts,
1682
and osteoclasts mediated by stromal ceUs in BM. (Am J Pathol 1995, 147:1682-1692)
Interleukin-6 (IL-6) is a multifunctional cytokine,1 and its effect on stimulating osteoclastgenesis has been demonstrated by in vitro and ex vivo experiments.2`S This cytokine is secreted from bone marrow stromal cells and osteoblasts. The secretion is promoted by parathyroid hormone and suppressed by estrogen.6-9 Examinations in ovariectomized osteoporosis models suggested that IL-6 plays an important role in enhanced bone resorption or bone mass reduction.10'11 Furthermore, it has been reported that ovariectomized IL-6 gene knockout mice do not show either enhanced bone resorption or reduced bone mass.12 Involvement of IL-6 has been reported not only in osteoporosis but also in other bone lesions such as Paget's disease and bone lesion associated with multiple myeloma.13,14 Moreover, IL-6 causes proliferative effects on precursors of granulocytes and monocytes in bone marrow,15-18 where it has been recognized as the source of osteoblasts and osteoclasts. Thus IL-6 has many effects on bone and bone marrow. In the present study, we have produced human IL-6 transgenic mice (hlL-6 tgm) to elucidate the effect of sustained overexpression of hlL-6 on bone and bone marrow. Thus we report on cellular interrelationships among hematopoietic Accepted for publication August 17, 1995. Address reprint requests to H. Kitamura, Preliminary Drug Evaluation Laboratory, Fuji-Gotemba Research Laboratory, Chugai Pharmaceutical Co., Ltd., 1-135, Komakado, Gotemba City, Shizuoka, 412, Japan.
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cells, osteoblasts, osteoclasts, and stromal cells in bone marrow.
Materials and Methods Production of hIL-6 tgm The 3.3-kb Sphl-Xhol fragment (Ld-IL-6) containing human IL-6 cDNA fused with H-2Ld promoter was microinjected into the pronucleus of fertilized eggs from C57BL/6J (Clea Japan Inc., Tokyo, Japan) as described.19 The plasmid containing human IL-6 cDNA and H-2Ld promoter was generously provided by Prof. T. Kishimoto (Institute for Molecular and Cellular Biology, Osaka University, Japan). Integration of the transgene was screened by Southern blot analysis of EcoRI digested tail DNA using a32P-labeled Taql-Banll fragment of human IL-6 cDNA as a probe. For experiment 1,atotalof 11, 10, and 13micefromhlL-6tgm;anda total of 9, 1 1, and 13 mice from normal littermates were submitted for examination at 9, 1 1, and 14 weeks old, respectively. For experiment 2, a total of 3, 3, and 4 mice from both hlL-6 tgm and normal littermates were submitted for analysis of bone marrow cell population with colony formation assays at 5, 10, 15 weeks old. Regular diet for rodents (CE-2 consisting of 1.78% Ca, 1.33% inorganic phosphorus (Pi), 200 IU/100 g vitamin D3, Clea, Japan) and tapwater were given ad libitum. They were kept under specific pathogen-free conditions.
Experiment 1 Serum and Urine Biochemical Analysis Blood for serum samples was collected via cardiac puncture under ether anesthesia. At 14 weeks old, mice were placed into individual metabolic cages for 24 hours from the day before sacrifice, and urine samples were collected. The urine samples were centrifuged, and their supernatants were assayed as described. Concentrations of Ca, Pi, creatinine (Crea) were measured from both serum and urine samples. Serum alkaline phosphatase (ALP), tartrate resistant acid phosphatase (TRAP), and blood urea nitrogen (BUN) were also measured. Ca was measured using an assay kit based on the o-cresolphthalein complexon method (Ca Test Wako, Wako Pure Chemicals, Osaka, Japan).20 Other chemistries were measured with COBAS FARA II automatic analyzer (Nippon Roche, Tokyo, Japan). Serum human IL-6 level was measured with an enzyme-linked immunosorbent assay system using hIL-6 specific monoclonal antibodies.21
Histological Examination Left femurs were removed and fixed with 20% neutral buffered formalin. Femurs were embedded in paraffin, and 4 ,um thick sections were cut and stained with hematoxylin and eosin (H&E). Tibiae were obtained only from 14-week-old mice and subjected to osteoid staining.22 Histopathological examination and histomorphometric analyses were performed with the H&Estained specimens. The latter analysis was measured with a microscope imaging analyzer ARGUS-10 (Hamamatsu Photonics, Hamamatsu, Japan). Length of cancellous bone surface (BS), area of cancellous bone (BV), area of examined tissue (TV), length of osteoclast surface (OcS), length of eroded surface (ES), and number of osteoclasts (OcN) were measured or counted. Histomorphometry was performed in seven different fields of femoral distal metaphysis under a 20x objective lens. Only secondary spongiosa was examined by morphometry. Definitions of ES, OcN and OcS are as follows: ES, rough bone surface with little cellular adhesion except for osteoclasts; OcN, number of cells that have one or more nuclei and a foamy eosinophilic cytoplasm and are attached to the bone surface; and OcS, length of ES demonstrating osteoclast adhesion. As a parameter of bone mass, fractional bone volume (BV/TV) was estimated. As parameters of bone resorption and osteoclast formation, fractional eroded surface (ES/BS), fractional osteoclast surface (OcS/BS), and mean osteoclast number (OcN/ BS) were also estimated. These parameters were calculated according to the following formulas.23
(=m')
BV BV/TV (%) = TV (jtm')
x 100
ES/BS (%)
=
ES (gm) BS (,um) x
OcS/BS (%)
=
OcS (,um) BS (,um)
OcN/BS (count/mm)
=
100 100
OcN (count) BS
(cm)
1000
Bone Measurement Removed femurs were fixed with 70% ethanol, and soft tissues were subsequently removed. Bone length was measured as distance from greater trochanter to medial condyle using a vernier caliper.
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granulocytes, monocytes, and their common progenitor cells (CFU-G, -M, and -GM, respectively) were counted microscopically.
Bone mineral density (BMD) as a parameter of bone measured by single photon absorptiometry with DCS-600 (Aloka, Tokyo, Japan). mass, was
Experiment 2
CFU in Fibroblastic Assay of Bone Marrow Cells
Colony Forming Unit in Culture Assay of Bone Marrow Cells
Mononuclear bone marrow cells that were used in the CFU-C assay were also used in the CFU in fibroblastic (CFU-F) assay. Mononuclear bone marrow cells were suspended with 10% FCS in a-modified minimal essential medium (GIBCO, Grand Island, NY) and 8.0 x 105 cells were placed into a well of a six-multiwell plate (Corning). Culture media were changed on day 4, and after that, every 3 days. After 14 days of culture, the cells were fixed for 30 seconds with ice-cold methanol (90% v/v) containing formalin (9.99% v/v) and acetic acid (0.01% v/v). Subsequently they were washed with distilled water, and ALP cytochemistry was performed. ALP activity was detected with naphthol AS-BI (Sigma Chemical Co., St. Louis, MO) as substrate and fast blue RR salt (Sigma Chemical Co.) as coupler.25 Stained specimens were observed microscopically at 10OX magnification, and the colony number was counted. Clusters of adherent cells that consisted of >10 cells were regarded as a colony. Colonies that contained ALP-positive (ALP') cells were counted as ALP+ colonies.
The colony forming unit in culture (CFU-C) assays were performed using a modified method of Bradley and Metcalf.24 Briefly, both femurs were removed from mice aseptically, bone marrow was flushed out with 10% fetal calf serum (FCS; Moregate, Melbourne, Australia) in Iscove's modified Dulbecco's medium (IMDM; Boehringer Mannheim, Mannheim, Germany). 1.0 x 105/ml of mononucleated bone marrow cells was suspended in 0.3% bacto-agar (DIFCO, Detroit, MI) in 20% FCS/IMDM, which contained 10 ng/ml of recombinant murine granulocytemacrophage colony-stimulating factor (INTERGEN, Purchase, NY), 20 ,ug/ml transferrin (Boehringer Mannheim), and 20 ,ug/ml soybean-lipids (Boehringer Mannheim). A volume of 0.5 ml of this suspension was placed into a well of a 24-multiwell plastic plate (Corning, Corning, NY) and cultured at 370C in 5% C02 in humidified air for 6 days. The cultured coagulated suspensions were collected, dried, and stained with the May-Giemsa method. The number of Table 1.
Results of Serum Biochemical Analyses
Age Sex (weeks) Female
Male
Ca (mg/dl)
Pi (mg/dl)
ALP (U/I)
TRAP (U/I)
BUN (mg/dl)
Crea (mg/dl)
7.49 ± 0.60 9.01 ± 0.41** 8.04 ± 0.68 8.22 ± 1.06 8.10 ± 0.63 10.4 ± 1.17** 7.41 ± 0.73 9.18 ± 0.71** 9.82 ± 0.66 9.48 ± 0.78 8.28 ± 0.21 9.48 ± 0.49**
9.37 ± 0.90 11.7 ± 1.36* 11.5 ± 2.24 10.7 ± 0.93 8.12 ± 0.79 10.7 ± 2.79* 9.01 ± 0.82 11.2 ± 0.59 11.1 ± 1.32 10.4 ± 0.93 8.53 ± 0.76 10.5 ± 1.75
280 ± 19.4 137 ± 33.6** 249 ± 25.2 197 ± 24.8* 254 ± 45.4 50.1 ± 18.7** 216 ± 27.8 94.4 ± 16.9** 102 ± 59.8 62.8 ± 23.5 196 ± 16.0 36.2 ± 3.80**
1.80 ± 0.51 1.60 ± 0.52 3.32 ± 0.67 2.68 ± 1.00 4.25 ± 0.50 4.30 ± 1.13 1.33 ± 0.13 1.70 ± 0.76 2.73 ± 0.49 2.16 ± 0.92 4.56 ± 0.62 3.58 ± 0.63*
33.4 ± 5.19 24.8 ± 4.50* 30.7 ± 4.50 26.1 ± 3.14 24.3 ± 4.69 54.1 ± 32.9* 32.5 ± 1.07 23.2 ± 1.68** 25.7 ± 3.41 22.6 ± 5.47 25.4 ± 4.16 53.0 ± 30.2
0.68 ± 0.08 0.62 ± 0.08 0.86 ± 0.05 0.81 ± 0.10 0.43 ± 0.04 0.48 ± 0.12 0.63 ± 0.09 0.53 ± 0.06* 0.76 ± 0.13 0.65 ± 0.05 0.44 ± 0.04 0.64 ± 0.04**
Status
Normal (n = 5) hlL-6 tgm (n = 5) 11 Normal (n = 5) hlL-6 tgm (n = 5) 14 Normal (n = 8) hlL-6 tgm (n = 7) 9 Normal (n = 4) hIL-6 tgm (n = 6) 11 Normal (n = 6) hlL-6 tgm (n = 5) 14 Normal (n = 5) hlL-6 tgm (n = 6) 9
Numbers represent means ± SD. *,
Table 2.
Results of Urine Ca/Crea and Pi/Crea Ratio Assay
Sex
Status
Female
Normal hlL-6 tgm Normal hlL-6 tgm
Male
Significant difference from same-sex normal littermates (*P < 0.05; **P < 0.01).
Ca (mg/dl) 8.00 13.8 8.00 9.08
± ± ± ±
4.92 5.35 1.91 4.21
Pi (mg/dl) 144 145 168 163
± ± ± ±
24.8 42.4 16.0 23.3
Crea (mg/dl) 25.6 22.3 37.6 25.4
± ± ± ±
5.30 10.7 7.26 10.5
Ca/Crea 0.32 0.71 0.21 0.41
± ± ± ±
Numbers represent means ± SD. *: Significant difference from same-sex normal littermates (*P < 0.05).
0.21 0.30* 0.04 0.23
Pi/Crea 5.66 7.23 4.57 7.38
± ± ± ±
0.39 2.15 0.73 2.83
Bone and Bone Marrow of hIL-6 Transgenic Mice 1685 AJP December 1995, Vol. 14 7, No. 6
Serum and Urine Pi Although the serum Pi concentration tended to be raised in female hIL-6 tgm except at 11 weeks old, it was not evident in the male counterparts. The urinary ratio of Pi/Crea at 14 weeks old was also higher in hIL-6 tgm.
Results Experiment 1 Serum and Urine Biochemical Analysis Results of serum and urine biochemical analysis are shown in Tables 1 and 2.
Serum and Urine Ca
Serum ALP and TRAP Interestingly, serum ALP decreased in hlL-6 tgm of both sexes as the mice became older until it fell to 1/5 of the levels observed in normal littermates at 14 weeks old. In contrast, the serum TRAP concentration in hlL-6 tgm remained at levels similar to those observed in normal littermates.
hlL-6 tgm showed higher serum Ca concentrations compared with those of normal littermates at ages between 9 and 14 weeks old. Urinary Ca/Crea ratio at 14 weeks old showed higher excretion of Ca in hlL-6 tgm. Although these changes occurred in male mice, they were prominent in female mice. Results of Assay for Serum hlL-6 Concentration
Table 3.
Status Normal
hlL-6 tgm
Sex
9 weeks old
Serum hlL-6 concentration (pg/ml) 1 1 weeks old
Female Male Female Male
ND ND 202.4 + 223.9 288.1 ± 228.8
ND ND 919.7 + 461.7 603.0 + 122.2
14 weeks old
ND ND 2170.0 ± 856.1 1418.1 + 474.5
Numbers represent means -- SD. ND: Not detected.
WIA."D
"'
t: vM MMhO,*L
.,*,..s
It4
I.'-, t9
I._ 'Ai.:
'4'W5 '
;
'
j i
t.
* i1^,t,
'T
,
'
E.
.5: _.5.
Figure 1. Photomicrograph of bone marrow in femoral diaphvsis. (a) Normal littermate; (b) hIL-6 tgm. hIL-6 tgm showed marked increase of neutropbils and mild increase of megakaryocytes and immature myeloid cells. 14 weeks old, male, H&E, original magnification X80.
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Kitamura et al
im;~ ~ F'
AJP December 1995, Vol. 14 7, No. 6
BUN and Crea
Temporal profiles of BUN and the serum Crea concentration were similar. In hlL-6 tgm, levels were slightly lower than the normal littermates at 9 and 11 weeks old. However, at 14 weeks old, hlL-6 tgm showed higher BUN and serum Crea concentrations than normal littermates at levels, which suggested the onset of renal failure in this group.
Serum hlL-6 Concentration
Results of the assay for the serum hlL-6 concentration are shown in Table 3. Serum hlL-6 concentration in hlL-6 tgm remained highly elevated in both sexes. This result also verified the hlL-6 overexpression in hlL-6 tgm at all ages. Furthermore, normal littermates did not produce detectable hlL-6 at any of the ages within this experimental design.
Histological Examination
In bone marrow of hlL-6 tgm, an increase of neutrophils (neutrophilia), immature myeloid cells (not iden-
tified), and megakaryocytes were observed (Figure 1b) at 9 weeks old. In particular, neutrophilia became more evident with aging. Such bone marrow produced a histologically monotonous appearance because of the presence of numerous neutrophils. Cells of other hematopoietic lineages such as erythroblast islets were hardly observed. The increase of immature myeloid cells and megakaryocytes in hlL-6 tgm was mild, and there was no apparent difference with regard to these cells at any of ages tested. A very small number of plasma cells were observed in the bone marrow of hlL-6 tgm. In the bone tissue of hlL-6 tgm, several histological changes were observed and mainly apparent at 14 weeks old. Cuboidal osteoblasts were decreased at the bone surface of primary and secondary spongiosa and cortical bone in hlL-6 tgm (Figure 2b). Some parts of this bone surface were not covered, and the others were covered only with lining cells. The suppression of primary spongiosa formation and hypertrophic disorder of growth plate chondrocytes were also observed in hlL-6 tgm together with a decrease of osteoblasts. In the junction between the J
la t
..
%
Sr
lia~~~~~~
.~~~~~w
Ve '.
J.,~~~~~~~~,
Figure 2. Photomicrograph of distalfemoral metaphysis. (a) Normal littermate; (b) hIL-6 tgm. Cuboidal osteoblasts (arrowheads) are seen along bone surface in normal littermate. These cells were not observed in bIL-6 tgm. Hypertrophic failure of chondrocytes (arrow) was also seen in growth plate (GP) of bIL-6 tgm. 14 weeks old, male, H&E, original magnification X 40.
Bone and Bone Marrow of hIL-6 Transgenic Mice
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AJP December 1995, Vol. 14 7, No. 6
growth plate and primary spongiosa of such mice, osteoclasts were also decreased. These findings at 11 weeks old were milder than those observed at 14 weeks old. In the specimens with osteoid staining, osteoid volume in primary and secondary spongiosa and cortical bone was decreased in hlL-6 tgm (Figure 3b). The distribution of this decrease coincided with the diminished presence of osteoblasts. Results of histomorphometric analysis are summarized in Table 4. Parameters for the osteoclastgenesis and bone resorption, OcN/BS, OcS/BS and ES/BS were lower in hlL-6 tgm as compared with those of normal littermates at all ages tested. Values for BV/TV, a parameter of bone volume, were not
obviously decreased in hlL-6 tgm at any of the ages tested. Bone Measurement Results of bone length and BMD are shown in Figure 4. Femurs obtained from hlL-6 tgm were shorter than those of normal littermates at all ages. Although it seemed that BMD in hlL-6 tgm was slightly lower than that in corresponding normal littermates, this was not significantly different. The accuracy of BMD was verified by comparison between actual ash weight and bone mineral content as determined by single photon absorptiometry using the same bone
*0
:.. ; A,4w .
--:fi
.4
*'$--
Figure 3. Photomicrograph ofproximal tibial metaphysis. (a) Normal littermate; (b) hIL-6 tgm. Thickly stained osteoid area (arrowheads) observed in hIL-6 tgm. GRP growth plate. 14 weeks old, female, osteoid staining, original magnification X32.
Table 4. Status
Female Normal hlL-6 tgm Male Normal hlL-6 tgm
hardly
Results of Bone Histomorphometry 9 weeks old
Sex
was
11 weeks old
ES/BS (%) OcS/BS (%) OcN/BS (Imm) BVfTV (%) 11.1 ± 3.93 8.45 ± 9.16 ± 1.41 8.47 ± 10.3 ± 1.62 9.67 ± 9.36 ± 2.80 8.01 ±
Numbers represent means
±
2.36 2.00 ± 0.78 1.05 1.56 ± 0.24 2.04 2.17 ± 0.46 2.37 1.34 ± 0.45*
25.4 25.6 27.2 26.3
± ± ± ±
4.37 7.55 1.32 7.60
ES/BS (%) 16.2 ± 8.64 ± 11.0 ± 5.71 ±
2.66 2.21*^ 3.82 1.71*
14 weeks old
OcS/BS (%) OCN/BS (/mm) BV/TV (%) 15.2 ± 8.039.17 ± 4.73 ±
1.89 2.32 ± 0.26 1.79** 1.42 ± 0.25^* 2.82 1.31 ± 0.09 2.31* 0.894 ± 0.42
18.5 ± 28.7 ± 22.9 ± 21.9 ±
SD. *, **: Significant difference from same-sex normal littermates (*P < 0.05; **P < 0.01).
4.79 8.47 4.74 5.66
ES/BS (%) 17.3 ± 8.92 ± 7.48 ± 5.02 ±
OcS/BS (%) OcN/BS (/mm)
4.77 13.2 ± 4.68 2.24 ± 1.38^* 6.91 ± 1.69** 1.34 ± 3.38 5.71 ± 2.85 1.36 ± 1.71 2.17 ± 1.37* 0.509 ±
0.71 0.28* 0.61 0.30*
BVflV (%) 11.5 20.3 15.1 13.4
± 4.28 ± 3.07** ± 2.08 ± 2.86
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Kitamura et al
AJP December 1995, Vol. 14 7, No. 6
I-
E
O Normal female
*
0 Normal male a
O NomMal
hIL-6 tgm female
* hlL-6 tgm IC
* hIL-4 tgm male
*01c
0
. U1
m age
(week old)
age (week old)
ol Normal
female
*
hlL4 tgm female
C2
Normal male
*
hIL-6 tgm male
E S000~4O00 400-
*
Nomnal
.
hILL-Stgm
e
E
24.4
II.
T*-
25.1
E
rn
27.0
200 11
age
(week oid) 5
Figure 4. Summary of bone measurements. BMD tended to be lower in hIL-6, but there was no significant difference (NSD) from normal littermates (A). Femurs of hIL-6 tgm were shorter than normal littermates (B). The numbers represent the means ± SD $ Significant differencefrom the same-sex nonnal littermates ("P < 0.05, **P < 0.01). 10000-
E
c c
E:
4000T*
.
TI
hlL- tgm
50*
6000T* 43.1
Experiment 2 Results of CFU-C assay were shown in Figure 5. The colony numbers of CFU-GM, -G, and -M were increased in hlL-6 tgm. The ratios of colony numbers of respective precursors (hlL-6 tgm/normal littermates) were: GM, 1.29 to 1.81; G, 1.81 to 2.01; and M, 1.72 to 2.05, as measured between 5 and 15 weeks old. The proportion of the CFU-GM, -G, and -M in hlL-6 tgm was similar to that in normal littermates.
Nonmal
G
T
T
CFU-C Assay
15
52.9
E
sample, and there was a very high correlation between them (r2 = 0.91; data not shown).
10
age (week old)
5. 50. T ~~~~T
20000 5
age
10 (week old)
15
Figure 5. Summary of CFU-C assay ofbone marrow cells. GM, colonies consisting ofboth granulocyte and monocyte precursors. G, colonies of granulocytic precursors. M, colonies of monocytic precursors. All types of examined colonies increased in hIL-6 tgm. Numbers (O%) above bars indicate proportion of the colony versus total colony numbers. 7Tis proportion showed no obvious alteration between hIL-6 tgm and normal littermates. # Significant difference from the age matched normal littennates (9q P < 0.05, *$. P < 0.01).
CFU-F Assay Results of CFU-F assay were summarized in Figure 6. In normal littermates, total colony number and ALP' colonies, which contained precursors of osteoblasts, increased with time. In hlL-6 tgm, colony number and proportion of ALP+ colonies significantly decreased at 15 weeks old (Figure 7). Total
colony number in hIL-6 tgm at 15 weeks old tended to decrease. The individual colonies of hlL-6 tgm consisted of a smaller number of cells compared with the normal littermate counterparts; thus the colony size in hlL-6 tgm was smaller than that of normal littermates.
Bone and Bone Marrow of hlL-6 Transgenic Mice 1689 AJP December 1995, Vol. 14 7, No. 6
o Norman hIL-4tgm
200-
IE
T2"l;1
E
0
0T
10
5
15
age (week old)
100-
o Normal hIL-S6tgn
B
4! u~~~~~~4.4 55~~~~.2 252.5
c
50
10
T 14.7
5
10
15
age (week old)
Figure 6. Summaty of CFU-F assay of bone marrow coells. Total colony number (A) tended to decrease in hIL-6 tgm at 15 weeks old. ALFP colony number (B) were significantly decreased in hIL-6 tgm at 15 weeks old. Numbers (%) above bars indicate proj o?rtion of ALP+ colonies versus total colony numbers. qT: Signjficant difference from the age-matched normal littermates ( 9/ P < 0.
