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*
Department of Immunology, Schering-Plough Research Institute, Kenilworth, NJ 07033;
Department of Drug Safety and Metabolism, Schering-Plough Research Institute, Lafayette, NJ 07848; and
DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1
were elevated, and the number of circulating neutrophils was increased.
In addition, ubiquitous expression of p19 resulted in constitutive
expression of acute phase proteins in the liver. Surprisingly,
liver-specific expression of p19 failed to reproduce any of these
abnormalities, suggesting specific requirements for production of
biologically active p19. Bone marrow transfer experiments showed that
expression of p19 by hemopoietic cells alone recapitulated the
phenotype induced by its widespread expression, pointing to hemopoietic
cells as the source of biologically active p19. These findings indicate
that p19 shares biological properties with IL-6, IL-12, and G-CSF and
that cell-specific expression is required for its biological
activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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from Th1
and NK cells (reviewed in Ref. 6). To investigate the biological properties of p19, we generated transgenic animals expressing it ubiquitously or in a tissue-specific fashion. Phenotypic analysis of these transgenic animals indicates that p19 has biological properties related to IL-6, G-CSF, and IL-12 and that its expression from hemopoietic cells is a prerequisite for its biological activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 0.5-kb cDNA encoding p19 was cloned as an EcoRI
fragment into an expression vector containing the human CMV
enhancer/chicken 
-actin promoter and the rabbit -globin
polyadenylation signal (7). For liver specific expression,
the cDNA for p19 was cloned into an expression vector containing the
promoter for human 1-antitrypsin
(HAT,2 bp 1–1976, GenBank
accession number K02212) and 145 bp of a transcriptional enhancer from
the human 1-microglobulin/bikunin gene (bp
2163–2308) (Fig. 5
A). Transgenes were separated from vector
sequences by zonal sucrose gradient centrifugation as described
(8). Fractions containing the transgenes were pooled,
microcentrifuged through Microcon-100 filters (Amicon, Bedford, MA),
and washed five times with microinjection buffer (5 mM Tris-HCl (pH
7.4), 5 mM NaCl, 0.1 mM EDTA).
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DNA containing the transgene was resuspended in microinjection
buffer to a final concentration of 1–5 ng/ml and microinjected into
mouse eggs (C57BL/6J x DBA/2 (B6D2) F2; The
Jackson Laboratory, Bar Harbor, ME). The surviving eggs were
transplanted into oviducts of ICR (Sprague Dawley) foster mothers,
according to published procedures (9). By 10 days of life,
a piece of tail from the resulting animals was clipped for DNA
analysis. Founders carrying the p19 gene under control of the human CMV
enhancer/chicken -actin promoter (Cp19) were identified by PCR
amplification of a segment of the transgene using primers
5'-GCCCTCCTCCAGCCAGAGGAT-3' and
5'-CCAGCCACCACATTCTGATAGGCAGCCTGCAC-3'. As an internal
control for the amplification reaction, primers for the endogenous low
density lipoprotein gene were used: 5'-ACCCAAGACGTGCTCCCAGGATGA-3'
and 5'-CGCAGTGCTCCTCATCTGACTTGT-3'. These primers amplify a 200-bp
segment of the transgene and a 397-bp segment of the low density
lipoprotein gene, respectively. To identify founders carrying
the p19 gene under control of the HAT promoter (Hp19), we used the PCR
primers 5'-CCGTTGCCCCTCTGGAT-3' and 5'-GATTCATATGTCCCGCTGGTG-3'. As an
internal control for this amplification, reaction primers for the
endogenous ZP3 gene were used: 5'-CAGCTCTACATCACCTGCCA-3'
and 5'-CACTGGGAAGAGACACTCAG-3'. These primers amplify a 327-bp
segment of the transgene and a 511-bp segment of the ZP3
gene, respectively. PCR conditions were: 95°C, 30 s; 60°C,
30 s; 72°C, 60 s, for 30 cycles. The resulting transgenic
mice were kept under specific pathogen-free conditions. Experiments
were performed following the guidelines of the Schering-Plough Animal
Care and Use Committee.
RNA analysis
RNA was extracted from tissues using RNA STAT-60, following
specifications from the manufacturer (Tel-Test, Friendswood, TX). Total
RNA (20 µg) was denatured and blotted onto Biotrans membrane (ICN
Biomedicals, Costa Mesa, CA). Transgene expression was assessed by
hybridization to randomly labeled (Stratagene, La Jolla, CA) p19 cDNA.
Acute phase gene expression in liver was assessed by Northern blot
hybridization of total RNA with 32P-labeled PCR
segments of the murine hemopexin gene (PCR primers:
5'-GGATGCCCGTGACTACCTTCGTAT-3' and 5'-GGGCCAGGAAACCTCTGT-3'), of the
murine 1-acid glycoprotein (AGP-1; PCR
primers: 5'-CCAGTGTGTCTATAACTCCACCC-3' and
5'-GACTGCACCTATCCTTTTTCCA-3') or of the murine haptoglobin gene (PCR
primers: 5'-AGAGTATAGCCCAACCCTTCC-3' and
5'-GAGAATAGTACAGTGCCCGAGAA-3'). A PCR segment of the murine -actin
gene was amplified using specific primers purchased from Stratagene,
labeled as described above, and used for Northern blot
hybridization.
ELISA
ELISA kits for murine IL-2 (sensitivity, <3 pg/ml), murine
IL-1 (sensitivity, <3 pg/ml), murine IFN- (sensitivity, <2
pg/ml), murine TNF- (sensitivity, <5.1 pg/ml), and murine IL-12 p40
(sensitivity, <4 pg/ml) were purchased from R&D Systems (Minneapolis,
MN). ELISA kits for murine IL-6 (sensitivity, <8 pg/ml) and murine
serum amyloid A (SAA; sensitivity, <0.23 µg/ml) were purchased from
Biosource International (Camarillo, CA). ELISA kits for murine IL-1
(sensitivity, <6 pg/ml) were purchased from Endogen (Cambridge, MA).
Assays were performed according to the manufacturer’s
instructions.
Levels of insulin-like growth factor-1 (IGF-1) in mouse serum were determined using a RIA for human IGF-1 that also recognizes murine IGF-1. Serum samples were acid-ethanol extracted according to instructions provided by the manufacturer (Nichols Institute, San Juan Capistrano, CA).
Histology
Mouse tissues were fixed by immersion in 10% phosphate-buffered formalin. Formalin-fixed tissues were routinely processed at 5 µm and were stained with hematoxylin and eosin (H&E). The number of megakaryocytes in transgenic and nontransgenic spleens was determined by counting the number of megakaryocytes in at least five different optical fields for each spleen.
Hematology
Blood samples were collected from the infraorbital sinus into sterile, evacuated tubes with added EDTA (Vacutainer Systems, Becton Dickinson, Rutherford, NJ). Hematological values were determined with an automated system (Cell-Dyn 3500; Abbott Laboratories, Abbott Park, IL). Platelet counts were performed manually when the instrument was unable to provide accurate platelet counts due to excessive clumping or excessively large platelets. Blood smears were stained with modified Wright-Giemsa stain (Hema-Tek Stain Pack; Bayer, Elkhart, IN) using an automated stainer (Hema-Tek 2000; Bayer). Bone marrow smear were obtained from the sternum, fixed in methanol, and stained with Wright-Giemsa stains using a Midas II stainer (EM Diagnostics Systems, Gibbstown, NJ).
Bone marrow transfers
Bone marrow cells were flushed from femurs of transgenic and control mice under sterile conditions. Single-cell suspensions (10 million cells) were injected into the tail veins of lethally irradiated (1100 rad for 5 min) B6D2F1 mice (6–8 wk old).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To express p19 in transgenic animals, we initially used the Cp19
transgene (Fig. 1A). The
enhancer/promoter cassette used directs expression of the transgene to
virtually all organs (10). Twenty-four founder mice
(referred to as Cp19) were generated; of these, 10 died during the
first 3 wk of life before data could be collected. Of the remaining 14
founders, 3 grew normally but 11 were small and failed to thrive (Fig. 1B). Reduced body weight was evident in the first 10 days of
life (average weight for Cp19, 5.1 ± 1.9 g,
n = 8; controls, 8.6 ± 1.4g, n =
23; p = 0.0007). Four of the 11 small founders
(founders 25, 36, 51, 88) were severely runted, appeared moribund, and
were sacrificed before weaning. To determine whether there was a
correlation between the appearance of this phenotype and transgene
expression, we performed Northern blot analysis of skeletal muscle RNA
(Fig. 1C). Transgene expression was detected in all 11 Cp19
founders showing stunted growth, whereas no expression was detected in
controls or in the 3 remaining, normally growing founders. All seven
expressing Cp19 founders that survived beyond weaning age were clearly
affected by the expression of the transgene; all had impaired growth, a
swollen abdomen, and ruffled fur. These animals were infertile, and
none survived beyond 90 days of age. Thus, ubiquitous expression of p19
resulted in stunted growth, infertility, and death.
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Tissues were collected from 10 of the 11 Cp19 founders described
in Fig. 1
, routinely fixed, processed to slides, and examined
microscopically. Tissues collected from age- and gender-matched
nontransgenic littermates were used as reference. Minimal to moderate
inflammation (Fig. 2), sometimes
associated with epithelial hyperplasia, was detected in all 10 Cp19
animals (age 15–85 days) in one or multiple sites, including the
esophagus, stomach, small intestine, large intestine, skin, lungs,
liver, and pancreas. In general, the inflammatory infiltrates consisted
of lymphocytes and macrophages, sometimes accompanied by varying
numbers of neutrophils. In the esophagus (Fig. 2, A and
B), stomach, and intestines (Fig. 2C), the
infiltrates were minimal to moderate, multifocal, and primarily
localized in the epithelium, lamina propria, and submucosa and were
often associated with epithelial hyperplasia. The hyperplasia resulted
in lengthening of intestinal glands and shortening or loss of villi in
the small intestine (Fig. 2C). Inflammation in the skin was
multifocal, involved the epidermis and dermis, and was sometimes
associated with acanthosis and/or ulceration (Fig. 2D). In the lungs (Fig. 2, E and F),
findings consisted of peribronchial/perivascular mononuclear cell
infiltrates; neutrophils were not a prominent component of the
pulmonary inflammation. In addition to the inflammation, the epithelium
lining bronchi and bronchioles often had minimal to mild hyperplasia,
sometimes with eosinophilic intracytoplasmic material. Inflammation in
the liver consisted of minimal to mild periportal inflammatory
infiltrates (data not shown). Pancreatic inflammatory infiltrates were
minimal and consisted primarily of lymphocytes (data not shown).
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H). The splenic
capsule was sometimes thickened (Fig. 2H). The cortex of
lymph nodes was sometimes sparsely cellular and lacked secondary
follicles. Microscopic changes were not observed in the skeletal
muscle, heart, kidney, and brain, despite high levels of transgene
expression in these organs (Fig. 1 and data not shown). Cp19 mice are anemic and have increased numbers of neutrophils in the peripheral blood
The effect of p19 on peripheral blood was analyzed in three independent founders and compared with data from three nontransgenic littermates. All Cp19 mice examined had mild to moderate microcytic hypochromic anemia. The mean hematocrit values in the Cp19 animals were 37–70% lower than that of controls (Cp19, 23.9 ± 8.3%; controls, 47 ± 2.4%, p = 0.029). Microscopic analysis of blood smears revealed erythrocytes of abnormal shape (poikilocytosis) and/or fragments of erythrocytes (schistocytosis). Slight to moderate polychromasia and variation in the size of erythrocytes (anisocytosis) suggested the presence of regeneration. The presence of microcytosis (small average RBC size) and hypochromia (diminished erythrocyte hemoglobin concentration) could be a direct effect of p19 activity or a secondary effect of the multiorgan inflammation in these animals.
The number of neutrophils in the blood was increased 3- to 11-fold over
the highest neutrophil count in control animals (Table I). This increase in peripheral blood
neutrophils, which is typical of inflammation, was associated with
infiltration of neutrophils into various tissues. Accordingly, the
myeloid component (as assessed by the granulocytic/erythroid ratio) was
increased in the bone marrow relative to the erythroid component.
Interestingly, the number of circulating platelets in Cp19 animals was
slightly increased over the number found in control littermates (Cp19,
1708 ± 599; control, 821 ± 32 x
103 cells/µl; p = 0.12).
Although these differences were not statistically significant, the
presence of excessive clumping and platelets of unusual morphology in
Cp19 blood smears suggested increased platelet production. To determine
whether the increased number of platelets in Cp19 founders was caused
by accelerated platelet production by megakaryocytes or by an increase
in the number of megakaryocytes, we examined the spleen and bone marrow
of Cp19 mice microscopically. In these tissues, megakaryocytes were
enlarged, but their numbers were not increased (Cp19 (n
= 11), 7.5 ± 5.8; control (n = 5), 8.1 ±
3.2 per optical field; p = 0.81), suggesting that the
mild thrombocytosis observed in Cp19 blood resulted from accelerated
production of platelets by megakaryocytes.
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To test whether the systemic inflammation seen in Cp19 animals was
associated with altered expression of proinflammatory cytokines, we
determined the concentrations of IL-1, TNF-, IL-6, IFN-, and
IL-12p40 in the peripheral blood. Concentrations of TNF- were
significantly increased in 7 of 10 founders tested (Fig. 3), and concentrations of IL-1 were
increased in 2 of 3 founders tested (292 and 518 pg/ml).
IL-1 concentrations in serum of unchallenged control littermates ranged
from 0 to 70 pg/ml. The levels of TNF- and IL-1 observed in Cp19
mice were within the range of those observed in control mice after
induction of acute inflammation by LPS (M. T. Wiekowski,
unpublished observation).
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were increased in 3 of 13
founders tested (Fig. 3). Another cytokine involved in an inflammatory
response is IL-12, a heterodimeric cytokine formed by association of
the subunits p40 and p35 (6, 11, 12). Therefore, we
determined the concentration of p40 in serum of eight Cp19 founders.
With the exception of a single Cp19 founder, the levels of p40 did not
differ from controls (Fig. 3C). This indicated that
overexpression of p19 in Cp19 animals did not elevate the
concentrations of circulating p40.
Surprisingly, IL-6 protein could not be detected in the peripheral
blood of Cp19 animals, despite the high circulating concentrations of
TNF- and IL-1, cytokines that directly induce IL-6 production
(13). This was especially surprising considering the fact
that IL-6 is expressed during systemic inflammation (14, 15).
Acute phase protein genes are chronically expressed in the livers of Cp19 animals
During inflammation, genes encoding acute phase proteins are
up-regulated in the liver. Because Cp19 mice exhibit a phenotype
characterized by systemic inflammation, we investigated whether the
expression of acute phase genes was altered in their livers. As shown
in Fig. 4A, the acute phase
genes AGP-1, haptoglobin, and hemopexin were highly expressed in the
liver of all four transgenic founders tested, whereas no expression of
these genes was detected in nontransgenic livers. To test whether the
concentration of acute phase proteins was also increased in the
circulation, blood from Cp19 founders was tested for the presence of
SAA. The average level of circulating SAA (248 ± 159 µg/ml,
n = 10) was significantly increased over the levels
found in controls (8 ± 5.1 µg/ml, n = 8,
p = 0.05) (Fig. 4B). These results indicate
that acute phase liver genes are chronically expressed in Cp19
animals.
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Growth impairment caused by chronic inflammatory conditions
(16, 17) or by overexpression of cytokines in transgenic
animals (18) is associated with a decrease in the
circulating levels of IGF-1, a hormone that regulates postnatal growth
(19) and influences fertility (20). To test
whether the impaired growth of Cp19 animals was associated with reduced
levels of IGF-1, serum samples of transgenic animals were assayed for
IGF-1. In all founders tested, the amount of IGF-1 in the serum was
reduced to 12–14% of the concentrations found in nontransgenic,
age-matched littermates (Table II). This suggests that
overexpression of p19 may directly or indirectly reduce IGF-1
concentrations, resulting in impaired growth and infertility of
transgenic animals (20).
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The infertility and premature death seen in Cp19 transgenic
animals precluded further analysis of the biological function of p19.
Therefore, we attempted to generate another transgenic model expressing
p19 using a tissue-specific promoter. To this end, we made transgenic
animals carrying the p19 gene under the control of the liver-specific
human 1-antitrypsin promoter (Hp19 animals).
We generated eight founders, from which seven transgenic lines were
derived. Transgene expression was detected by Northern blot analysis in
all mice analyzed, but not in controls (Fig. 5B). The two highest
expressing lines were expanded, and a detailed analysis of growth
patterns, blood parameters, expression of acute phase genes, and
histology of several organs was performed. Surprisingly, these
transgenic animals grew normally and had no signs of hepatic (Fig. 5, C and D) or systemic inflammation (data not
shown). In addition, we were unable to detect any changes in the
peripheral blood cell composition or in the expression of acute phase
genes (data not shown). These results suggest that there are
requirements for the biological activity of p19 that are not satisfied
by its production in liver cells.
Transplantation of transgenic hemopoietic cells into wild-type mice results in the development of multiorgan inflammation
The absence of a phenotype in mice overexpressing p19 in liver and
the observation by Oppmann et al. (1) that p19 is
expressed by a subset of hemopoietic cells led us to examine whether
overexpression of p19 by hemopoietic cells would be sufficient to
induce a phenotype similar to the one described for Cp19 animals. We
observed previously that the CMV/-actin promoter targets expression
of transgenes to a variety of cells within the immune system, including
T and B lymphocytes and dendritic cells (21). Thus, we
generated a new set of Cp19 founders and transferred their bone marrow
(Fig. 6A) into lethally
irradiated wild-type recipient mice. The health of these Cp19 bone
marrow recipients deteriorated within 35–66 days posttransfer, as
judged by the appearance of ruffled fur and inflamed skin around the
snout and ventral neck. In contrast, recipients of wild-type bone
marrow did not develop an obvious phenotype. Analysis of the bone
marrow recipients showed p19 expression in the bone marrow (data not
shown) and spleens of Cp19, but not control, bone marrow recipients
(Fig. 6A). In Cp19 bone marrow recipients, the acute phase
liver genes hemopexin and AGP-1 were highly expressed (not shown), and
the serum levels of SAA were elevated (Fig. 6B), but again
no IL-6 could be detected in circulation (data not shown). As in Cp19
donor animals, skin, lung, liver, and the gastrointestinal tract were
inflamed in recipients of Cp19 bone marrow, but not in wild-type bone
marrow recipients. Perivascular/peribronchial infiltrates of
lymphocytes and macrophages and slight epithelial hyperplasia were
observed in the airways of Cp19 bone marrow recipients (Fig. 6, C and D). These results indicate that p19
produced by bone marrow cells is biologically active and can induce a
phenotype of systemic inflammation similar to that obtained by
ubiquitous expression of p19.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). For example, runting or failure to
thrive has been reported for transgenic animals expressing IL-6 in skin
(22), astrocytes (23), neurons
(18), and in the airway epithelium (24); and
for transgenic animals expressing oncostatin M in the skin
(25). Similar to observations in mice overexpressing IL-6,
Cp19 mice had reduced levels of IGF-1, which may have contributed to
their failure to thrive and their reduced fertility (18).
It is unclear whether the reduced levels of IGF-1 were directly caused
by p19 or caused by other factors secreted during systemic
inflammation.
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and IL-1.
Similar findings have been reported in transgenic mice expressing the
p35/p40 IL-12 heterodimer in brain (12). Inflammation,
usually in the form of mesangioproliferative glomerulonephritis, has
also been observed in mice overexpressing IL-6 in liver
(26) and plasma cells (27). This condition,
however, usually develops after several months of chronic exposure to
IL-6, presumably as a function of the high circulating levels of Igs.
This localized inflammation differs from the systemic inflammation
observed in CP19 mice, suggesting that the mechanisms mediating
inflammation in the Cp19 mice may be different from those induced
by IL-6.
Neutrophilia has also been shown as a result of transgenic expression
of IL-6 (26) and G-CSF (5, 28), another
closely related cytokine. However, G-CSF-induced neutrophilia was not
associated with other phenotypic changes as described here for Cp19
animals. Interestingly, the degree of EMH observed in the spleen of
Cp19 animals was disproportionate to the systemic inflammation seen in
these animals, raising the intriguing possibility that p19, like G-CSF,
may have a direct effect on hemopoiesis (29). Likewise, a
direct negative effect of p19 on erythropoiesis cannot be ruled out,
because the Cp19 mice are anemic. Anemia has also been observed in
animals overexpressing IL-6 (32) and in animals repeatedly
injected with IL-12 (11). An indirect mechanism
contributing to the anemia observed in the Cp19 mice could be the
dysregulated cytokine production. For instance, TNF- and IL-1,
cytokines that are elevated in the Cp19 mice, have been shown to play a
role in anemia of chronic disorders (35, 36), a microcytic
hypochromic anemia often seen in humans with inflammatory disease.
In Cp19 animals, acute phase liver genes like AGP-1, haptoglobin, hemopexin, and SAA were chronically expressed, and platelet production was increased. Because IL-6 is the primary inducer of an acute phase response (reviewed in Ref. 30) and functions as a megakaryocyte differentiation factor (31, 32), the phenotype described here could be caused by p19-mediated up-regulation of IL-6 expression. However, neither IL-6 protein nor IL-6 mRNA (data not shown) could be detected in Cp19-transgenic mice. These results suggest that p19 may function directly in the induction of an acute phase response and megakaryocyte differentiation.
In vitro, p19 is secreted in complex with the p40 subunit of IL-12. This p19-p40 heterodimer has now been named IL-23 (1). Apparently, this association is not only necessary for secretion but also seems essential for its biological function, because purified p19 is biologically inert in vitro. These findings have important implications for the interpretation of the phenotypes described here. Expression of p40 is normally restricted to monocytes, macrophages, and dendritic cells in the mouse (33). We hypothesize that the phenotype observed in mice expressing p19 in hemopoietic cells is the function of the simultaneous production of p40, and we suggest that the absence of a phenotype in the animals expressing p19 in liver is due to lack of coexpression of p40 (34). Our inability to detect p40 in the serum of Cp19 animals may indicate either that p19/p40 complexes are found at very low levels in circulation or that they act locally, in an autocrine or paracrine manner. It is also possible that p19 produced by hemopoietic cells has biological activities that are independent of its dimerization with p40.
TNF- and IL-1 are also known inducers of IL-6 expression
(13). Thus, it was surprising that no IL-6 could be
detected in Cp19 animals despite the high concentrations of circulating
TNF- and IL-1. This intriguing and unexpected result suggests that
p19 may have a negative effect on IL-6 expression by a yet unidentified
mechanism.
Overexpression of p19 in Cp19 animals did not always result in an
increase of IFN- expression. This in vivo result differs from the in
vitro results obtained by Oppmann et al. (1), who reported
induction of IFN- by T-cells after IL-23 (p19/p40) treatment.
Unfortunately, this discrepancy could not be satisfactorily resolved
because of the short life span of the transgenic mice, which precluded
analysis of specific immune responses. Thus, the function of p19 on
expression of IFN- in vivo and its functional relationship to IL-12
remains to be determined in mice deficient for p19 and in
transgenic mice expressing p19 conditionally.
Our results show that overexpression of p19 in vivo induces a phenotype resembling that observed on overexpression of the structurally related cytokines IL-12, IL-6, and G-CSF. Further studies will be necessary to understand how expression of p19 leads to these phenotypes and the molecular nature of the receptor(s) mediating these responses.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: HAT, human 1-antitrypsin; SAA, serum amyloid A; IGF-1, insulin-like growth factor-1; H&E, hematoxylin and eosin; EMH, extramedullary hemopoiesis; AGP-1, 1-acid glycoprotein.
Received for publication October 27, 2000. Accepted for publication April 9, 2001.
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