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Originally published online as doi:10.1189/jlb.0506304 on September 14, 2006

Published online before print September 14, 2006
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(Journal of Leukocyte Biology. 2006;80:1445-1453.)
© 2006 by Society for Leukocyte Biology

Transgenic expression of CSF-1 in CSF-1 receptor-expressing cells leads to macrophage activation, osteoporosis, and early death

Suwen Wei, Xu-Ming Dai and E. Richard Stanley1

Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, USA

1 Correspondence: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. E-mail: rstanley{at}aecom.yu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CSF-1 is the primary mononuclear phagocyte and osteoclast growth factor. Autocrine regulation by CSF-1 has been reported in macrophages during inflammatory responses and in neoplastic cells. To investigate whether inflammatory disease or neoplasia was the dominant consequence of autocrine regulation by CSF-1 in CSF-1 receptor (CSF-1R)-expressing cells, we created mice that express CSF-1 under the control of the CSF-1R promoter/first intron driver [transgene TgN(Csf1r-Csf1)Ers (TgRC) mice], which have reduced thymic size, a short lifetime, and low body weight and develop osteoporosis. In 4-week-old TgRC mice, osteoclast numbers are elevated, and macrophage densities are increased in bone marrow, spleen, liver, and brain. Cultured TgRC macrophages express CSF-1 and proliferate without exogenous CSF-1 and in the presence of neutralizing antimouse CSF-1 antibody. Compared with macrophages from nontransgenic littermates, TgRC macrophages exhibit a stellate morphology, express elevated mRNAs for proinflammatory cytokines, and despite a lower, steady-state cytokine secretion, secrete elevated levels of inflammatory cytokines in response to LPS, indicating that TgRC macrophages are functionally primed through the CSF-1R. Thus, autocrine regulation of CSF-1R-expressing cells by CSF-1 leads to a severe phenotype that emphasizes the importance of the known, local production of CSF-1 in inflammatory disease.

Key Words: inflammation • autocrine regulation • cytokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CSF-1, also known as M-CSF, is the primary regulator of the development, survival, proliferation, and differentiation of tissue macrophages and together with the receptor activator of NF-{kappa}B (RANK) ligand, regulates osteoclast production and function [1 ]. Normal expression of the CSF-1 receptor (CSF-1R) is largely restricted to macrophages, osteoclasts, certain dendritic cells, decidual cells, and cells of the trophoblast [2 , 3 ]. Local and humoral regulation of these cells by CSF-1 is mediated by the distinct but overlapping actions of the secreted glycoprotein, the secreted proteoglycan, and the cell-surface, membrane-spanning isoforms of CSF-1, which are expressed by a wide variety of cell types [4 5 6 7 8 ].

CSF-1 not only regulates macrophage survival and proliferation but also macrophage activation [2 ]. Compared with macrophages derived from monocytes by culture with GM-CSF, CSF-1-induced, monocyte-derived macrophages are distinct in their cell-surface antigen expression and exhibit higher Fc{gamma}R-mediated phagocytosis, reactive oxygen species production, and sensitivity, stronger suppressor activity, and resistance to Mycobacterium tuberculosis. CSF-1 also stimulates macrophage chemotaxis and cytokine production [9 ]. Although priming of monocytes/macrophages with CSF-1 down-regulates expression of many TLRs, it is without effect on TLR4 [10 ] and increases expression of another LPS receptor, CD14 [11 ]. Thus CSF-1 enhances cytokine production in response to LPS but suppresses the CpG DNA response [10 ]. CSF-1 also induces the expression of CD16 (Fc{gamma}RIII) on the CD14+CD16+ subset of human monocytes [11 ], which exhibit a more activated, macrophage-like character. CD16+ monocytes are elevated in a variety of infectious and chronic inflammatory conditions [2 ].

CSF-1 regulation of macrophages and osteoclasts plays an important role in chronic inflammatory disease and cancer. In mouse models of arthritis [12 ], atherosclerosis [13 ], obesity [14 ], and autoimmunity [15 ], the degree of severity of disease is reduced greatly in CSF-1-deficient mice compared with their littermate controls. In cancer, circuIating CSF-1 is elevated in myeloid and lymphoid leukemias [16 ] and in cancers of the female reproductive system [17 ]. In breast cancer, an increased accumulation of tumor-associated macrophages is correlated with poor prognosis [18 19 20 ], and production of CSF-1 by tumor cells contributes to osteoclastogenesis from tumor-associated macrophages, leading to tumor-associated osteolysis [21 ].

CSF-1 plays an autocrine role in the development of radiation-induced, acute myeloid leukemia in mice [22 ] and in the mouse c-myc retroviral induction of clonal, monocyte-macrophage tumors [23 ]. Although autocrine growth control by CSF-1 in the normal, steady-state condition is not common, it has been reported in myoblast proliferation [24 ]. However, monocyte/macrophage production of CSF-1 mRNA has also been reported in stimulated conditions [25 26 27 ], and a CSF-1/CSF-1R autocrine loop is indicated in the microglial activation observed in Alzeheimer’s disease [28 ] and in renal allograft rejection [29 ].

These studies indicate that autocrine regulation by CSF-1 or elevation of its local production may contribute significantly to disease development in a number of chronic diseases and in cancer. To determine the relative importance of autocrine regulation by CSF-1 for the development of chronic inflammatory disease versus neoplasia, we created mice in which the CSF-1R-expressing cells express CSF-1. We show that these mice develop a crippling and lethal disease involving osteoporosis and macrophage accumulation in many tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant DNA constructs, transgenic mice, and genotyping
To assemble the transgene TgN(Csf1r-Csf1)Ers (TgRC) construct (see Fig. 1A ), an 8-kb ApaI fragment, containing the mouse CSF-1R promoter and first intron (GenBank MGI:1339758), in which the two ATG sites located in Exon 1 (corresponding to Exon 2 of human CSF-1R [30 ]) were mutated to AGT and GTG, respectively, was inserted into the ApaI site of a Bluescript plasmid (Stratagene, La Jolla, CA). The entire mouse CSF-1 cDNA (Exons 1–8; GenBank MGI:1339753) [31 ] with the CSF-1 ATG start codon was then cloned into the SalI site downstream of the CSF-1R promoter fragment, and the SV40 polyriboadenylic acid sequence subsequently inserted into the NotI site at the 3' end of the CSF-1 cDNA. The DNA insert, containing the TgRC expression cassette, was excised for microinjection into FVB/NJ oocytes, and production of transgenic mice was carried out as described for the generation of TgC mice [4 ]. Mice were genotyped by PCR of tail DNA with the following primers: forward (tgRC-F3), 5'-ATAGAGTTGGAAGCTGATTGAAGG; reverse (tgRC-R3), 5'-GGTCTACAAATTCAAAGGCAATCT; expected product, 380 bp (see Fig. 1A ). To confirm transgenic expression, total mRNAs were extracted from tissues and cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA) and subjected to RT-PCR using the One-Step RT-PCR kit (Qiagen, Valencia, CA) with the following primers: forward (cfms-exon1F, CSF-1R Exon 1), 5'-GTCCTGCTGCTGGCCACAGTTTGG, reverse (tgRC-R3), expected product, 281 bp (see Fig. 1A ). Mouse β-actin (forward, 5'-CGTGGGCCGCCCTAGGCACCA; reverse, 5'-TTGGCCTTAGGGTTCAGGGGGG) was used as the internal standard.


Figure 1
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  		Figure 1. Transgene construction and expression, the phenotype of TgRC mice, and the autocrine behavior of TgRC macrophages. (A) The TgRC construct designed to drive expression of CSF-1 in CSF-1R-expressing cells. The construct is comprised of 4.4 kb of the mouse Csf1r promoter and first intron joined to the mouse Csf1 cDNA. Also shown are the genotyping (tgRC-F3 and tgRC-R3) and expression (cfms-exon 1F and tgRC-R3) primers. (B) Four-week-old, wild-type (WT) and TgRC3/+ (RC) littermates. (C) RT-PCR expression of TgRC chimeric mRNA in tissues and macrophages. DW, Distilled water; Control, cells or tissues from nontransgenic littermates. (D) CSF-1-independent macrophage colony formation by TgRC7 BMM. (E) Increased CSF-1 mRNA expression in TgRC7 PM and BMM by Q-PCR.

 
Peritoneal macrophage (PM) and bone marrow-derived macrophage (BMM) cultures
PM were obtained as described previously [5 ], cultured for 4 days in {alpha}-MEM medium with 15% FCS containing 36 ng/ml human recombinant (hr)CSF-1 (a gift from Chiron Corp., Emeryville, CA), photographed, and lysed to extract RNA. Day 5 BMM, prepared as described [32 ], were cultured for 5 days in 120 ng/ml hrCSF-1 prior to RNA extraction. To test for CSF-1-independent growth, Day 5 BMM were plated in triplicate at 2 x 105 cells/ml in a six-well tissue-culture plate (Falcon, Franklin Lakes, NJ), cultured with CSF-1 overnight, and washed three times with PBS prior to a subsequent culture in the absence of exogenous CSF-1 for an additional 2 days. Resulting colonies were fixed and stained with 0.2% methylene blue in 50% methanol and photographed using a Fujifilm LAS-3000 imaging system (Fujifilm Life Science, Japan). BMM cultured in this manner were also used to test whether the CSF-1-independent growth of tgRC macrophages could be blocked by exogenous anti-CSF-1 antibody. TgRC BMM without CSF-1 and control BMM in the presence of 1.2 ng/ml mouse L cell CSF-1 [33 ] were cultured for 3 days with a 1/200 dilution of rabbit antimouse CSF-1 antiserum or prebleed serum. Attached cells were fixed and stained with 0.2% methylene blue in 50% methanol, and the cells in five low-power fields were counted. Phagocytosis assays were carried out as described previously [34 ] using sheep erythrocytes optimized with rabbit IgG.

Radiography, immunohistochemistry, and histochemistry
Radiography was carried out as described previously [5 ]. For histology, mice were killed with CO2 and immediately perfused with 2% paraformaldehyde and 0.05% glutaraldehyde buffer, pH 6.8 [35 ], and selected tissues were removed and fixed at 4°C overnight in the same fixative. Femurs were decalcified further in Immunocal (Decal Corp., Congers, NY) before embedding. Paraffin-embedded tissue sections at a 5-µm thickness were stained with H&E using standard protocols. Immunohistochemical staining with rat antimouse F4/80 antibody (a gift from Dr. David A. Hume, Centre for Molecular Biology and Biotechnology, University of Queensland, St. Lucia, Queensland, Australia) was carried out as described previously [36 ]. Staining for tartrate-resistant acid phosphatase (TRAP) was carried out as described previously [37 38 39 ]. The images were captured with a Zeiss Axiophot upright microscope through an attached digital camera.

Analysis of mRNA expression
Quantitative RT-PCR (QRT-PCR) reactions using a DNA Engine Opticon 2 real-time system (MJ Research Inc., South San Francisco, CA) were performed in triplicate using the QuantiTectTM Syber® Green PCR kit (Qiagen). Primers targeted different exons or intron–exon boundaries to avoid coamplification of contaminating genomic DNA or were otherwise adopted from previous publications [40 ]. Total RNA (2.5 µg) was reverse-transcribed by SuperScriptTM III RT (Invitrogen) in a 20-µl reaction mixture according to the manufacturer’s instructions. A RT-minus control (no enzyme) was included as a negative control. cDNA reaction mixture (1 µL) was diluted further, 30-fold, in distilled water, and 2 µl diluted cDNA was used as the template in a final PCR reaction volume of 20 µl. Thermocycling was initiated by a 15-min incubation at 94°C, followed by 40 cycles at 94°C for 15 s, 55°C for 30 s, and 72°C for 30 s, and a single fluorescence reading was taken at the end of each cycle. Each run was monitored with a melting curve analysis to confirm the specificity of amplification and lack of primer dimers. Comparative threshold cycle (Ct) values were determined by the Opticon2 software using a fluorescence threshold manually set to 0.001 for all runs and exported into a MS Excel workbook (Microsoft Inc., Redmond, WA) for further analysis. In each QRT-PCR reaction, a housekeeping gene β-actin was used as an internal control to normalize the minor variations in the starting amount of RNA or differences in efficiency of cDNA synthesis and PCR amplification. A relative quantification method, 2–{Delta}{Delta}Ct [41 ], was used to quantify the results.

Analysis of secreted cytokines was carried out using BMM, which were cultured in CSF-1 and stimulated with and without LPS as described previously [42 ]. Only a proportion BMM from chimeric TgRC7 mice was capable of growth factor-independent growth (see Fig. 1D ). To enrich for these CSF-1-expressing cells, TgRC7 BMM were first cultured for 48 h without exogenous CSF-1 and then expanded by an additional 5 days of culture in hrCSF-1 and compared with nontransgenic littermate control BMM, which had been cultured with CSF-1 for 7 days. Both BMM preparations were seeded into six-well (5x105 cells/well) culture dishes and incubated overnight at 37°C, prior to a medium change to 1.5 ml serum-free {alpha}-MEM (Invitrogen) containing 120 ng/ml CSF-1, supplemented with 100 µg/ml endotoxin-free BSA, with or without LPS (1 µg/ml) for 24 h. All cells were incubated with CSF-1 as a standard condition [42 ]. Duplicate supernatants were collected and incubated in duplicate with a mouse inflammation array (Ray Biotech Inc., Norcross, GA). Antibodies to the following cytokines were arrayed: B lymphocyte chemoattractant, CD30 ligand, eotaxin, eotaxin-2, Fas ligand, fractalkine, G-CSF, GM-CSF, IFN-{gamma}, IL-1{alpha}, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12p40p70, IL-12p70, IL-13, IL-17, IFN-inducible T cell-{alpha} chemoattractant, keratinocyte-derived chemokine, leptin, LPS-induced CXC chemokine (LIX), lymphotactin, MCP-1, monokine induced by IFN-{gamma}, MIP-1{alpha}, MIP-1{gamma}, RANTES, stromal cell-derived factor 1, T cell activation 3 (TCA-3), thymocyte-expressed chemokine, tissue inhibitor of metalloproteinase 1 (TIMP-1), TIMP-2, TNF-{alpha}, soluble TNF receptor I (sTNFRI), and sTNFRII. Following the instructions of the manufacturer, chemiluminescence was measured by using a Fujifilm LAS3000 imager and quantified with MultiGauge software provided by the manufacturer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth retardation and early death of TgRC mice expressing CSF-1 in CSF-1R-expressing cells
The fragment of the mouse CSF-1R gene containing 4.4 kb of the promoter and the entire first intron, which confers essentially a wild-type CSF-1R expression pattern in mice [30 ], was used to create a transgene [TgN-(Csf1r-Csf1) Ers (TgRC), Fig. 1A ], driving the expression of full-length CSF-1 in CSF-1R-expressing cells of TgRC mice. The full-length CSF-1 cDNA used encodes all three biologically active CSF-1 isoforms: the secreted glycoprotein, the secreted proteoglycan, and the membrane-spanning, cell-surface CSF-1 glycoprotein [4 5 6 ].

A summary of the gross phenotypic characteristics of the seven TgRC founder mice obtained from three rounds of pronuclei injections is presented in Table 1 . Four founder mice (TgRC1, -2, -4, and -7) possessed a similar phenotype. At 2 weeks of age, they appeared normal and healthy, with no obvious petechia, bleeding, or neoplasia. By 3 weeks of age, they had a lower-than-normal body weight. TgRC7 was killed at 3.3 weeks of age. By 6 weeks of age, of the remaining three, two, like TgRC7, dragged one hind leg, and all had ruffled fur, and they died at 6, 8, and 9.5 weeks of age. Another founder (TgRC5) had above-normal body weight, was healthy, but did not transmit the transgene, and yet another (TgRC6) had normal body weight, was healthy, and transmitted the transgene to progeny, and these progeny, like their founder, were similarly unaffected. We assume that the transgenes in each founder are integrated at unique sites, each with the potential of being affected by transgene integration or conversely, of affecting transgene activity. In TgRC5 and TgRC6, the transgenes appear to have been silenced, but integration of TgRC5 also prevented male germline transmission. The remaining founder mouse (TgRC3) had above-normal body weight, was healthy, and sired 12 progeny, of which two possessed the transgene and a phenotype similar to the TgRC1, -2, -4, and -7 founder mice, indicating that this transgene was silenced in the founder and then activated in the progeny, or alternatively, the founder was a mosaic, with expression in the germline. These two mice, TgRC3-1 and TgRC3-2, were killed at 4 weeks of age, as their death seemed imminent as a result of their low weight (Table 1 , Fig. 1B ), sickly appearance (Fig. 1B) , and lack of mobility. As most transgenic founders possessed a similar phenotype, and the progeny of the one normal founder to which the transgene was transmitted also exhibited these traits, we can conclude that the disease phenotype was a direct result of expression of the transgene rather than a mutation introduced into the germline by integration of the transgene. The disease phenotype was investigated histologically in TgRC3-1 and TgRC3-2, in which the transgene was expressed in the germline. Additional data were obtained from the chimeric TgRC7 founder mouse, killed at 3.3 weeks of age as a result of impending death. Mice from TgRC3 and TgRC7 lines but not nontransgenic littermate mice had a significantly decreased thymus size.


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  		Table 1. Phenotypes of TgRC Founders and Progeny

 
Increased CSF-1 mRNA production in tissues and macrophages of TgRC mice and autocrine regulation by CSF-1 in TgRC macrophages
The expression of the transgene was confirmed by RT-PCR of mRNA from selected tissues of the TgRC7 chimeric founder mouse (Fig. 1C) . Transgenic CSF-1 mRNA was expressed in brain, liver, spleen, kidney, stomach (all known to contain macrophages), as well as PM and BMM. Sequence analysis of the RT-PCR products from brain confirmed expression of the chimeric mRNA (data not shown).

The survival and proliferation of cultured BMM are CSF-1-dependent [43 ]. In contrast to nontransgenic littermate BMM, BMM from the founder TgRC7 formed colonies in the absence of exogenous CSF-1 in culture media (Fig. 1D) , suggesting that the proliferation of macrophages was supported by the local production of CSF-1 by TgRC transgene expression in macrophages. Using a radioimmunoassay, which only detects biologically active mouse CSF-1 [33 , 44 ], CSF-1 was detected in the medium conditioned for 3 days by TgRC7 BMM (0.12 ng/ml) but not wild-type BMM. Real-time QRT-PCR analysis revealed a 20-fold increase in CSF-1 mRNA expression in PM derived from the TgRC7 founder mouse compared with PM from wild-type mice and a corresponding 806-fold increase in BMM (Fig. 1E) .

Autocrine regulation by CSF-1 can be dependent [22 , 23 ] or independent [24 ] of secreted CSF-1. To determine which mechanism was involved in TgRC macrophages, the effects of a neutralizing antimouse CSF-1 antiserum on the proliferation of TgRC macrophages growing in the absence of exogenous CSF-1 and of control BMM growing in the presence of exogenous mouse CSF-1 were assessed over a 3-day period. Compared with the large reduction in attached macrophages observed in the control BMM cultures receiving antibody versus prebleed serum (27±3 c.f. 104±8, mean±SD for five fields, P≤0.001, Student’s t-test), there was no significant difference in cell numbers between the TgRC7 macrophages receiving antibody (71±9) and those receiving prebleed serum (77±8; P=0.326), indicating that release of CSF-1 into the medium was not necessary for autocrine regulation in this system.

Despite the expression of CSF-1 by TgRC macrophages, the plasma CSF-1 concentration in these TgRC mice was within the normal range (data not shown), indicating that TgRC-regulated expression of CSF-1 only elevated local CSF-1 concentrations. These results indicate that there is autocrine regulation by CSF-1 within a proportion of BMM in the TgRC mice. Although local levels of CSF-1 may be elevated in the vicinity of macrophages and other cells expressing the CSF-1R, this local production would be insufficient to elevate the systemic CSF-1 concentration significantly, as the turnover and steady-state concentration of circulating CSF-1 are relatively high [45 ].

TgRC mice exhibit an osteoporotic phenotype
Four-week-old TgRC3-1 and TgRC3-2 mice, expressing the transgene in their germline (Table 1) , were subjected to histologic and X-radiographic analyses. Compared with nontransgenic littermate control mice, the TgRC3 mice showed a marked radiolucency as a result of decreased bone density with a decreased thickness of cortical bones in the femurs (Fig. 2A ), humeri, ulnas, radii (Fig. 2B) , and tail vertebrae (Fig. 2C) , as well as a decreased thickness of the growth plate (Fig. 2A , arrows). Consistent with the X-ray analysis, histological examination of the femurs in TgRC3 mice also revealed decreased thickness of cortical bones (Fig. 2D) , decreased growth-plate cartilage (Fig. 2E) , and decreased trabecular bone (Fig. 2E and 2F) compared with the nontransgenic control mice. Consistent with their osteoporosis, there was an increase in the staining of TRAP-positive osteoclasts on the endosteal surfaces of the trabecular and cortical bone of the TgRC3 mice compared with the littermate control mice (Fig. 2F and 2G) .


Figure 2
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  		Figure 2. TgRC mice are osteoporotic with increased numbers of osteoclasts. (A–C) Radiograms of 4-week-old TgRC3/+ and wild-type littermate femurs (A), forelimbs (B), and tails (C). Arrows in A indicate growth plate. (D and E) H&E staining of (D) mid-diaphyseal femoral cross-sections, showing reduced cortical bone thickness (bars) and (E) longitudinal sections of the subepiphyseal regions of the distal femurs of the same mice, in which the extent of growth plates is indicated by double-headed arrows. Original bars, 200 µm. (F and G) TRAP-stained, hematoxylin-counterstained, longitudinal sections of the subepiphyseal regions of the distal femurs of the same mice showing (F) decreased trabecular bone (original bar, 200 µm) and (G) increased TRAP staining (original bar, 50 µm) in sections from the TgRC3/+ mice. Boxed areas in F are those shown in G.

 
Tissues of TgRC mice express elevated macrophage densities
There was no significant difference in the complete blood cell counts of TgRC3 and littermate control mice (data not shown). Compared with nontransgenic littermate control mice, the numbers of F4/80+ macrophages in TgRC3 mice were increased greatly in the bone marrow of the long bones (Fig. 3B ) and also increased in the red pulp of spleen (Fig. 3C and 3D) . In addition, there was a decreased density of megakaryocytic cells in the bone marrow (Fig. 3A , arrows; mean±SD, 27.3±3.4 cells/mm2 in TgRC3 vs. 48±5.2 cells/mm2 in littermate control mice, P=0.001) and an increased density in the spleens (Fig. 3D , arrows; 39.1±5.9 cells/mm2 in TgRC3 vs. 17.5±3.9 cells/mm2 in littermate control mice, P=0.001) of TgRC mice. Also, there were increased F4/80+ macrophage numbers in the livers of TgRC3 mice (599±81 cells/mm2 in TgRC3 vs. 322±44 cells/mm2 in littermate control mice, P=0.05), and these exhibited a more dendritic morphology than macrophages in nontransgenic littermate control livers (Fig. 4A and 4B ). In contrast to the macrophages from littermate control mice, TgRC3 macrophages were not distributed uniformly throughout the liver but were present in dense clusters (Fig. 4A) . Similar to these findings in liver, the numbers of F4/80-positive microglia in the brain were increased in the meninges (Fig. 4C and 4D) and parenchyma (Fig. 4E and 4F) , where again, they appear in clusters of high density. These observations suggest that the local production of CSF-1 increases the local survival and/or proliferation of macrophages.


Figure 3
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  		Figure 3. F4/80+ macrophage densities and megakaryocyte numbers in bone marrow and spleens of TgRC mice. (A) H&E staining of longitudinal sections of the femoral diaphyses of 4-week-old TgRC3/+ and wild-type littermates. Arrows indicate megakaryocytes. Original bar, 200 µm. (B) The same sections stained with the macrophage-specific anti-F4/80 antibody and counterstained with hematoxylin. Original bar, 100 µm. (C and D) F4/80 antibody-stained sections of spleens of the same mice counterstained with hematoxylin, showing (C) increased F4/80+ staining (original bar, 200 µm) and (D) increased numbers of megakaryocytes (arrowed; original bar, 50 µm) by TgRC3/+ spleens.

 

Figure 4
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  		Figure 4. F4/80+ macrophages in livers and brains of TgRC3/+ mice. (A) Low-power (original bar, 200 µm) images and (B) higher-power (original bar, 50 µm) images of areas boxed in (A) of F4/80+-stained sections of liver from 4-week-old wild-type and TgRC3/+ littermates, counterstained with hematoxylin. (C and D) Similarly stained sections of the meninges of the brains of the same mice. (C) Original bar, 100 µm. (D) Higher-power images of areas boxed in C. Original bar, 50 µm. (E and F) Similarly stained sections of brain parenchyma from the same mice. (E) Original bar, 200 µm. (F) Higher-power images of areas boxed in E. Original bar, 50 µm.

 
TgRC macrophages are stellate and more responsive to stimulation with LPS
Compared with the normal, elongated, bipolar morphology of littermate control macrophages, when TgRC7 PM (Fig. 5A ) and BMM (Fig. 5B) were cultured in the presence of CSF-1, they exhibited a highly stellate morphology with multiple protruding pseudopods. When activated in the absence of CSF-1, TgRC7 BMM were somewhat less stellate than in the presence of CSF-1 (data not shown). However, there was no difference in the phagocytic indices (erythrocytes ingested per cell) of TgRC7 and wild-type BMM (for cells cultured without CSF-1 for 24 h; wild-type, 17.7±1.4; TgRC7, 14.8±1.8; P>0.05, and similar results were obtained for cells cultured with CSF-1). Measurement of mRNAs to cytokines known to be involved in macrophage activation by QRT-PCR revealed that compared with wild-type mice, mRNAs for IL-1β and IL-6 were elevated 39 ± 3.5-fold and 234 ± 11.6-fold (mean±SE), respectively, in BMM of TgRC7 mice, and mRNAs for IL-6, IL-10, and IFN-{gamma} were elevated 4 ± 0.6-fold, 10 ± 1.4-fold, and 3 ± 0.7-fold, respectively, in PM from TgRC7 mice. The increased expression of inflammatory cytokine mRNAs is consistent with an activated or primed state of TgRC7 macrophages.


Figure 5
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  		Figure 5. Phase-contrast morphology and cytokine mRNA expression of PM and BMM from 3.3-week-old TgRC7 mice. Phase-contrast photomicrographs of (A) PM and (B) BMM cultured with CSF-1. Original bar, 50 µm. (C) Relative levels of secreted cytokines in TgRC7 and nontransgenic littermate BMM, with and without LPS stimulation. Cytokine secretion was measured with respect to internal standards on anticytokine blots (error bars indicated range)*, RC7 – LPS significantly different from RC7+LPS, P ≤ 0.05; {dagger}, WT – LPS significantly different from WT + LPS, P ≤ 0.05.

 
As CSF-1 is known to prime macrophages for responses to activating agents such as LPS [46 ], we examined the effect of LPS treatment for 24 h on cytokine release by CSF-1-expressing BMM from TgRC7 mice, compared with BMM from nontransgenic littermate control mice. The LPS responses of most cytokines examined (Materials and Methods) were similar in TgRC7 and littermate control BMM (data not shown), and in contrast to the elevated levels of some proinflammatory cytokine mRNAs, secretion of cytokines was generally, slightly lower in unstimulated TgRC7 than in littermate control BMM (e.g., Fig. 5C ). In TgRC7 macrophages, LPS increased the secretion of IL-1{alpha}, IL-1β, IFN-{gamma}, IL-3, GM-CSF, RANTES, IL-2, TCA-3, LIX, IL-17, IL-4, and IL-13 (Fig. 5C) , which except for IL-4 and IL-13, are predominantly inflammatory. Following LPS stimulation of littermate control BMM, secretion of these same cytokines was less increased, not altered, or decreased compared with unstimulated, control cells. Furthermore, TgRC7 BMM failed to decrease the secretion of three inflammatory cytokines, which were decreased by LPS treatment of wild-type BMM (MCP-1, MIP-1{alpha}, and MIP-1{gamma}; Fig. 5C ). Thus, the majority of the cytokines in which the LPS-induced secretion was increased or not decreased in TgRC7 relative to wild-type BMM was proinflammatory cytokines. These patterns of LPS-induced cytokine release are consistent with a primed state of TgRC macrophages.

The mediation of the LPS response involves TLR4, TLR2, and CD14 [47 ]. We therefore measured the relative concentrations of mRNAs encoding these LPS receptors. TgRC7 mRNAs for TLR4 were decreased 0.26 ± 0.01-fold and 0.03 ± 0.02-fold, respectively, in BMM and PM. TgRC7 mRNAs for TLR2 were also decreased 0.29 ± 0.20-fold and 0.53 ± 0.11-fold, respectively, in BMM and PM. TgRC7 mRNAs for CD14 were increased 1.79 ± 0.26-fold in BMM and 1.04 ± 0.07-fold in PM. The reduction in TgRC TLR4 and TLR2 mRNA levels may contribute to the failure of the LPS reduction of MIP-1{gamma}, MIP-1{alpha}, and MCP-1 secretion in TgRC7 BMM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that inappropriate expression of CSF-1 in CSF-1R-bearing cells in TgRC mice expressing transgenes, in which CSF-1 expression is driven by the CSF-1R promoter, results in an autocrine regulation of these cells by CSF-1, which is apparently intracellular. This autocrine regulation leads to a rapidly debilitating disease involving reduced growth rate, severe osteoporosis associated with an increase of osteoclasts, tissue macrophage accumulation and activation, reduced thymus size, and early death. Associated with disease development, cultured macrophages from TgRC mice exhibited an atypical, stellate morphology, elevated proinflammatory cytokine mRNA expression, and lower, steady-state cytokine secretion than littermate control macrophages but upon stimulation with LPS, were primed, secreting higher levels of inflammatory cytokines than macrophages from control mice. The majority of TgRC chimeric mice was dying prior to sacrifice or had already died within the first 2 months of life. In the one instance where germline transmission occurred, it was from a chimeric founder without overt disease (TgRC3), whose transgenic progeny all exhibited severe disease, requiring euthanasia at 4 weeks of age. As they did not survive to reproductive age, it was not possible to establish a TgRC line. Nevertheless, the development of disease in TgRC mice is consistent with the reported autocrine regulation of macrophages by CSF-1 in some chronic diseases associated with macrophage activation [2 ].

Striking aspects of the disease in TgRC mice are the increased numbers of tissue macrophages and osteoclasts and the osteoporotic condition. The increase in tissue macrophages is consistent with the role of CSF-1 as the primary regulator of macrophage production, and the osteoporotic condition reflects the well-established role of CSF-1 with RANK ligand in osteoclastogenesis [1 ]. Relevant to our observations in TgRC mice, a recent study of the mechanism of TNF-{alpha}-induced osteolysis indicates that the local bone marrow stromal cell production of CSF-1 plays a major role in disease development [48 ]. Supracalvarial injection of TNF-{alpha} stimulated CSF-1 production by bone marrow stromal cells, which in turn, induced expression of the RANK in osteoclast progenitor cells, which was associated with an increase in osteoclast production. In the same study, CSF-1-regulated osteolysis was also shown to be an important component of inflammatory, arthritis-induced osteoclastogenesis. Thus, the osteoclastogenic and osteoporotic condition of TgRC, in which there is local production of CSF-1, is consistent with these observations, which indicate the importance of CSF-1 in osteolytic aspects of inflammatory disease. In the present study, the crippled phenotype of TgRC mice may well have resulted from their osteoporotic condition.

The phenotype of TgRC mice was distinct from the phenotypes of CSF-1-deficient mice injected daily with CSF-1 for periods of up to 3 months [35 ] or wild-type mice expressing secreted CSF-1 driven by the CSF-1 promoter and first intron [6 ], which exhibited elevated levels of circulating CSF-1. These mice exhibited even higher levels of macrophages in some tissues, e.g., liver, than those observed in TgRC mice (ref. [35 ]; unpublished observations). However, in other tissues, e.g., bone marrow, their tissue macrophage densities were lower, they were not severely osteoporotic, and their cultured macrophages did not exhibit the stellate appearance of TgRC macrophages. It is normal that circulating CSF-1 is tightly regulated by sinusoidally located macrophages, in particular, the Kupffer cells of the liver, which clear circulating CSF-1 by CSF-1R-mediated internalization and degradation [45 ]. As Kupffer cell density is regulated by the levels of circulating CSF-1, this is an efficient, homeostatic-feedback mechanism. Increases in circulating CSF-1 result in increased Kupffer cell numbers and increased clearance of CSF-1, whereas decreased levels of circulating CSF-1 decrease Kupffer cell numbers, resulting in a decreased CSF-1 clearance rate. Furthermore, increased, circulating CSF-1 is expected to have less effect in certain regions, where access to systemic CSF-1 is restricted, such as the bone marrow [49 ]. In contrast, local production of CSF-1 by CSF-1R-expressing cells in TgRC mice would not be subject to such regulation, and in these mice, CSF-1R-expressing cell accumulation is not affected by compartmental boundaries. In addition, local or autocrine production leads to a more primed phenotype, and together, these properties contribute to disease.

As the inflammatory cascade induces extensive, apoptotic death in lymphoid tissues, the decreased thymus size in the TgRC mice is a relevant aspect of the disease phenotype. Thymic involution of offspring has been observed in rats following administration of IL-1{alpha} or IL-1β, postnatally [50 ] or to pregnant mothers [51 ]. Similarly, exposure to the microbial toxins, LPS and vomitotoxin, which are known to activate the proinflammatory cytokines IL-1β, TNF-{alpha}, and IL-6, induces a dramatic depletion of thymic lymphocytes, which is largely IL-1-dependent [52 ]. Furthermore, fetal thymus involution in preterm labor patients is strongly associated with funisitis, the histologic manifestation of fetal inflammatory response syndrome [53 ]. We are unaware of any studies reporting increased production of IL-1 in response to elevated CSF-1 alone. However, in the context of bacterial infection and given the LPS-primed state of TgRC7 macrophages, TgRC might contribute directly to thymic involution through such a mechanism. Alternatively, decreased thymus size could result from other secondary effects. The decreased frequency of megakaryocytes in the bone marrow and their increase in the spleen may be a result of the increased macrophage numbers in the bone marrow.

CSF-1 can prime macrophage activation by LPS [46 ]. Cultured TgRC macrophages exhibited an activated, stellate morphology compared with nontransgenic macrophages. They also expressed higher levels of the mRNAs for the proinflammatory cytokines IL-1β, IL-6, and IFN-{alpha}. However, for all three of these cytokines and most of the others examined, the levels secreted by TgRC BMM were lower than the levels secreted by littermate control BMM. Thus, in terms of their cytokine secretion profile, TgRC macrophages do not appear to be more activated than control BMM. However, upon LPS stimulation, TgRC BMM, compared with control BMM, exhibited an increased capacity to release cytokines in response to LPS. These LPS studies, carried out in the presence of exogenous CSF-1, demonstrate a special role of autocrine regulation by CSF-1 in priming the cytokine secretion response to CSF-1 over and above that seen with exogenous CSF-1. This increased priming of TgRC macrophages is likely to have contributed significantly to TgRC disease. How autocrine regulation in macrophages leads to their altered morphology and primed state is of considerable interest.

These studies demonstrate that autocrine/paracrine regulation by CSF-1 in CSR-1R-bearing cells leads to an inflammatory disease that is consistent with reports of autocrine regulation by this growth factor in chronic inflammatory diseases. Our failure to detect neoplastic lesions histologically indicates that inflammatory disease is the dominant outcome of autocrine regulation in CSF-1R-bearing cells. The rapid onset of this disease probably leaves insufficient time for the occurrence of the independent, secondary events likely to be required for neoplastic transformation of the autocrine-regulated CSF-1R-expressing cells. Owing to the early death of mice exhibiting germline transmission of the transgene prior to attaining reproductive age, a system involving inducible expression of the TgRC transgene would be useful as a disease model, for example, in determining the efficacy of CSF-1/CSF-1R-targeted therapies for inflammatory diseases.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant CA32551 (E. R. S.), the Albert Einstein College of Medicine Cancer Center Grant 5P30-CA13330, an American Society of Hematology Fellow Scholar Award (X-M. D.), and a Leukemia and Lymphoma Society Special Fellow Award (X-M. D.). We thank Dr. Xiao-Hua Zong and Ranu Basu for technical assistance, Sayan Nandi for CSF-1 radioimmunoassays, and Drs. Violeta Chitu, Paul Jubinsky, Fiona Pixley, and Yee-Guide Yeung for reviewing the manuscript. We thank Dr. Meinrad Busslinger for assistance in mutating the ATG sites in the CSF-1R promoter and Dr. Dianne Cox for assistance with the phagocytosis assays. We thank members of the Albert Einstein College of Medicine Transgenic, Histopathology, DNA Sequencing, and Analytical Imaging facilities for assistance in different aspects of the work.

Received May 5, 2006; revised July 26, 2006; accepted August 15, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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