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Department of Life Science, Center for Biofunctional Molecules, Pohang University of Science and Technology, Kyungbuk, Korea;
Laboratory of Molecular Oncology, Korea Cancer Center Hospital, Seoul, Korea; and
Department of Microbiology, College of Natural Science, Pusan National University, Pusan, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The physiological function of IL-12p40 in vivo is still unclear, and, therefore, further investigations are needed to define its role in the immune system. In addition, the fact that mouse IL-12p40 can be produced in a 5- to 90-fold excess over IL-12p70 both in vivo and in vitro has led us to speculate the existence of additional immunological roles of IL-12p40 in a point of the efficiency of the immune system. To investigate the potential roles of IL-12p40 in vivo, we demonstrated that the expression of IL-12p40 induces macrophage recruitment in a tumor model system and serves as a chemoattractant for peritoneal macrophages in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to ten-week-old female Buffalo (BUF/N) rats and BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). These animals were maintained in a positive pressure facility (one-way flow) and were fed autoclaved food and water.
Cell lines and Abs
RH7777, rat hepatoma cells of BUF/N rat, and COS-7 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA). These cells were maintained in DMEM that was supplemented with 10% heat-inactivated FBS, 3.7 g/L sodium bicarbonate, and 2.5 g/L HEPES. Mouse anti-rat CD4 (CD134), mouse anti-rat CD8 (CD8b), mouse anti-rat NK (NKR-P1A), and mouse anti-rat macrophage (macrophage subset) mAbs were purchased from PharMingen (San Diego, CA). These reagents specifically react with activated CD4+ T lymphocytes, peripheral T cytotoxic/suppressor cells, NK cells/small subset of T lymphocytes, and macrophages, respectively. Rabbit anti-IL-12Rß1 and rabbit anti-cyclin E polyclonal Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Expression plasmids
Mouse IL-12p40 cDNA was inserted into the inducible mammalian expression vector, pUHD10-3, to construct pUHD10-3/p40. The pUHD10-3 plasmid, kindly provided by Dr. H. Bujard (University of California, Berkeley, CA) carries the regulatory region with human CMV minimal promoter and heptamerized upstream tet-operator, as described (22). The IL-12p40 expression plasmid, pCIN/p40-1/313 (wt) was constructed by inserting cDNA of mouse IL-12p40 gene into pCI-neo plasmid (Promega, Madison, WI). Deleted forms of the mouse IL-12p40 gene were created by PCR amplification of the desired nucleotides. To construct expression vectors of IL-12p40 deletion mutants, pCIN/p40-1/263, 1/273, 1/283, 1/293, 1/303, 1/308, 1/310 (the numbers indicate the amino acid residue of the IL-12p40 protein contained in the mutant products), common sense primer, including ATG translation start codon and different antisense primers (for pCIN/p40-1/263: 5'-AAAGGATCCTACTCTGTCTCC-3', 1/273: 5'-AAAGGATCCTAGAGGAACGCA-3', 1/283: 5'-AAAGGATCCTAGCATTGGACT-3',1/293:5'-AAAGGATCCTACTGAGCTTGC-3', 1/303: 5'-AAAGGATCCTACTTGCTGCAT-3', 1/308: 5'-AAAGGATCCTAGGGAACACAT-3',1/310:5'-AAAGGATCCTACCTGCAGGGA-3'), were used to amplify each deleted form of IL-12p40 gene. Amplified genes were directly inserted into the pGEM T Easy vector (Promega), and then each of them was subcloned into the XhoI and EcoRI site of pCI-neo. The resulting plasmids, including pCIN/p40-1/313 (wt) and pCI-neo as a negative control, were transfected into COS-7 cells by calcium phosphate method and cultured in CHO-SFMII medium for 48 h. The quantity of IL-12p40 and mutant IL-12p40 was measured in the supernatants of transfected cells using ELISA kit (Genzyme, Cambridge, MA).
Preparation of engineered RH7777 cells
To prepare a tumor cell line that inducibly expresses IL-12p40, pUHD10-3/p40 was cotransfected with a pUHD172-1 neo at a 4:1 ratio into RH7777 cells using calcium phosphate method. The plasmid, pUHO172-1 neo, provided by Dr. H. Bujard, encodes a neomycin phosphotransferase and carries the regulatory region with human CMV promoter/enhancer and the rtTA-gene (22). RH7777/neo cell, as a negative control, was also prepared by cotransfection with pUHD10-3 and pUHD172-1 neo. These cells were cultured in the media containing G418 (350 µg/ml) (Sigma, St. Louis, MO), and resistant clones were selected. The resistant clones were plated at a density of 105 cells per well in a 6-well plate and were incubated with various concentrations of doxycycline (dox)4 (0–104 ng/ml; Sigma) for 48 h. The supernatants of the transfected clones were quantified for mouse IL-12p40 expression using an ELISA kit.
Tumor challenge
BUF/N rats were s.c. injected into a leg with 5 x 106 engineered RH7777 cells (RH7777/p40 or RH7777/neo) in 0.4 ml of serum-free media. The local tumor growth was determined by measuring the diameter of tumors with calipers every 2 days. Rats were administered with or without dox (200 µg/ml) in drinking water that contained 5% sucrose, as previously described (23, 24). Specific protocols are presented in the legends of individual experiments.
Histological and immunohistochemical analyses
Tumors were removed from rats and fixed in 4% buffered formalin, dehydrated, cleared, and embedded in paraffin wax using standard histological procedures. The paraffin blocks of tissue were sectioned serially at 7 µm, affixed to gelatin-coated slides, and stored at room temperature. The prepared serial sections were stained with hematoxylin and eosin for histological studies. For immunohistochemical analyses, slides were deparaffinized by xylene and rehydrated through a decreasing series of ethanol solutions (100%, 90%, 70%, water). Endogenous peroxidase activity and nonspecific binding were blocked by 3% H2O2 treatment for 10 min and PBS that contained 5% normal horse serum for 60 min. The slides were then incubated for 24 h in a humid chamber with 1:10 diluted mouse mAbs (CD8b, CD134, NKR-P1A, and macrophage subset), respectively. The slides were then washed with PBS and incubated for 2 h with an anti-mouse biotinylated Ab diluted 1:200 in 5% normal horse serum. After washing with PBS, the slides were incubated for 1 h with streptavidin and biotinylated HRP (Novostain super ABC kit; Novocastra, Claremont, Newcastle, U.K.), and washed in PBS. The color reaction was performed in diaminobenzidine solution (Vector stain ABC kit; Vector Laboratories, Burlingame, CA).
Macrophage cultures and chemotaxis assay
Resting peritoneal macrophages (PM) were harvested as previously described (25) by washing the peritoneal cavity of BUF/N rats and BALB/c mice with RPMI 1640 medium containing 10% heat-inactivated FBS and were seeded in a tissue culture dish. After incubation for 9 h at 37°C in a humidified CO2 incubator, nonadherent cells were removed by washing three times with medium, and the remaining adherent cells were used in the following experiment. Chemotaxis was assayed with a modification of the 96-well microchemotaxis assay, as previously described (26). For the assay, recombinant mouse (rm)IL-12p40 homodimer (R&D Systems, Minneapolis, MN), rmIL-12p70 heterodimer (R&D Systems), rmIL-2 (PharMingen), and BSA (Sigma) were diluted in PBS with 0.2% BSA to several concentrations. Wild-type IL-12p40 and its deletion mutants were obtained from supernatants of COS-7 cell transfected with pCIN/p40–1/313 (wt), 1/263, 1/273, 1/283, 1/293, 1/303, 1/308, and 1/310, and diluted to a final concentration of 100 ng/ml with CHO-SFMII medium containing 0.2% BSA. The culture supernatant from pCI-neo transfectant, a negative control, was used without dilution. Specimens were placed in the lower wells of a 24-well culture plate and overlaid on a chamber with an 8-µm pore size polycarbonate membrane (Nalge Nunc International, Roskilde, Denmark). In all chemotaxis assays used in this study, the PM suspension (106 cells/ml) that was diluted in HBSS medium with 0.2% BSA was added to the top wells of the chamber. After incubation for 2 h at 37°C, the cells on the upper surface of the membrane were wiped off, and the cells on the lower surface of the membrane were fixed in 4% buffered formalin and stained with hematoxylin. Chemotactic activity was represented as the number of cells that migrated across the membrane.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the in vivo role of IL-12p40, a rat hepatoma cell line
(RH7777) was transfected with a vector that expresses mouse IL-12p40
under inducible conditions and modified RH7777 clone (RH7777/p40),
which could express IL-12p40 only after dox treatment was selected. As
a negative control, RH7777/neo was also established by transfection of
a control vector that does not contain the IL-12p40 gene. The
expression of IL-12p40 in RH7777/p40 was efficiently induced and
gradually increased in proportion to the dox concentration (Fig. 1
). In contrast, RH7777/neo did not
express IL-12p40 in the presence or absence of dox. Irrespective of dox
treatment, the morphology and the growth kinetics between RH7777/p40
and RH7777/neo were indistinguishable from those of parental RH7777
cells in vitro, which indicated that neither expression of IL-12p40 nor
dox treatment had any cytotoxic effects on cell growth.
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Without dox treatment, the injection of RH7777/p40 and RH7777/neo
cells into syngeneic BUF/N rats resulted in no difference of tumor
formation between two groups of animals. However, when dox was supplied
soon after tumor cell injection, only 2 of 18 rats of the
RH7777/p40-injected group developed tumors, whereas all
RH7777/neo-injected rats, as a control, developed tumors (Fig. 2A). In addition, we could
observe that there were inflammatory immune responses, such as redness
and swelling, at the injection sites of the RH7777/p40 group, but not
at those of the RH7777/neo group, between 5 and 15 days postinjection
of tumor cells in the presence of dox. It was surprising that IL-12p40
itself appeared to have an antitumor effect, considering the fact that
IL-12p40 has been thought to be an antagonist of IL-12. To further
investigate the activity of IL-12p40, tumors 
0.5 cm in diameter were
established in the absence of dox after injection of RH7777/p40 or
RH7777/neo. When dox was supplied to the RH7777/p40 group, the growth
of established tumors was retarded for 8–12 days after dox treatment
(Fig. 2B). The tumors then regressed and eventually
disappeared. In addition, the redness and swelling was observed at the
tumor sites of the rats for 10 days after dox treatment, suggesting
that IL-12p40 expression may induce an inflammatory reaction in tumor
sites. As expected, the tumors of the RH7777/neo group still continued
to grow after dox treatment. When the tumor-regressed rats were
challenged with parental RH7777 cells at 1 mo after tumor regression,
75% of them were also protected from tumor development, whereas all
naive rats formed tumors (Fig. 2C). These results
suggest that a memory immune response against the parental tumor cells
is also generated in the tumor-regressed rats.
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To identify the immune cells involved in IL-12p40-mediated tumor
regression, both histological and immunohistochemical analyses were
conducted at 1, 3, 7, and 14 days after the initiation of dox treatment
on rats bearing tumors that were up to 0.5 cm in diameter. Histological
analyses revealed that in RH7777/p40-injected rats, a few inflammatory
cells were located in the margin of the tumor injection site at day 1,
and that tumor-infiltrating cells began to appear around tumor mass at
day 3 (Fig. 3A, a
and b). At day 14, necrosis as well as numerous infiltrating
inflammatory cells were observed at the tumor site (Fig. 3Ac). In contrast, the RH7777/neo group exhibited the
typical features of active hepatoma and few infiltrating inflammatory
cells, even at day 14 (Fig. 3Ad). To examine the kinetic
profile of infiltrating inflammatory cells that are associated with
tumor regression, immunohistochemical analyses were performed using
mouse mAbs to CD8+ T, CD4+
T, NK cell, and macrophage-specific surface markers. At day 3, mouse
macrophages, but not other immune cells, were recruited into the margin
of the tumors (Fig. 3B, a, e,
i, and m). At day 7, macrophages were the
predominant cell type infiltrated into the tumors, although low levels
of additional cell types, such as CD8+,
CD4+ T cells, and NK cells, were observed (Fig. 3B, b, f, j, and
n). These results, together with the growth retardation of
the established tumors of RH7777/p40 group up to 8–12 days after dox
treatment, suggest that infiltrating macrophages are predominantly
involved in the initial stages of tumor regression. At day 14,
CD8+, CD4+ T cells, and NK
cells, as well as macrophages, had infiltrated into the necrotic tumor
sites (Fig. 3B, c, g, k,
and o). It is likely that these cells are associated with
the tumor regression that was observed 12–18 days after dox treatment.
In RH7777/neo group, inflammatory cells, such as T, NK cells, and
macrophages, were not observed at any time point up to day 14 (Fig. 3B, d, h, l, and
p).
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To examine whether the macrophage recruitment into tumors is
directly caused by IL-12p40, an in vitro chemotaxis assay for PM from
BUF/N rat, as described in Materials and Methods, was
performed using a rmIL-12p40 homodimer. BSA and rmIL-2, which has been
known as a macrophage activator, were used as controls in these
experiments. As the concentration of rmIL-12p40 in the lower chemotaxis
chamber was increased from 1 ng/ml to 1000 ng/ml, PM began to migrate
across the polycarbonate membrane in a concentration-dependent manner
(Fig. 4A). However, neither
rmIL-2 nor BSA had measurable chemotactic activity for PM. The heat
treatment of rmIL-12p40 dramatically decreased its chemotactic activity
for rat PM, indicating that the activity of IL-12p40 is dependent on
its conformation (Fig. 4B). The similar chemotactic effect
of IL-12p40, at a concentration of 1–1000 ng/ml, was also observed
with primary PM from BALB/c mouse and RAW264.7 cells, mouse macrophage
cell line (data not shown). To exclude the possibility that the
chemotactic function of IL-12p40 is an indirect effect mediated by
other cytokines or chemokines released from IL-12p40-treated
macrophages, we examined the chemotactic activity of IL-12p40 in the
presence of Ab against IL-12Rß1 subunit (anti-IL-12R). This Ab,
but not anti-cyclin E, was able to reduce the IFN-
production
from mouse PM treated with 150 pg/ml of IL-12p70 at the concentration
of 20 µg/ml (data not shown). The anti-cyclin E is an
isotype-matched Ab obtained from the same species. Anti-IL-12R, but not
anti-cyclin E, appeared to inhibit the chemotactic activity of
IL-12p40 (Fig. 4C). As controls, the PM chemotaxis by both
fMLP, known as a strong chemoattractant, and BSA was not affected in
the presence or absence of anti-IL-12R and anti-cyclin E. These
data indicate that macrophage chemotactic activity is a direct effect
of IL-12p40.
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from splenocytes treated with IL-12p70 (12), it
was of interest to characterize whether IL-12p40 also has an agonistic
or antagonistic activity on IL-12p70-mediated PM chemoattraction. In
the presence of 1 ng/ml of rmIL-12p70, the effect of rmIL-12p40 at
various concentrations was observed in terms of macrophage
chemoattraction (Fig. 4
D). The rmIL-12p40 at relatively low
concentrations (0.1–10 ng/ml) does not appear to inhibit the
chemotactic activity of IL-12p70. Interestingly, at higher
concentrations of rmIL-12p40 (10–1000 ng/ml), the similar chemotactic
pattern was shown, regardless of the presence or absence of rmIL-12p70.
As expected, when mouse PM were cotreated with 150 pg/ml of rmIL-12p70
and various concentrations of rmIL-12p40 (0–600 ng/ml), the increase
in the concentration of rmIL-12p40 gradually decreased the IFN-
secretion from rmIL-12p70-treated PM (data not shown), which is
consistent with a previous result observed in splenocytes
(12). Taken together, our data indicated that IL-12p40
appears to, by itself, have a PM chemotactic activity, but acts as an
antagonist in the IFN- release from IL-12p70-treated PM. C-terminal portion of IL-12p40 is responsible for PM chemotaxis and IL-12 receptor binding
To further confirm the macrophage chemotactic activity of
IL-12p40, seven different C-terminal deleted forms of the IL-12p40 gene
were generated. The C-terminal deletion mutants of IL-12p40 (amino acid
1–310, 1–308, 1–303, 1–293, 1–283, 1–273, and 1–263) were
obtained by transient transfection of these deleted forms into COS-7
cells, quantified by using IL-12p40 ELISA kit, and used for chemotaxis
assay. The p40-1/310, 3 amino acids-deleted mutant, still retained the
chemotactic activity similar to that of p40-1/313, wild type of
IL-12p40. In contrast, deletion of more than only 5 amino acids from
C-terminal of IL-12p40 significantly reduced chemotactic activity for
mouse PM (Fig. 5A). A similar
result was also observed when primary rat PM were used (data not
shown). As shown in Fig. 4C, PM chemotaxis by IL-12p40
appeared to be mediated by the binding to its receptor. Therefore,
it was necessary to determine whether C-terminal portion of IL-12p40
affected the receptor binding. Because IL-12p40 was known to reduce the
IL-12p70-induced IFN- production by competitive binding to the IL-12
receptor, we examined whether C-terminal deletion mutants of IL-12p40
inhibit the IFN- production from mouse PM in the presence of
IL-12p70. Only p40-1/310 mutant appeared to reduce the IFN-
production as much as wild-type IL-12p40, while the other mutants did
not inhibit the IFN- production by IL-12p70 (Fig. 5B).
Taken together, these results suggest that C-terminal portion of
IL-12p40 is associated with its receptor binding and consequently plays
a role in mediating the PM chemotaxis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been demonstrated that IL-12p40 is produced as monomer and homodimer in large excess over IL-12p70 (11, 12, 13, 14, 15). The mouse IL-12p40 has been reported to inhibit IL-12-mediated responses by competitive binding to IL-12 receptor with an affinity similar to that of IL-12p70 (12, 16). Also, there is an in vivo role of IL-12p40 as a IL-12p70 antagonist, in which IL-12p40 delayed the allograft rejection of cardiac myoblast (18), and IL-12p40 transgenic mice showed the increased susceptibility to the malaria infection (20). However, it was recently reported that IL-12p40 may have additional in vivo roles that are not related to the biological antagonist of IL-12p70 (21, 27). Piccotti et al. (21) showed that IL-12p40 homodimer exacerbated, rather than delayed, cardiac allograft rejection , which partially agreed with our results. These unexpected results were explained in the sequential report that CD4+ T cells are likely to provide requisite help for the environment for signaling by IL-12p40 and then, CD8+ T cells differentiated by CD4+ T cells are developed by the IL-12p40 (27). In our immunohistochemical analyses, CD4+ and CD8+ T cells were not detected in the tumors generated by the injection of IL-12p40-expressing tumor cells until these tumors began to regress, indicating that IL-12p40 does not respond to CD4+ and CD8+ T cells but chemoattracts macrophages to induce inflammation in the initial stage. Therefore, it is suggested that IL-12p40 may be initially involved in enhancing nonclassical immune responses, leading to the induction of tumor regression.
Recently, we reported that IL-12p70 has chemotactic activity for
macrophages in vitro (28). This activity was maximum at
the concentration of 0.1 ng/ml IL-12p70 and gradually decreased at
higher concentrations. In contrast, it is of interest to note that
IL-12p40 showed the enhanced chemotactic activity as the concentration
of IL-12p40 was increased to 1000 ng/ml, which is maximum concentration
used in this assay condition, indicating that the chemotactic activity
of IL-12p40 for macrophages is concentration-dependent. In the
infection of foreign pathogens, the amount of IL-12p40 was known to be
produced up to 90 times more than that of IL-12p70
(13, 14, 15). Although IL-12p40 was shown to have an
antagonistic effect of IL-12p70-mediated biological activity
(12), it is unlikely that the presence of IL-12p40
inhibits the chemotactic activity of IL-12p70. It may be impossible to
directly demonstrate the potential antagonistic activity of IL-12p40,
since both of them appear to have similar chemotactic activity. In
summary, it is suggested that IL-12p40 appears to be an antagonist of
IL-12p70 in terms of IFN- induction, but acts as a macrophage
chemoattractant together with IL-12p70 to induce the initial immune
responses.
There are several reports that nonlymphoid immune cells migrated by cytokine or chemokine augmented tumor immunogenicity (29, 30, 31, 32, 33), which is similar to the observation of our study. TCA3, one of ß chemokines, was shown to recruit neutrophils and macrophages into tumors accompanied by tumor necrosis, suggesting that the chemotactic activity of TCA3 appeared to induce the tumor regression in the end (29). Based on these reports, it can be speculated that chemotactic activity of IL-12p40 may be closely associated with IL-12p40-mediated tumor regression.
Taken together, it would be carefully reevaluated that IL-12p40 may be a therapeutically useful antiinflammatory agent in the therapy of autoimmune disease and the induction of tolerance in transplantation procedures, since IL-12p40 has a potential to initiate inflammatory immune response via macrophage chemoattraction.
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Acknowledgments |
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Footnotes |
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2 S.J.H. and C.H.L. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Young Chul Sung, Department of Life Science, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang, Kyungbuk, 790-784, Korea. E-mail address:
4 Abbreviations used in this paper: dox, doxycycline; PM, peritoneal macrophages; rm, recombinant mouse.
Received for publication August 21, 1998. Accepted for publication June 25, 1999.
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References |
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 F. Ethuin, C. Delarche, S. Benslama, M.-A. Gougerot-Pocidalo, L. Jacob, and S. Chollet-Martin Interleukin-12 increases interleukin 8 production and release by human polymorphonuclear neutrophils J. Leukoc. Biol., September 1, 2001; 70(3): 439 - 446. [Abstract] [Full Text] [PDF]
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K. H. Baek, S. J. Ha, and Y. C. Sung A Novel Function of Phosphorothioate Oligodeoxynucleotides as Chemoattractants for Primary Macrophages J. Immunol., September 1, 2001; 167(5): 2847 - 2854. [Abstract] [Full Text] [PDF]
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 L. Del Rio, A. J. Buendia, J. Sanchez, M. C. Gallego, M. R. Caro, N. Ortega, J. Seva, F. J. Pallares, F. Cuello, and J. Salinas Endogenous Interleukin-12 Is Not Required for Resolution of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection in Mice Infect. Immun., August 1, 2001; 69(8): 4808 - 4815. [Abstract] [Full Text] [PDF]
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U. Grohmann, M. L. Belladonna, C. Vacca, R. Bianchi, F. Fallarino, C. Orabona, M. C. Fioretti, and P. Puccetti Positive Regulatory Role of IL-12 in Macrophages and Modulation by IFN-{{gamma}} J. Immunol., July 1, 2001; 167(1): 221 - 227. [Abstract] [Full Text] [PDF]
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M. J. Walter, N. Kajiwara, P. Karanja, M. Castro, and M. J. Holtzman Interleukin 12 p40 Production by Barrier Epithelial Cells during Airway Inflammation J. Exp. Med., January 29, 2001; 193(3): 339 - 352. [Abstract] [Full Text] [PDF]
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S. W. Lee, M. K. Song, K. H. Baek, Y. Park, J. K. Kim, C. H. Lee, H.-K. Cheong, C. Cheong, and Y. C. Sung Effects of a Hexameric Deoxyriboguanosine Run Conjugation into CpG Oligodeoxynucleotides on Their Immunostimulatory Potentials J. Immunol., October 1, 2000; 165(7): 3631 - 3639. [Abstract] [Full Text] [PDF]
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H. Kahlert, E. Grage-Griebenow, H.-T. Stuwe, O. Cromwell, and H. Fiebig T Cell Reactivity with Allergoids: Influence of the Type of APC J. Immunol., August 15, 2000; 165(4): 1807 - 1815. [Abstract] [Full Text] [PDF]
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S. Epelman, T. F. Bruno, G. G. Neely, D. E. Woods, and C. H. Mody Pseudomonas aeruginosa Exoenzyme S Induces Transcriptional Expression of Proinflammatory Cytokines and Chemokines Infect. Immun., August 1, 2000; 68(8): 4811 - 4814. [Abstract] [Full Text] [PDF]
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A. Pagenstecher, S. Lassmann, M. J. Carson, C. L. Kincaid, A. K. Stalder, and I. L. Campbell Astrocyte-Targeted Expression of IL-12 Induces Active Cellular Immune Responses in the Central Nervous System and Modulates Experimental Allergic Encephalomyelitis J. Immunol., May 1, 2000; 164(9): 4481 - 4492. [Abstract] [Full Text] [PDF]
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) cells and RH7777/p40 (
) were
plated at a density of 105 cells per 60 mm dish and treated
with dox at the indicated concentration for 48 h. The amount of
IL-12p40 in each cell supernatant was measured by ELISA. The results
are the mean ± SD of four independent experiments.
), rmIL-2 (
), or BSA (
) in rat (BUF/N) PM
chemoattraction was assessed (A). The chemotactic
activity of each protein boiled for 15 min for rat PM was compared with
the intact form at a concentration of 100 ng/ml (B).
Mouse PM were incubated with 20 µg/ml of anti-IL-12Rß1 (
) for 20 min at 37°C, and the PM suspension was
used for the examination of chemotactic activity by 1 µg/ml of
rmIL-12p40. A total of 5 nM fMLP and 0.2% BSA were used as positive
and negative reference chemoattractants, respectively
(C). To examine the effect of IL-12p40 on PM
chemoattraction by IL-12p70, mouse PM chemoattraction by rmIL-12p40 at
the indicated concentrations was assessed in either presence (