Tumor-induced modulation of macrophage class II MHC molecule mRNA expression

Tumor-induced modulation of macrophage class II MHC molecule mRNA expression

Molecular Immunology, Vol. 30, No. 10, pp. 91 l-920, 1993 Printed in Great 0 Britain. TUMOR-INDUCED MODULATION OF MACROPHAGE MOLECULE mRNA EXPRES...

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Molecular Immunology, Vol. 30, No. 10, pp. 91 l-920, 1993 Printed

in Great

0

Britain.

TUMOR-INDUCED

MODULATION OF MACROPHAGE MOLECULE mRNA EXPRESSION

CLASS

0161-5890/93 %6.00 + 0.00 1993 Pergamon Press Ltd

II MHC

DAVID ASKEW,* CAROL J. BURGER and KLAUS D. ELGERT~ Department

of Biology, Microbiology and Immunology Section, Virginia Polytechnic State University, Blacksburg, VA 24061-0406, U.S.A. (First received 8 September

1992; accepted in revised form 4 February

Institute

and

1993)

Abstract-Class II MHC protein expression in macrophages (M$) is reduced during tumor growth. Because regulation of class II MHC proteins occurs during transcription, tumor growth may suppress class II MHC protein expression by suppressing mRNA. The decrease in class II mRNA may result from (i) a decrease in MI$ responsiveness to an inducing agent, such as interferon-y (IFN-y), or (ii) an increase in M4 sensitivity to suppressing agents, such as prostaglandin E, (PGE,). To determine how tumors induce suppression of class II mRNA, M$ were cultured in the presence of IFN-y with or without other factors, and Northern blot analyses were performed. Unstimulated normal host (NH) or tumor-bearing host (TBH) M4 do not express detectable class II mRNA. The addition of IFN-y induces class II mRNA expression in NH and TBH M4, but class II mRNA expression is significantly lower in TBH M#. Kinetic studies suggested that NH M4 class II mRNA is induced faster and in greater amounts than TBH M4 class II mRNA. There is a decrease in M4 class II mRNA stability during tumor growth that may account for the decreased induction by IFN-y. Lipopolysaccharide (LPS) suppresses class II mRNA induction in both NH and TBH IFN-y-treated M& but TBH Md are more sensitive to its suppression. PGE, and tumor-necrosis factor-a (TNF-a), two factors produced by LPS-stimulated M$J, were tested for their ability to modulate class II mRNA expression in NH and TBH IFN-y-treated Mr$. PGE, suppressed class II mRNA expression in both NH and TBH M$. The addition of TNF-a to IFN-y-treated M$ suppressed class II mRNA in NH M4 but, surprisingly, had an additive effect on IFN-y-induced class II mRNA expression. TNF-a did not induce class II mRNA expression in TBH M4 in the absence of IFN-y. The cause of the reduced class II mRNA expression during tumor growth is a decreased response to IFN-y and an increased sensitivity to PGE,. This change may cause the observed suppression mediated by TBH M4.

M4 populations induce NK cell function through the release of interferon-y (IFN-y). The release of PGE, or IFN-7 suggests that class II expression can be regulated by M4 alone, as IFN-7 induces class II mRNA (Steeg et al., 1982; Cao et al., 1989; Faris and Zwilling, 1991), while PGE, suppresses class II mRNA induction (Steeg et al., 1982; Snyder et al., 1982). The importance of PGEz in regulating class II expression also is seen in the amounts of PGE, produced by different M+ populations. Peritoneal M4 produce more PGE, than splenic M+ (unpublished observation), which may account for lower class II expression in peritoneal MI$ compared to splenic M+ (Yurochko et al., 1989; Yurochko et al., 1990a). In TBH mice treated with indomethacin, a cyclooxygenase inhibitor, there is an increase in the percentage of class II+ M4 (Nelson et al., 1990a). This increase is associated with tumor regression and blockage of metastasis. If class II- M@ are a prime cause of immunosuppression, regulation of class II expression may be a major mechanism of tumor influence over the immune system. Several immune molecules, such as IFN-?/ (Steeg et al., 1982; Cao et al., 1989; Faris and Zwilling, 1991), interleukin (IL)-3 (Frendl and Beller, 1990), and IL-4 (Cao et al., 1989) are known to up-regulate class II expression in Md. Some inhibitors of class II expression include

INTRODUCTION

Earlier work in our laboratory found a correlation between a decrease in macrophage (M4) class II MHC protein expression and immunosuppression (Garner et al., 1986; Yurochko et al., 1989, 1990a). In addition, we have shown that class II- M4 are the primary source of suppression and that tumor-bearing host (TBH) class II- M4 are more suppressive than their normal host (NH) counterparts (Alleva et al., 1993). Others have found an increase in the number of class II- M$ during tumor growth associated with immunosuppression (Nelson et al., 1990a). The immunosuppression was attributed to prostaglandin E, (PGE,) production. Nelson et al. (19906) showed that class II M4 suppress NK cell function through the release of PGE,, while class II+ *Present address: Department

of Immunology, St Jude Children’s Research Hospital, Memphis, TN 38101-0318, U.S.A. j-Author to whom correspondence should be addressed. Abbreviations: M4, macrophages; NH, normal host; TBH, tumor-bearing host; FBS, fetal bovine serum; ECL, enhanced chemiluminescence; DEPC, diethylpyrocarbonate; PGE,, prostaglandin E,; LPS, lipopolysaccharide; TNF-a, tumor necrosis factor-a ; IFN-y, interferon-y ; IFN-P, interferon-/?; IL, interleukin; Act. D, actinomycin D. 911

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lipopolysaccharide (LPS) (Steeg et al., 1982; Koerner et al., 1987), tumor-necrosis factor-a (TNF-y) (Hoffman and Weinberg, 1987; Zimmer and Jones, 1990; Melhus et al., 1991), and PGE, (Snyder et al., 1982; Zimmer and Jones, 1990). The fact that TBH M@ produce more PGE, than their normal counterparts (Denbow et al., 1984; Malick et al., 1987; Parhar and Lala, 1988) may account for their decreased class II expression, Because TBH M4 have decreased responses to GM-CSF as measured by functional ability and class II expression (Walker et al., 1992a), TBH M+ may have a decreased responsiveness to IFN-y. If TBH class II- M4 are the key to tumor-induced immunosuppression, the mechanisms that control the expression of class II in TBH M$ may provide information to reverse the immunosuppression. Many reports show that class II expression in MQ, is transcriptionally regulated (Benoist and Mathis, 1990). Regulation of transcription is based on several &-acting regions and associated DNA binding proteins (Benoist and Mathis, 1990; Lafuse, 1991; Glimcher and Kara, 1992). Some regions essential for constitutive class II expression in B cells, and required for IFN-y-induced class II expression in M4, have been identified. The ability to regulate class II expression in TBH Md may prove beneficial because IFN-), is able to reduce suppression mediated by class II- M$ (Alleva et al., 1993). To measure the ability of NH and TBH M$J to express class II, M$J were cultured in the presence of IFN-7 with or without the addition of other class II modulating factors. Total RNA was isolated and class II mRNA levels were measured using Northern blot analysis. By measuring the responses of NH and TBH M4 it may be possible to identify the mechanism by which tumor-induced suppression occurs. MATERIALS

AND METHODS

Animals Eight to 12 week-old male BALB/c mice (Harlan Sprague-Dawley, Madison, WI) received intramuscular injections of a single-cell suspension of 4 x lo5 methylcholanthrene-induced transplantable fibrosarcoma cells in one hind leg. These injections led to palpable tumors in 10-14 days. Mice with no tumors (NH; day 0) or with tumors (TBH; day 21 post tumor cell injection) were used. TBH mice were in an immunological suppressed state that was not caused by nonspecific inflammation (Garner et al., 1987). M@ collection

and culturing

Elicited peritoneal M4 were harvested 4 days after sterile thioglycollate injection, incubated on tissue culture plates for 2 hr (37°C 5% CO?) in RPMI-1640 medium (Hazelton, Denver, PA), and washed to remove nonadherent cells. Two x lo7 NH and TBH M4 were incubated per plate. The M$ were then incubated with IFN-), in combination with TNF-cr (both kind gifts from Genentech, San Francisco, CA), LPS (Sigma, St Louis, MO), PGE, (Sigma), or indomethacin (Sigma) in com-

plete RPMI-1640 medium for indicated times. During mRNA stability experiments, IFN-y-treated NH or TBH M4 were cultured with 2 pgg/ml actinomycin D (Act. D, Sigma) at indicated times. The M4 were washed three times with PBS and were ready for total RNA isolation. Complete RPMI-1640 medium included 10% heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY), 4 x 10-j M 2-mercaptoethanol (Sigma), 50mg/l gentamicin (Gibco), 25 mM NaHCO, and 25 mM HEPES (Research Organics, Cleveland, OH). RNA

extraction

and analysis

Total RNA was isolated using guanidinium isothiocyanate and cesium chloride (MacDonald et al., 1987). For Northern blotting analysis, 20 pg of total RNA was added per sample onto a 1.4% agarose gel with 10% formaldehyde. Ethidium bromide-stained ribosomal bands were used as internal loading controls to ensure that equal amounts of RNA were analyzed. The RNA was transferred to nitrocellulose overnight by blotting. The class IT probe for the IA /? chain, 1.1 kb Cfo I insert of plap-1 (a kind gift from Dr J. G. Seidman; Robinson et al., 1983), was isolated using a plasmid isolation kit (Qiagen, Studio City, CA). The probe contained cDNA from the 3’ region containing the second domain to the 3’ untranslated region. The plasmid was digested with Cfo, loaded onto a 1.0% agarose gel, and the insert isolated using GeneClean (BiolOl, La Jolla, CA). The insert was labelled using enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL) protocol and hybridized overnight with the nitrocellulose at 37°C. The nitrocellulose was washed twice for 20 min at 37 C in 6 M urea and 0.5% SSC and twice for 5 min at 20°C using 2X SSC. ECL detection reagents were added to the nitrocellulose and exposed on ECL-film. The relative amounts of mRNA and rRNA (18s) were measured by densitometry (620 Video Densitometer; BioRad, Richmond, CA). The relative areas for mRNA and rRNA were compared to ensure that we account for any differences in loading. Results throughout are representative experiments done in triplicate. RESULTS

Induction

of class II mRNA

by IFN-y

To determine if TBH M$ have a decreased response to class II inducing agents, NH and TBH M4 were cultured with IFN-y. Neither unstimulated NH nor TBH M4 constitutively express class II mRNA, but IFN-?/ (200 U/ml) addition induced class II expression in both (Fig. 1). Class II mRNA expression was lower in TBH M4. Indomethacin had no affect on class II expression in NH and TBH M4, suggesting that the decrease in class II mRNA expression is through a decrease in TBH M$ response to IFN-y and not by IFN-y induction of PGEz production. To measure the kinetics of class II induction, M4 were incubated for 8, 16 and 24 hr in the presence of 200 U/ml IFN-?/ (Fig. 2). The amounts of class II induced in TBH Md were lower than in NH M$ for all times tested. To determine if tumor growth causes

Class II expression

in macrophages

a decrease in M4 class II mRNA stability, NH TBH Mc$ were cultured in the presence of Act. D. half-life of NH M4 mRNA was 4 hr (Fig. 3), while half-life of TBH MC/I mRNA was only about (Fig. 4).

and The the 2 hr

Suppression of class II mRNA induction by LPS Although TBH Mc$ have a decreased response to IFN-y, other mechanisms may be involved. To determine if TBH M&J are more sensitive to suppression, NH and TBH IFN-y-treated M4 were cultured in the presence of known class II suppressing agents. MC#J were incubated with 200 U/ml of IFN-y and either 10 or 100 ng/ml of LPS for 24 hr. LPS reduced class II mRNA expression in both NH and TBH M4 (Fig. 5), but there was no detectable class II mRNA in TBH M4 at either dose. The addition of 1 pgg/ml LPS could not completely block NH M$J class II mRNA expression (not shown). Factors produced by LPS-stimulated M+ were tested for their suppression of class II mRNA.

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One factor produced by LPS-stimulated MC#Jthat suppresses class II mRNA expression is PGE, (Steeg et al., 1982). PGE,-mediated induction

suppression

of

class

II

mRNA

To directly measure PGE, affects on class II mRNA IFN-y-treated NH and TBH M4 were expression, cultured with PGE, . PGE, (0.1 and 1.O PM) was added to IFN-y-treated Mb and cultured for 24 hr. Both concns suppressed class II expression in NH and TBH Mqj (Fig. 6); however, TBH M4 were more susceptible to PGE,-mediated suppression. Because LPS-treated TBH M4 produced less PGE, than LPStreated NH Mc$, and because indomethacin could not completely block LPS-mediated suppression (not shown), PGE, may not be the only source of suppression. To determine if other factors are involved in LPS-mediated suppression, TNF-cr was added to IFNy-treated MC+.

6

6 Fig. 1. Tumor growth decreases IFN-?/ induced class II mRNA expression. (A) NH and TBH Mrj were cultured for 24 hr in the presence of RPMI-1640 medium alone, 200 U/ml of IFN-y, or 200 U/ml IFN-), and lo-’ M indomethacin. Total RNA was extracted from M4 as described in Materials and Methods. Lane 1, NH M4; lane 2, NH M&J with 200 U/ml IFN-y; lane 3, NH M+ with 200 U/ml IFN-y and indomethacin; lane 4, TBH Mq5; lane 5, TBH M4 with 200 U/ml IFN-y; and lane 6, TBH M4 with 200 U/ml IFN-y and indomethacin. The arrow shows the position of 18 S rRNA. The probe hybridized to a 1.4 kb fragment of RNA. The mRNA/rRNA ratio for lanes 1 to 6 are: 0.0, 2.1, 1.0, 0.0, 1.2, and 1.1. All results for this figure and others are representative experiments repeated at least three times. (B) Ethidium bromide staining of total RNA showing relative amounts of RNA added to each lane.

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Fig. 2. Kinetics of class II mRNA expression in NH and TBH M#. (A) NH and TBH M+ were cultured in the presence of 200 U/ml of IFN-y for 8, 16 or 24 hr. Total RNA was extracted from M# as described in Materiais and Methods. Lane 1, NH M# after 8 hr incubation with 200 U/ml IFN-y; lane 2, NH Mq5 after 16 hr incubation with 200 U/ml IFN-I,; lane 3, NH Mq5 after 24 hr incubation with 200 U/ml IFN-y; lane 4, TBH Mb after 8 hr incubation with 200 U/ml IFN-1;; lane 5, TBH M$ after 16 hr incubation with 200 U/ml IFN-19; and lane 6, TBH MQ, after 24 hr incubation with 200 U/ml IFN-y. The mRNA/rRNA ratio for lanes 1-6 are: 0.5, 1.4, 1.4, 0.3, 0.6, and 1.3. (B) Ethidium bromide staining of total. RNA.

Modulation of chss II m.RNA expression by TNF-cr Because TNF-& suppresses class II mRNA (Hoffman and Weinberg, 1987; Zimmer and Jones, 1990; Melhus et al., 1991), TBH M4 may be more sensitive to TNF-amediated suppression. The addition of 10 and 100 U/ml TNF-a to IFN-y-treated M4 suppressed class II mRNA induction in NH MC#J at both concns. Surprisingly, TNF-a enhanced class II mRNA levels in TBH M4 at 10 and 100 U/ml (Fig. 7). The effects of higher concns of TNF-a: (1000 U) did not differ significantly from 100 U. TBH Md, were cultured with 100 and lOOOU/ml of TNF-cl alone, but TNF-a was unable to induce class II expression in TBH Mb (not shown). DISCUSSION

Regulation of MHC class 11 gene expression occurs through the involvement of several c&-acting DNA sequences and their associated trans-activating DNAbinding proteins (Benoist and Mathis, 1990; Lafuse,

1991; Glimcher and Kara, 1992) that are essential for both constitutive expression in B cells and IFN-), induction in M#. Several DNA-binding proteins have been identified, but have not been fully studied. There is evidence that some of the proteins can enhance transcription while others block transcription. IFN-), induction of class II genes requires de nmo synthesis of new protein (Amaldi et al., 1989) which was reported to be a rvans-activating protein. In addition to transcriptional regulation, there is evidence that post-transcriptional regulation also occurs (Gravallese et al., 1991). Because we have shown an increase in class 11~. M& and an increase in suppression mediated by class II- M4 (Yurochko ef uf., 1990b; Alleva et al., 1993). tumor growth may alter class II mRNA regulation leading to immunosuppression. We have shown that TBH M4 have altered responses to cytokines (Yurochko et al., 1990b; Walker et al., 1992a; Walker ef al., 19923), so the decrease in class II induction in TBH M4 may result from an altered

Class II expression

in macrophages

response to IFN-y. The inability of TBH M4 to express class II mRNA to the same extent as in NH MC+supports this conclusion (see Fig. 1). Of interest was the decrease in class II mRNA stability during tumor growth (see Figs 3 and 4). Although other evidence showed that class II expression is transcriptionally regulated (Benoist and Mathis, 1990), the decrease in mRNA stability contributes to the suppressive effects of tumor growth on class II expression. The kinetics of class II induction suggest

during

tumor

growth

915

that TBH Mc$ respond to IFN-y slower and this slower response leads to lower levels of class II mRNA (see Fig. 2). LPS-mediated suppression of MC#Jclass II expression occurs through transcriptional regulation (Koerner et al., 1987; Figeiredo et al., 1989), and is mediated by PGE, (Steeg et al., 1982). Although LPS suppressed class II induction in TBH MC#J more than in NH MC#J(see Fig. 5), LPS-stimulated TBH MC#Jproduce less PGE, than NH M4 (unpublished observations). Although

Fig. 3. Stability of IFN-y-treated NH M&J. (A) NH M4 were cultured with IFN-y for 24 hr then incubated with 2 pg/ml Act. D for 0, 4, 8, or 12 hr. Total RNA was extracted from M4 as described in Materials and Methods. Lane 1, NH Mb, with 0 hr Act. D; lane 2, NH M$J after 4 hr Act. D; lane 3, NH MqS after 8 hr Act. D; and lane 4, NH M4 after 12 hr Act. D. The mRNA/rRNA ratio for lanes 14 are: 2.2, 1.2, 0.6, and 0.3. (B) Ethidium bromide staining of total RNA.

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Fig. 4. Stability of IFN-y-treated ‘i3H M$. (A) NH Md were cultured with IFN-y for 24 hr then incubated with 2 pg/ml Act. D for 0, 2, 4, or 6 h. Total RNA was extracted from M4 as described in Materials and Methods. Lane 1, NH M4 with 0 hr Act. D; lane 2, NH M$ after 2 hr Act. D; lane 3, NH Mc$ after 4 hr Act. D; and lane 4, NH M4 after 6 hr Act. D. The mRNA/rRNA ratio for lanes 1 to 3 are: 2.4, 1.0, and 0.6. (B) Ethidium bromide staining of total RNA.

TBH

Mc$ are more

susceptible

to PGE,-mediated

sup-

pression (see Fig. 6), other factors cannot be ruled out, because indomethacin could not eliminate LPS-mediated suppression (not shown). In addition to acting transcriptionally, there is some evidence that LPS is able to decrease expression of class II mRNA in B cells other than through transcription initiation (Gravallese et al., 199 1). The overall decrease in class II mRNA expression in LPS- and PGE,-treated TBH M4 may be a combi-

nation of decreased transcription and decreased mRNA stability. That is, because class II mRNA is less stable in TBH Mq5, the greater decrease after LPS and PGE, treatment can be explained as an accentuation of the effect of these agents on NH M+ without having to invoke any additional transcriptional mechanisms. In contrast, TNF-cr may reverse the instability of this mRNA. Although associated with suppression of class II in-

Class II expression

in macrophages

duction in M4 (Hoffman and Weinberg, 1987; Zimmer and Jones, 1990; Melhus et al., 1991) there is some evidence that TNF-cr can enhance class II expression (Chang and Lee, 1986). At first glance, our results seem conflicting because TNF-cr suppresses class II mRNA induction in NH M4 but enhances class II mRNA expression in TBH M4 (see Fig. 7). Recently, it was discovered that TNF-c( can enhance class II expression in immature M4 but suppresses class II in mature Mb (Watanabe and Jacob, 1991). Suppression of class II mRNA in M$ has been attributed to decreased transcription (Melhus et al., 1991). Because we had earlier determined that TBH Md are immature (Garner et al.. 1987) the ability of TNF-cr to enhance class II in TBH Md is expected. Because TNF-c( functions through PGE, (Lehmmann et al., 1988), TNF-c( -induced suppression of class II may occur through the same mechanism as PGE,. A TNF-a-responsive sequence required for induction of class II, called the T box, has been identified in M4 cell lines (Freund et al., 1989). TNF-cr induces the

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expression of a NF-k-B-like protein (NF-TNF), which is responsible for increased expression of invariant chain mRNA and may bind the promoters of MHC molecules (Pessara and Koch, 1990). Induction of class II mRNA through the T box occurs only in immature cells, not in mature cells. Although TNF-a alone can induce class II in M4 cell lines (Freund et al., 1989) TNF-c( alone could not induce class II mRNA in TBH M4. In addition to affecting transcription rates, TNF-cr could act to stabilize TBH M& class II mRNA. We previously showed that TNF-cr, produced by M4, stimulates PGE, production, which down-regulates TNF-a production (unpublished observations). Enhancement of M4 class II expression by TNF-a may be short-lived once PGE, levels increase. The elimination of PGE, may allow TBH Mb to express class II after TNF-cr treatment. TNF-a-treated class II TBH M$ enhance T-cell function when PGE, production is blocked (Alleva et al., 1993). The question is: do class II- M4 produce suppressive factors, or do existing suppressor M$ produce factors

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Fig. 5. LPS-mediated suppression of class II mRNA expression in NH and TBH M+. (A) NH and TBH M4 were cultured for 24 hr with 200 U/ml of IFN-y combined with 10 or 100 ng/ml or LPS. Total RNA was extracted from M4 as described in Materials and Methods. Lane 1, NH M4 with 200 U/ml IFN-7; lane 2, NH M$J with 200 U/ml IFN-y and 10 ng/ml LPS; lane 3, NH Md with 200 U/ml IFN-y and 100 ng/ml LPS; lane 4, TBH M4 with 200 U/ml IFN-7; lane 5, TBH M4 with 200 U/ml IFN-y and 10 ng/ml LPS; and lane 6, TBH M$ with 200 U/ml IFN-y and 100 ng/ml LPS. The mRNA/rRNA ratio for lanes l-6 are: 4.9, 2.3, 0.5, 0.9, 0.0, and 0.0. (B) Ethidium bromide staining of total RNA.

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6 Fig. 6. PGE,-mediated suppression of class II mRNA expression. (A) NH and TBH M4 were cultured for 24 hr in the presence of 200 U/ml of IFN-y with 0, 0.1, or 1.O PM PGE, Total RNA was extracted from Mb as described in Materials and Methods, Lane 1, NH M$ with 200 U/ml IFN-y; lane 2, NH M4 with 200 U/ml IFN-1; and 0.1 PM PGEz; lane 3, NH M#J with 200 U/ml IFN-y and 1.0 PM PGEz; lane 4, TBH M$ with 200 U/ml IFN-7; lane 5, TBH M4 with 200 U/ml IFN-y and 0.1 PM PGE,; and lane 6, TBH M4 with 200 U/ml IFN-y and 1.O PM PGE,. The mRNA/rRNA ratio for lanes 1 to 6 are: 2.5, 2.0, 1.2, 0.6,0.4, and 0.1. (B) Ethidium bromide staining of total RNA.

that inhibit class II induction? IFN-p can induce suppression mediated by class II M4, but class II+ M4 must be present (Boraschi and Niederhuber, 1982). It is likely that any population of M4 can become suppressive, and one consequence of the suppression is loss of class II expression. The production of suppressing agents by Mb dictates that M$J become class III. When M4 are divided into subpopulations based on FcRI expression (Szabo et al., 1990) FcRI M4 are the primary antigen-presenting cells and are class II+. The FcRI+ M4 are not efficient at antigen presentation and are mostly class III. It is the FcRI’ M4 that produce PGE, and TNF-U, which suppress class II expression in NH M4 (Snyder et al., 1982; Hoffman and Weinberg, 1987; Zimmer and Jones, 1990; Melhus et al., 1991). If suppressive M+ by their nature become class III, can restoration of class II expression in TBH M4 enhance the immune response and eliminate the tumor? Although TBH MI$ do not respond to IFN-y like NH

M4 do, IFN-y will reduce class II M4-mediated suppression (Alleva et al., 1993) and TNF-c( will convert TBH class II- M$ to an enhancing population if PGE, production is inhibited (unpublished observations). We have shown that GM-CSF could increase class II expression in NH M4 but not in TBH M4 (Walker et al., 1992~). GM-CSF-treated NH class II M4 could enhance alloreactivity, but GM-CSFtreated TBH class II Mb suppressed alloreactivity (Walker et al., 1992~). The ability of IFN-fl to induce M4 -mediated suppression (Boraschi and Niederhuber, 1982) corresponds to IFN-/?-mediated class II mRNA suppression (Kitari et al., 1988). The importance of class II expression in M4 enhancing or suppressing functions may provide a key to understanding immune regulation and the mechanisms of tumor-induced suppression. It may be possible to intervene in the suppression and activate the immune system to attack the tumor.

Class II expression

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Fig. 7. Modulation of class II mRNA levels in TNF-a-treated NH and TBH M+. (A) NH and TBH M$ were cultured in the presence of 200 U/ml IFN-y for 24 hr with 0, 10, or 100 U/ml of TNF-cr. Total RNA was extracted from M$ as described in Materials and Methods. Lane 1, NH M4 with 200 U/ml IFN-y; lane 2, NH M4 with 200 U/ml IFN-y and 10 U/ml TNF-c(; lane 3, NH Mb with 200 U/ml IFN-y and 100 U/mlTNF-c(; lane 4, TBH M4 with 200 U/ml IFN-y; lane 5, TBH M4 with 200 U/ml IFN-y and 10 U/ml TNF-cr; and lane 6, TBH M+ with 200 U/ml IFN-y and 100 U/ml TNF-c(. The mRNA/rRNA ratio for lanes l-6 are: 2.5, 0.1, 0.1, 0.2, 1.2, and 2.0. (B) Ethidium bromide staining of total RNA.

REFERENCES Alleva D. G., Burger C. J. and Elgert K. D. (1993) Tumor growth increases Ia- macrophage synthesis of tumor necrosis factor-a and prostaglandin E,: Changes in macrophage suppressor activity. J. Leukocyte Biol. 53, (in press). Amaldi I., Reith W., Berte C. and Mach B. (1989) Induction of HLC Class II genes by IFN-y is transcriptional and requires a trans-acting protein. J. Immun. 142, 999-1004. Benoist C. and Mathis D. (1990) Regulation of Major Histocompatibility Complex Class-II genes: X, Y and other letters of the alphabet. A. Rev. Immun. 8, 681-715. Boraschi D. and Niederhuber J. E. (1982) Regulation of macrophage suppression and cytotoxicity by interferon: Role of Ia-bearing macrophages. J. Zmmun. 129, 1854-1858. Cao H., Wolff R., Meltzer M. and Crawford R. (1989) Differential regulation of class II MHC determinants on macrophages by IFN-y and IL-4. J. Immun. 143,3524-353 1. Chang R. J. and Lee S. H. (1986) Effects of interferon-y and tumor necrosis factor-u on the expression of an Ia antigen on a murine macrophage cell line. J. Zmmun. 137, 2853-2856. Denbow C. J., Conroy J. M. and Elgert K. D. (1984) Macro-

phage-derived prostaglandin E modulation of the mixed lymphocyte reaction: An anomaly of increased production and decreased T cell susceptibility during tumor growth. Cell. Immun. 84, l-13. Faris M. and Zwilling B. S. (1991) Characterization of the induction of persistent I-A expression by macrophages from Beg’ mice. J. Leukocyte Biol. 49, 289-293. Figueiredo F., Koerner T. J. and Adams D. 0. (1989) Molecular mechanisms regulating the expression of class II histocompatibility molecules on macrophages: Effect of inductive and suppressive signals on gene transcription. J. Zmmun. 143, 3781-3786. Frendl G. and Beller D. (1990) Regulation of macrophage activation by IL-3. I. IL-3 functions as a macrophage-activating factor with unique properties, inducing Ia lymphocyte function-associated antigen-l but not cytotoxicity. J. Immun. 144, 3392-3399. Freund Y. R., Dedrick R. L. and Jones P. P. (1989) Cis-acting sequences required for class II gene regulation by interferony and tumor necrosis factor-a in a murine macrophage cell line. J. exp. Med. 171, 1283-1299. Garner R. E., Malick A. P. and Elgert K. D. (1986) Variations in macrophage antigen phenotype: A correlation between Ia

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D. ASKEW et al.

antigen reduction and immune dysfunction during tumor growth. J. Leukocyte Biol. 40, 561-574. Garner R. E., Malick A. P., Yurochko A. D. and Elgert K. D. (1987) Shifts in macrophage (M&) surface phenotypes during tumor growth: Association of Mac-2+ and Mac-3+ M# with immunosuppressive activity. Cell. Immun. 108, 255-268. Glimcher L. H. and Kara C. J. (1992) Sequence and factors: A guide to MHC Class-II transc~ption. A. Rev. Zmmun. 10, 1349. Gravallese E. M., Darling J. M., Glimcher L. H. and Boothby M. (1991) Role of lipopolysaccharide and IL-4 in control of tran~ription of the class II Aa gene. J. ~~~~~. 147, 2377-2383. Hoffman M. and Weinberg J. B. (1987) Tumor necrosis factor-u induces increased hydrogen peroxide production and Fc receptor expression, but not increased Ia antigen expression by peritoneal macrophages. f. Leukocyte Rio/. 42, 704-707. Kitaura M., Kato T., Inaba K., Sakata T., Watanabe Y., Kawadw Y. and Muramatsu S. (1988) Down-regulation of macrophage Ia mRNA expression by Interferon (IFN)-or and IFN-fi mediated de nooo synthesized protein. Cell. Immun. 114, 347-358. Koemer T. J., Hamilton T. A. and Adams D. 0. (1987) Suppressed expression of surface Ia on macrophages by lipopolysaccharide: Evidence for regulation at the level of accumulation of mRNA. J. Immun. 139, 239-243. Lafuse W. P. (1991) Molecular biology of murine Class II genes. Crit. Reo. rrnrn~~. 11, 167-194. Lehmmann V., Benninghoff B. and Droge W. (1988) Tumor necrosis factor-induced activation of peritoneal macrophages is regulated by prostaglandin E, and CAMP. J. ~mmun. 141, 587-591. MacDonald R. J., Swift G. H., Przybyla A. E. and Chigwin J. M. (1987) Isolation of RNA using guanidinium salts. Meth. Enzymol. 152, 219-232. Malick A. P., Elgert K. D., Garner R. E. and Adkinson N. F., Jr (1987) Prostaglandin E, production by Mac-2+ macrophages: Tumor-induced population shift. J. Leukocyte Biol. 42, 673-68 1. Melhus O., Koerner T. J. and Adams D. 0. (1991) Effects of TNFct on the expression of Class II MHC molecules in macrophages inducd by IFNy : Evidence for suppression at the level of transcription. J. Leukocyte Biol. 49, 21-28. Nelson J. A. S., Parhar R. S., Scodras J. M. and Lala P. K. (1990a) Down-regulation of macrophage I-A expression in tumor-bearing mice. J. Leukocyte Biol. 48, 394402. Nelson J. A. S., Parhar R. S., Scodras J. M. and Lala P. K. (1990b) Characterization of macrophage subsets regulating murine natural killer cell activity. J. Leukocyte Biol. 48, 382-393. Parhar R. S. and Lala P. K. (1988) Prostaglandin E,-mediated inactivation of various killer lineage cells by tumor-bearing host macrophages. J. Leukocyte Bioi. 44, 474-484.

Pessara U. and Koch N. (1990) Tumor necrosis factor-a regulates expression of the major histocompatibility complex class II-associated invariant chain by binding an NFKB-like factor to a promoter element. Molec. Cell. Biol. 10, 41464254. Phipps R. P., Stein S. H. and Roper R. L. (1991) A new view of prostaglandin E regulation of the immune response. Immun. Today 12, 349-352. Robinson R. R.? Germain R. N., McKean D. J., Mescher M. and Seidman J. G. (1983) Extensive polymorphism surrounding the murine Ia A/? chain gene. J. Immun. 131, 2025-203 1. Snyder D. S., Beller D. 1. and Unanue E. R. (1982) Prostaglandins modulate macrophage Ia expression. Nature 299, 163-165. Steeg P. S., Johnson H. M. and Oppenheim J. J. (1982) Regulation of murine macrophage Ia antigen expression by an immune interferon-like lymphokine: Inhibitory effect of endotoxin. J. Immun. 129, 2402-2406. Szabo G., Miller-Graziano C. L., Wu J.-Y., Takayama T. and Kodys K. (1990) Differential tumor necrosis factor production by human monocyte subsets. J. Leukocyte Biol. 47, 206-216. Walker T. M., Yurochko A. D., Burger C. J. and Elgert K. D. (1992a) Tumor growth changes the contribution of gran~ocyte-macrophage colony-stimulating factor during macrophage-mediated suppression of allorecognition. Zmmunobiology 185, 427439. Walker T. M., Yurochko A. D., Burger C. J. and Elgert K. D. (1992b) Cytokines and suppressor macrophages cause tumor-bearing host CD8 + T cells to suppress recognition of allogeneic and syngeneic MHC class II molecules. J. Leukocyte Biol. 52, 661-669. Watanabe Y. and Jacob C. 0. (1991) Regulation of MHC class II antigen expression: Opposing effects of tumor necrosis factor-a on IFN-y-induced HLA-DR and Ia expression depends on the maturation and differentiation stage of the cell. J. Immun. 146, 899-905. Yurochko A. D., Pyle R. H. and Elgert K. D. (1989) Changes in macrophage populations: Phenotypic differences between the normal and tumor-bearing host macrophages. Zmmunobiology 178, 416435. Yurochko A. D., Burger C. J. and Elgert K. D. (1990a) Two-color flow cytometric analysis of the expression of MAC and MHC class II antigens on macrophages during tumor growth. Cytometry 11, 725-735. Yurochko A. D., Burger C. J. and Elgert K. D. (1990b) Tumor modulation of autoreactivity: Decreased macrophage and autoreactive T cell interactions. CeN Immun. 127, 105-I 19. Zimmer T. and Jones P. P. (1990) Combined effects of tumor necrosis factor-a, prostaglandin E,, and corticosterone on induced Ia expression on murine macrophages. J. Immun. 145, 1167-1175.