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Cited by 87interferon and immune responsesImmune Response and InflammationGenomics and Chromatin Dynamics

Differential Expression and Distinct Functions of IFN Regulatory Factor 4 and IFN Consensus Sequence Binding Protein in Macrophages

Open access
Marecki, Sylvia, Atchison, Michael L, Fenton, Matthew J·1999·The Journal of Immunology
DOI 10.4049/jimmunol.163.5.2713
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Abstract

IFN regulatory factor 4 (IRF4) and IFN consensus sequence binding protein (ICSBP) are highly homologous members of the growing family of IRF proteins. ICSBP expression is restricted to lymphoid and myeloid cells, whereas IRF4 expression has been reported to be lymphoid-restricted. We present evidence that primary murine and human macrophages express IRF4, thereby extending its range of expression to myeloid cells. Here, we provide a comparative analysis of IRF4 and ICSBP expression and function in distinct cell types. These IRF proteins can form specific complexes with the Ets-like protein PU.1, and can activate transcription via binding to PU.1/IRF composite sequences. EMSA analysis revealed that murine macrophages contained both IRF4/PU.1 and ICSBP/PU.1 complexes, analogous to B cells. Over-expression of ICSBP in these macrophages activated transcription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 had no effect on this promoter. In contrast, over-expression of either IRF4 or ICSBP in both macrophages and NIH-3T3 fibroblasts suppressed transcription of the PU.1-independent H-2Ld MHC class I promoter. In NIH-3T3 fibroblasts, IRF4 and ICSBP also synergized with exogenous PU.1 to activate transcription of a PU.1/IRF-dependent promoter. Furthermore, both IRF4 and ICSBP activated transcription of the IL-1β promoter in both cell types. While this promoter is PU.1-dependent, it lacks any known PU.1/IRF composite binding sites. Synergistic activation of the IL-1β promoter by these IRF proteins and PU.1 was found to require PU.1 serine 148. Together, these data demonstrate that IRF4 and ICSBP are dichotomous regulators of transcription in macrophages.

AI snapshot

Written from the paper’s full text.

TL;DR

Macrophages express both IRF4 and ICSBP, which form complexes with PU.1 and regulate transcription in a promoter- and cell-type-specific manner. IRF4 and ICSBP are differentially regulated by IFN-γ and LPS and can act as either activators or repressors depending on the promoter context.

Key findings

  • Macrophages express IRF4 mRNA and protein: RT-PCR produced the expected 554-bp IRF4 product in RAW264.7 macrophages and primary murine peritoneal exudate macrophages, but not NIH-3T3 fibroblasts; IRF4 message in macrophages was 25–35% of that in primary B cells.
  • IRF4 and ICSBP formed specific complexes with PU.1 at a PU.1/IRF composite site; anti-PU.1 blocked binding of both IRF4 and ICSBP, while anti-IRF4 and anti-ICSBP supershifted complexes, and anti-IRF1/IRF2 did not.
  • IFN-γ increased nuclear ICSBP by ~26-fold after 6 h but did not alter nuclear IRF4 or PU.1; LPS increased nuclear IRF4 by ~3-fold after 6 h, with a concomitant decrease in cytosolic IRF4, while ICSBP was not measurably altered.
  • Promoter-specific regulation: ICSBP overexpression activated the (PU.1/IRF)4 reporter ~4-fold in RAW264.7 cells, whereas IRF4 did not; both IRF4 and ICSBP repressed the H-2Ld MHC class I promoter; IRF4 activated the IL-1β reporter >7-fold in a dose-dependent manner, while ICSBP gave a 40–50% increase.

Methods

  • Used RAW264.7 murine macrophages, NIH-3T3 fibroblasts, primary murine peritoneal exudate macrophages, primary murine splenic B cells, and human monocytes; cells were stimulated with LPS or IFN-γ at 100 ng/ml.
  • Main assays included EMSA with a PU.1/IRF composite oligonucleotide, Western blotting for IRF4, ICSBP, and PU.1, semiquantitative RT-PCR for IRF4/CD19/β-actin, and transient transfections with CAT or luciferase reporter plasmids.
  • Transfections used 4 µg total plasmid DNA (2 µg reporter, 1 µg each expression vector, balance empty vector), performed in triplicate and repeated at least three times; CAT and luciferase activities were measured from cell lysates.

Limitations

  • Stated: future studies are needed to identify the elements within the 3.7-kb IL-1β promoter fragment that mediate synergy; the effects of IRF4 deficiency on macrophage function have not been reported; the authors cannot fully explain why previous studies failed to detect IRF4 in macrophages.
  • Evident: many functional conclusions rely on overexpression in cell lines and reporter assays rather than endogenous gene regulation or loss-of-function in macrophages.
  • Evident: IRF4 detection used semiquantitative RT-PCR and macrophages expressed lower IRF4 message than B cells; nuclear translocation of IRF4 after LPS was inferred from Western blot fractionation, not directly imaged.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.4049/jimmunol.163.5.2713

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 52 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § Discussion

    Murine macrophages contain both IRF4/PU.1 and ICSBP/PU.1 complexes, similar to B cells.

    “EMSA analysis revealed that murine macrophages contained both IRF4/PU.1 and ICSBP/PU.1 complexes, analogous to B cells.”
    Quote in the paperNot a substantial claim
  • Findingown data
    § A composite PU.1/IRF site is functional in macrophages

    The (PU.1/IRF)4 reporter plasmid is constitutively expressed in RAW264.7 macrophages.

    “As shown in Fig. 6, the (PU.1/IRF)4 reporter plasmid was constitutively expressed in RAW264.7 macrophages, whereas mutation of the IRF binding site completely abolished this activity.”
    Quote in the paperSubstantial claim
  • Findingown data
    § IRF4 mRNA and protein are expressed in macrophages

    Macrophages express IRF4 mRNA.

    “Using RT-PCR, these primers generated a 554-bp product using cDNA synthesized from RAW264.7 cells and primary murine PEC (Fig. 2, lanes 2 and 4).”
    • Also stated in: § IRF4 mRNA and protein are expressed in macrophages · plain
      “From these data, we conclude that macrophages do express IRF4 transcripts, although at levels lower than those found in B cells.”
    Quote in the paperSubstantial claim
  • Findingown data
    § IRF4 and ICSBP can form specific complexes with PU.1 in macrophages

    Similar EMSA results were obtained with primary murine and human macrophage nuclear extracts.

    “In addition, when EMSA analysis was performed upon primary murine and human macrophage nuclear extracts, similar results were obtained (data not shown).”
    Quote in the paperNot a substantial claim
  • Findingown data
    § Discussion

    Over-expression of either IRF4 or ICSBP suppresses transcription of the PU.1-independent H-2Ld MHC class I promoter in macrophages and NIH-3T3 fibroblasts.

    “In contrast, over-expression of either IRF4 or ICSBP in both macrophages and NIH-3T3 fibroblasts suppressed transcription of the PU.1independent H-2L d MHC class I promoter.”
    Quote in the paperSubstantial claim
  • May combine two claims. 30
  • Classified as probably not a substantial claim. 20
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data
    § A composite PU.1/IRF site is functional in macrophages

    Over-expression of ICSBP has no effect on basal expression of RSV LTR or NF-κB-dependent E-selectin (ELAM-1) reporters.

    “Meanwhile, over-expression of ICSBP had no effect upon the basal expression of either the Rous sarcoma virus long terminal repeat, or the NF-B-dependent E-selectin (ELAM-1) reporters.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Functional synergy between PU.1 and IRF proteins

    In NIH-3T3 fibroblasts, neither IRF4 nor ICSBP alone activates the (PU.1/IRF)4 promoter, while PU.1 alone activates a low level.

    “As shown in Fig. 7, neither IRF4 nor ICSBP alone could activate the promoter, although PU.1 alone could activate a low level of transcription.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Effects of IRF proteins on a natural PU.1-dependent promoter

    Over-expression of ICSBP in RAW264.7 macrophages increases basal IL-1β reporter activity by 40-50%, and this increase is not further enhanced by higher ICSBP plasmid amounts.

    “we found that over-expression of ICSBP resulted in a consistent 40 -50% increase in basal transcriptional activity, which was not further enhanced by increasing the quantities of ICSBP expression plasmid used.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § IRF4 and ICSBP can form specific complexes with PU.1 in macrophages

    Mutation of the IRF binding site abolishes binding of PU.1/IRF complexes but does not affect PU.1 binding alone; mutation of the PU.1 site blocks binding of both PU.1 and IRF proteins.

    “Mutation of the IRF binding site abolished binding of the PU.1/IRF complexes, but did not affect the binding of PU.1 alone (data not shown), while mutation of the PU.1 site blocked the binding of both PU.1 and IRF proteins to the oligonucleotide (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § IRF4 and ICSBP can form specific complexes with PU.1 in macrophages

    Antibodies against PU.1 and ICSBP supershift the DNA-protein complexes, whereas antibodies against IRF1 and IRF2 do not.

    “Abs against PU.1 (lane 7) and ICSBP (lane 3) could supershift the DNA-protein complexes, whereas Abs against IRF1 (lane 5) and IRF2 (lane 6) did not supershift the complexes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § A composite PU.1/IRF site is functional in macrophages

    Reporter plasmids with mutated IRF or PU.1 binding sites lack basal activity and cannot be activated by ICSBP over-expression.

    “Reporter plasmids, in which either the IRF or PU.1 binding sites were mutated, lacked basal activity and could not be activated by over-expression of ICSBP.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    Primary murine and human macrophages express IRF4, extending its known expression range to myeloid cells.

    “We present evidence that primary murine and human macrophages express IRF4, thereby extending its range of expression to myeloid cells.”
    • Also stated in: § Discussion · plain
      “These data support the conclusion that macrophages produce IRF4 message.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § IRF4 and ICSBP can form specific complexes with PU.1 in macrophages

    Antibodies against PU.1 block binding of both PU.1 and ICSBP to DNA, indicating ICSBP cannot bind DNA without PU.1.

    “Abs that recognize PU.1 blocked the binding of both PU.1 and ICSBP to the DNA, suggesting that ICSBP could not bind to DNA in the absence of PU.1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    Over-expression of ICSBP in macrophages activates transcription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 does not.

    “Over-expression of ICSBP in these macrophages activated transcription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 had no effect on this promoter.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation
    § Differential expression of IRF4 and ICSBP in macrophages

    Cytosolic levels of IRF4 and ICSBP mirror their nuclear levels.

    “Cytosolic levels of IRF4 and ICSBP mirrored that observed in the nucleus (data not shown), consistent with previous findings in lymphocytes (13,36).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [13] 1997

      Not checked in the free run.

    • cites [36] 1995

      Not checked in the free run.

  • May combine two claims.

    Findingown data
    § A composite PU.1/IRF site is functional in macrophages

    Increasing amounts of ICSBP expression plasmid (100-2000 ng) cause dose-dependent activation of the (PU.1/IRF)4 reporter, whereas increasing IRF4 has no effect.

    “We also found that transfecting the cells with increasing amounts of the ICSBP expression plasmid (100 -2000 ng) resulted in dose-dependent activation of the reporter, whereas increasing amounts of IRF4 had no effect on reporter gene expression (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § LPS induces nuclear translocation of IRF4 in macrophages

    LPS stimulation increases nuclear IRF4 protein levels within 2 h and decreases cytosolic IRF4 in RAW264.7 macrophages.

    “As shown in Fig. 5A, levels of nuclear IRF4 protein increased within 2 h following LPS stimulation, with a concomitant decrease in the cytosolic levels of this protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § IRF4 and ICSBP can form specific complexes with PU.1 in macrophages

    Antibodies against PU.1 block binding of both PU.1 and IRF4 to DNA, indicating IRF4 cannot bind DNA without PU.1.

    “Like ICSBP, Abs that recognize PU.1 blocked the binding of both PU.1 and IRF4 to the DNA, indicating that IRF4 could not bind to DNA in the absence of PU.1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    Only IRF4 was capable of synergizing with the S148A PU.1 mutant, albeit at lower levels than wild-type PU.1.

    “only IRF4 was capable of synergizing with the S148A PU.1 mutant (albeit at lower levels than wild-type PU.1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    The IL-1β promoter can be activated by IRF4 and weakly by ICSBP in RAW264.7 macrophages.

    “Here, we showed that a natural promoter (i.e., IL-1␤) could be activated by IRF4, and weakly by ICSBP, in RAW264.7 macrophages (Fig. 9).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Effects of IRF proteins on a natural PU.1-dependent promoter

    Over-expression of IRF4 in RAW264.7 macrophages activates the IL-1β reporter in a dose-dependent manner, with more than 7-fold activation at the highest amount used.

    “over-expression of IRF4 activated the IL-1␤ reporter in a dose-dependent fashion, with more than 7-fold activation at the highest amount of IRF4 expression plasmid used.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    LPS stimulation induces nuclear translocation of IRF4, but not ICSBP, in macrophages.

    “Second, LPS stimulation appeared to induce nuclear translocation of IRF4, but not of ISCBP (Fig. 5).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given
    § Transient transfections

    Each single transfection experiment represents triplicate independent transfections, and data are expressed as average values ± SD.

    “Each single experiment represents triplicate independent transfections, and data are expressed as average values Ϯ SD.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § I

    ICSBP-deficient mice develop a chronic myelogenous leukemia-like syndrome, whereas IRF4-deficient mice do not.

    “For example, ICSBP-deficient mice spontaneously develop a chronic myelogenous leukemia-like syndrome, whereas IRF4-deficient mice do not (16).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] 1997

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § I

    ICSBP is constitutively expressed in macrophages and can be further up-regulated by IFN-γ, but not significantly by IFNαβ.

    “ICSBP is constitutively expressed in macrophages, although its expression can be further up-regulated by IFN-␥, but not significantly by IFN␣␤ (31)(32)(33).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites (31)(32)(33)

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § Cell lines and tissue culture conditions

    Endotoxin levels in all medium components were below 10 pg/ml final concentration.

    “Endotoxin levels in all medium components were Ͻ10 pg/ml final concentration, as indicated by BioWhittaker or measured by Limulus amebocyte lysate kit (BioWhittaker).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § IRF4 and ICSBP can either activate or repress transcription in a promoter-specific manner

    Only ICSBP can activate transcription of the PU.1-dependent (PU.1/IRF)4 reporter in both RAW264.7 and NIH-3T3 cell lines, whereas both IRF4 and ICSBP repress the H-2Ld promoter.

    “Together, these findings demonstrate that IRF4 and ICSBP can repress transcription of the PU.1-independent H-2L d promoter in both cell types, whereas only ICSBP can activate transcription of the PU.1-dependent (PU.1/IRF)4 reporter in both cell lines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § A composite PU.1/IRF site is functional in macrophages

    IRF4 over-expression in macrophages does not activate the (PU.1/IRF)4 reporter, contrasting with its ability to activate this reporter in fibroblast cell lines.

    “Moreover, these data also demonstrate that IRF4 over-expression in macrophages does not activate the (PU.1/IRF)4 reporter plasmid, which contrasts with its ability to activate this reporter in fibroblast cell lines (19,20).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [19] 1993

      Not checked in the free run.

    • cites [20] 1992

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § RNA isolation and RT-PCR

    The IRF4 primers do not share sequence similarity with ICSBP.

    “The IRF4 primers correspond to sequences that do not share sequence similarity with ICSBP.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given
    § Transient transfections

    All transfection experiments were repeated at least three times using different plasmid preparations, and a single representative experiment is shown.

    “All transfection experiments were repeated at least three times using different plasmid preparations, and a single representative experiment is shown.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § I

    IRF4 has been reported to be expressed only in lymphocytes, and its expression is not changed by IFN-γ.

    “IRF4 has been reported to only be expressed in lymphocytes (7,10,16,35,36), and its expression was not altered by IFN-␥ treatment (10,36).”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [7] 1995

      Not checked in the free run.

    • cites [10] 1996

      Not checked in the free run.

    • cites [16] 1997

      Not checked in the free run.

    • cites [35] 1996

      Not checked in the free run.

    • cites [36] 1995

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § I

    IRF4 can bind PU.1, and IRF4/PU.1 complexes are required for Ig light chain expression.

    “Previous studies reported that IRF4 could bind to PU.1, and that these IRF4/PU.1 complexes were essential for Ig light chain expression (19 -21).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [19] 1993

      Not checked in the free run.

    • cites [20] 1992

      Not checked in the free run.

    • cites [21] 1993

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § I

    LPS and IFN-γ act synergistically to increase ICSBP steady-state mRNA and protein levels.

    “Moreover, LPS and IFN-␥ synergistically enhance ICSBP steady-state mRNA and protein levels (34).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] 1999

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § I

    ICSBP expression is largely limited to immune cells, including lymphocytes and macrophages.

    “Unlike most other IRF family members, ICSBP expression is mainly restricted to cells of the immune system, including lymphocytes and macrophages (5).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] 1993

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation
    § IRF4 and ICSBP can either activate or repress transcription in a promoter-specific manner

    Previous studies reported that IRF4 and ICSBP act as transcriptional repressors in various cell types.

    “Previous studies have reported that IRF4 and ICSBP function as a transcriptional repressor in various cell types (5,10,18).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [5] 1993

      Not checked in the free run.

    • cites [10] 1996

      Not checked in the free run.

    • cites [18] 1997

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § I

    PU.1 stabilizes the binding of IRF4 and ICSBP to a composite PU.1/IRF motif in Ig and light chain enhancers in B cells.

    “PU.1 stabilizes the binding of both IRF4 and ICSBP to a composite PU.1/IRF motif found in both the Ig and light chain enhancers in B cells.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data
    § IRF4 mRNA and protein are expressed in macrophages

    Semiquantitative RT-PCR was performed in the linear range using logarithmic cDNA dilutions and densitometry.

    “These analyses were performed using semiquantitative methods, where logarithmic dilutions of cDNA were used as templates for PCR and then analyzed by densitometry to assure that amplification was performed in the linear range.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    One prior study reported that ICSBP/PU.1 complexes failed to activate the same (PU.1/IRF)4 reporter plasmid used in this study.

    “One study reported that ICSBP/PU.1 complexes failed to activate the same (PU.1/IRF)4reporter plasmid used also in our study.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] 1996

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning
    § Discussion

    IRF4 and ICSBP serve nonredundant functions in macrophages, which may be related in part to their ability to interact with PU.1.

    “Furthermore, such differences suggest that these IRF proteins serve nonredundant functions in macrophages which may, in part, be related to their ability to interact with PU.1.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    The function of ICSBP/PU.1 complexes has been previously examined in NIH-3T3 fibroblasts.

    “The function of ICSBP/PU.1 complexes has been previously examined in NIH-3T3 fibroblasts (35).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] 1996

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation
    § Discussion

    PU.1, ICSBP, and IRF1 functioned together to activate transcription of a reporter plasmid containing tandem copies of the gp91 phox composite element.

    “These investigators found that PU.1, ICSBP, and IRF1 functioned together to activate transcription of a reporter plasmid containing tandem copies of this composite element.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [24] 1998

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § Discussion

    IRF4 shares the capacity to synergize with PU.1 in NIH-3T3 cells, and serine 148 on PU.1 is required for both IRF proteins to activate transcription.

    “These data are consistent with earlier studies showing that IRF4 shares this capacity in NIH-3T3 cells, and that serine 148 on PU.1 is required for both IRF proteins to activate transcription (48).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] 1997

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data
    § Discussion

    RNA from primary thioglycollate-elicited macrophages lacks CD19 transcripts, indicating no B cell contamination.

    “The lack of CD19 transcripts in RNA prepared from primary thioglycollate-elicited macrophages demonstrates the lack of B cell contamination (Fig. 2).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § Effects of IRF proteins on a natural PU.1-dependent promoter

    The IL-1β promoter is regulated by both LPS and IFN-γ and requires a functional PU.1 binding site within the cap site-proximal promoter.

    “The IL-1␤ promoter has been previously shown to be regulated by both LPS and IFN-␥, and to require a functional PU.1 binding site within the cap siteproximal promoter (46).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] 1994

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation
    § Discussion

    Eklund et al. reported that the PU.1/IRF composite site within the gp91 phox promoter was positively regulated by ICSBP in U937 cells.

    “In contrast, Eklund et al. ( 24) recently reported that the PU.1/IRF composite site within the gp91 phox promoter was positively regulated by ICSBP in U937 cells.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [24] 1998

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    Mice deficient in IRF4 do not generate mature, functional lymphocytes.

    “Mice deficient in IRF4 do not generate mature, functional lymphocytes (16), although the effects of IRF4 deficiency on macrophage function have not yet been reported.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] 1997

      Not checked in the free run.

  • May combine two claims. The quote only partly states this claim.

    Interpretationreasoning
    § Discussion

    The semiquantitative RT-PCR approach is more sensitive than Northern blot, which likely explains detection of IRF4 transcripts in macrophages.

    “Our success in detecting these transcripts presumably resulted from the use of the more sensitive semiquantitative RT-PCR approach.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    Phosphorylation of ICSBP affects its ability to associate with other transcription factors.

    “Phosphorylation of ICSBP has been reported to affect its ability to associate with other transcription factors (18).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] 1997

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation
    § Discussion

    ICSBP represses transcription when bound to ISRE sequences but activates transcription when bound to PU.1.

    “Our data agree with these results, and support a model in which ICSBP functions to repress transcription when bound to ISRE sequences, but instead activates transcription when bound to PU.1.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Nelson et al. (5) 1993

      Not checked in the free run.

    • cites Eklund et al. (24) 1998

      Not checked in the free run.

44 of 44

  • The paper relies on this without support.

    CD19 is expressed specifically by B cells and not by macrophages.

    “To confirm that the PCR product generated from primary murine macrophages was not due to B cell contamination, we performed PCR using primers to detect expression of the B cell-specific gene CD19.”

  • The paper relies on this without support.

    NIH-3T3 cells do not express endogenous PU.1, IRF4, or ICSBP.

    “To study these interactions in isolation, we expressed these factors in fibroblast lines that do not intrinsically express PU.1, IRF4, or ICSBP.”

  • The paper relies on this without support.

    The IL-1β promoter fragment used lacks functional PU.1/IRF composite sites.

    “While this promoter is PU.1-dependent, it lacks any known PU.1/IRF composite binding sites.”
    “The IL-1␤ promoter is not known to contain functional PU.1/IRF composite sites”

  • The paper relies on this without support.

    Unstimulated RAW264.7 macrophages contain little phosphorylated PU.1.

    “i.e., cells that contain little phosphorylated PU.1”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • ICSBP and PU.1 activate transcription through EICE and related elements in IFNγ-stimulated macrophages.

    “Accumulating evidence indicates that ICSBP and PU.1 activate transcription through EICE and related elements in IFN𝛄 -stimulated macrophages, as reported for genes encoding the respiratory oxidases gp91 phox and p67 phox and interleukin-1 𝛃 (IL-1 𝛃 ) (13, 23, 30).”

    From Gamma Interferon Triggers Interaction between ICSBP (IRF-8) and TEL, Recruiting the Histone Deacetylase HDAC3 to the Interferon-Responsive Element (2002) · cites this paper as [30]

  • ICSBP associates with PU.1 and stimulates activity of the Ets/IRF composite element (EICE), which contains IRS and is present in immunoglobulin κ and λ, gp91phox, scavenger receptor, TLR-4, and IL-1β genes.

    “It also associates with PU.1 and stimulates activity of the Ets/IRF composite element (EICE), which also contains IRS and is present in the immunoglobulin (Ig) k and l , gp91 phox , scavenger receptor, toll-like receptor-4 (TLR-4), and interleukin (IL)-1 b genes. (5,23,26-30)”

    From Review: ICSBP/IRF-8: Its Regulatory Roles in the Development of Myeloid Cells (2002) · cites this paper as [27]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • IRF4 protein is assigned two molecular sizes

    Minor

    The results assign IRF4 a size of 42 kDa and ICSBP a size of 45 kDa, but the discussion calls the protein recognized by anti-IRF4 antibodies 45 kDa.

  • Claim of restricted ICSBP expression conflicts with ocular expression

    Minor

    The summary states that ICSBP expression is restricted to lymphoid and myeloid cells, while the text explicitly reports its expression in retinal epithelia and the ocular lens.

  • Serine 148 is called required despite residual synergy with S148A

    Minor

    The summary says PU.1 serine 148 is required for synergistic activation of the IL-1␤ promoter, whereas the results report partial synergy with the S148A mutant and IRF4.

3 of 3

The citation lists haven’t been fetched yet

References 52 · Cited by 87

Research works printed but not linked: 48

  1. 1998, Annu. Rev. Biochem., 227
  2. 1997, Cytokine Growth Factor Rev., 293
  3. 1995, J. Interferon Cytokine Res., 1037
  4. 1994, J. Exp. Med., 977
  5. 1993, Mol. Cell. Biol., 588
  6. 1994, Oncogene, 1423
  7. 1995, Genes Dev., 1377
  8. 1998, Mol. Cell. Biol., 2986
  9. 1998, J. Biol. Chem., 2714
  10. 1996, Mol. Cell. Biol., 1283
  11. 1998, Mol. Cell. Biol., 4639
  12. 1996, Cell, 307
  13. 1997, J. Exp. Med., 921
  14. 1997, J. Exp. Med., 1535
  15. 1997, J. Exp. Med., 1523
  16. 1997, Science, 540
  17. 1994, Proc. Natl. Acad. Sci. USA, 5046
  18. 1997, J. Biol. Chem., 9785
  19. 1993, Mol. Cell. Biol., 6452
  20. 1992, Mol. Cell. Biol., 368
  21. 1993, Science, 1622
  22. 1998, J. Mol. Biol., 1075
  23. 1999, EMBO J., 977
  24. 1998, J. Biol. Chem., 13957
  25. 1994, Mol. Cell. Biol., 373
  26. 1995, Mol. Immunol., 541
  27. 1998, Proc. Natl. Acad. Sci. USA, 6085
  28. 1994, Mol. Cell. Biol., 4408
  29. 1999, Invest. Opthalmol. Visual Sci., 976
  30. 1999, J. Biol. Chem., 9686
  31. 1998, J. Inflamm., 28
  32. 1992, J. Immunol., 801
  33. 1994, J. Immunol., 2270
  34. 1999, J. Immunol., 7417
  35. 1996, Genes Dev., 2335
  36. 1995, Nucleic Acids Res., 2127
  37. 1995, Nature, 163
  38. 1998, Infect. Immun., 28
  39. 1989, Nucleic Acids Res., 6419
  40. 1998, J. Immunol., 241
  41. 1997, J. Immunol., 1848
  42. 1992, Mol. Cell. Biol., 3439
  43. 1996, Proc. Natl. Acad. Sci. USA, 383
  44. 1995, J. Virol., 4173
  45. 1994, Mol. Cell. Biol., 360
  46. 1994, J. Immunol., 4444
  47. 1998, J. Immunol., 268
  48. 1997, Proc. Natl. Acad. Sci. USA, 127

Authors
Marecki, S., Atchison, M. L., & Fenton, M. J.
Affiliations
  • Boston University (US)
  • University of Pennsylvania (US)
Corresponding author
Marecki, Sylvia
ORCID
Fenton, Matthew J
Venue
The Journal of Immunology
Publisher
Oxford University Press (OUP)
Published
1999-09-01
Volume
163
Issue
5
Pages
2713-2722
ISSN
  • 0022-1767 (ISSN-L)
  • 1550-6606
DOI
10.4049/jimmunol.163.5.2713
OpenAlex
W1926072853
Semantic Scholar
8a9cec9cbe4880406d7dae6e372e288d03057054
Cited by
87
References
52
Citations by year
2024: 1 · 2023: 2 · 2022: 4 · 2021: 1 · 2018: 3 · 2017: 4 · 2016: 2 · 2015: 3 · 2014: 3 · 2013: 1 · 2012: 1
FWCI
2.05
Type
journal article
Language
en
Open access
Licence
academic.oup.com/pages/standard-publication-reuse-rights (vor)
Locations
  • The Journal of Immunology (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (5r01gm057053-04)
  • National Institute of General Medical Sciences (GM57053)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
IRF4, IRF8, interferon regulatory factors, macrophages, PU.1, transcriptional activation, myeloid cells, lymphoid cells, transcriptional synergy
MeSH terms
Interferon Regulatory Factor-8, Interferon Regulatory Factor-4, Proto-Oncogene Protein Spi-1, Animals, Biological Transport, Carrier Proteins, Cell Line, Cells, Cultured, DNA-Binding Proteins, Drug Synergism, Humans, Interferons, Lipopolysaccharides, Macrophages, Mice, Inbred BALB C, Nuclear Proteins, Promoter Regions, Genetic, Proto-Oncogene Proteins, Repressor Proteins, RNA, Messenger, Transcription Factors, Transcription, Genetic, Trans-Activators, Consensus Sequence, Interferon Regulatory Factors, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-08.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.

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