Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1993-05-28
2001-03-13
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S369000, C435S069520, C435S070300, C536S024100, C530S351000
Reexamination Certificate
active
06200805
ABSTRACT:
TECHNICAL FIELD
The present invention relates to stably-transformed human cell lines containing a reporter gene operatively linked to a promoter comprising an interleukin-4-responsive element. Among the responsive elements that can be used are the human Fc
&egr;
RII (CD23) interleukin-4-responsive element and the germline epsilon transcript promoter. This invention also relates to methods for the use of such transformed cell lines to identify agonists and/or antagonists of human interleukin-4.
BACKGROUND OF THE INVENTION
Interleukin-4 (IL-4) is a protein which affects a broad spectrum of hematopoietic cells [Strober et al., Pediatr. Res. 24:549 (1988)]. IL-4 enhances a number of activities including macrophage function, IgG4 and IgE production, and the proliferation of immunoglobulin-stimulated B cells, antigen-stimulated T cells and erythropoietin-stimulated red blood cell progenitors. It also increases the proliferation of IL-3-stimulated mast cells.
Together with IgE, mast cells play a central role in allergic reactions. Mast cells are granule-containing connective tissue cells which are located proximally to capillaries throughout the body, with especially high concentrations in the lungs, skin and gastrointestinal and genitourinary tracts. Following exposure to an antigenic substance, mast cells degranulate and release chemical mediators such as histamine, serotonin, heparin, prostaglandins etc. to produce an allergic reaction.
The Fc
&egr;
receptor II (Fc
&egr;
RII) functions in B cell differentiation and in IgE-mediated immunity. It is the low affinity receptor (10
7
-10
8
/M) for the Fc portion of IgE and is positioned with its amino terminus in the cytoplasm and its carboxyl terminus outside the cell [Kikutani et al., Cell 47:657 (1986)]. Fc
&egr;
RII, also known as CD23 antigen, is a B cell-specific differentiation antigen restricted to mature B cells expressing IgM/IgD. Fc
&egr;
RII is not found on immature bone marrow B cells, suggesting that it might be involved in the regulation of growth or differentiation of B cells.
An important role for Fc
&egr;
RII in allergic reactions and immunity to parasitic infection has also been suggested, because it is present on certain populations of eosinophils and monocytes. Furthermore, interleukin-4 (IL-4), which is known to be responsible for the isotype switching of B cells to IgE, has been shown to induce Fc
&egr;
RII expression on B cells [Defrance et al., J. Exp. Med. 165:1459 (1987)], monocytes [Vercelli et al., J. Exp. Med. 167:1406 (1988)], and Burkitt's lymphoma cell lines [Rousset et al., J. Immunol. 140:2625 (1988)]. The biological significance of CD23 induction on human B cells by IL-4 remains to be determined, but it has been indicated that truncated forms of CD23 can be secreted and can act as an IgE binding factor, which might be involved in the IgE-mediated immunity.
The induction of CD23 surface expression on human B and Burkitt's lymphoma (BL) cell lines by IL-4 is correlated with enhanced transcription of the specific mRNA (Rousset et al., supra). The specific promoter regulatory element for the IL-4 induction of CD23 expression in Jijoye cells (a BL cell line) has been defined by transiently expressing fusion genes with different portions of the CD23 promoter linked to a chloramphenicol acetyl transferase (CAT) reporter gene [Suter et al., J. Immunol. 143:3087 (1989)]. The genomic DNA element responsible for IL-4 induction of CD23 expression was located within the first 250 bp 5′ of the transcription initiation start site.
Human IL-4 induction of this DNA element of CD23 linked to a CAT reporter gene in the transient expression system was about 2 fold. To date, this transient study appears to be the only one using a reporter gene for evaluating CD23 regulation by human IL-4. Most of the studies regarding CD23 expression have used indirect immunofluorescence staining of the Fc
&egr;
RII protein.
The effector function of antibody molecules is determined by the constant region of the immunoglobulin (Ig) heavy chain (C
H
). Antibodies retain their specificity while their effector functions are changed by isotype switching at the DNA level.
In vitro studies using murine B cell lines indicate that Ig class switching is preceded by expression of the corresponding germline C
H
gene [Stavnezer et al., Proc. Natl. Acad. Sci. USA 85:7704 (1988)]. In in vitro studies of human B cells also, it has been shown that germline epsilon (&egr;) transcript synthesis precedes and is required for subsequent &egr; switching and IgE production [Gauchat et al., J. Exp. Med. 172:463 (1990)].
Rothman et al. [Mol. Cell. Biol. 11:5551 (1991)] have shown that induction of germline &egr; sequence transcription in an Abelson murine leukemia virus-transformed pre-B cell line is under the control of an IL-4-responsive element located at the promoter of germline &egr; transcripts. IL-4 is one of only two cytokines that are presently known to specifically induce germline &egr; sequence transcription.
Recent studies have shown that human germline &egr; RNA comprises, in addition to the C&egr; exons [Qiu et al., Eur. J. Immunol. 20:2191 (1990)], a germline &egr; exon located 3.5 kilobases upstream from C&egr; [Gauchat et al., supra; Jabara et al., J. Immunol. 145:3468 (1990)] and 5′ from S&egr;. Synthesis of this RNA in highly purified normal B cells (Gauchat et al., supra; Jabara et al., supra) and in EBV transformed human B cells (Jabara et al., supra) or Burkitt's lymphoma cells can be induced by IL-4.
Because of the stimulatory effects of IL-4 on IgE production and mast cell proliferation, antagonists of IL-4 may be useful for the treatment of allergies by decreasing mast cell growth and IgE production. Increasing evidence suggests, however, that IL-4 may also have beneficial therapeutic applications [see, e.g., Tepper et al., Cell 57:503 (1989)]. There is thus a need to identify both antagonists and agonists of IL-4.
The search for such agonists and antagonists would be facilitated by the availability of fast and effective in vitro screening systems.
SUMMARY OF THE INVENTION
The present invention fills this need by providing materials and methods for such screening.
More particularly, this invention provides human cell lines which have been stably transformed by a recombinant vector comprising a reporter gene operatively linked to a promoter comprising a human Fc
&egr;
RII IL-4-responsive element, which element has a nucleotide sequence defined by a subsequence of the sequence of SEQ ID NO: 1 and is delimited at the 5′ end by one of bases 1 to 298 and at the 3′ end by one of bases 507 to 678 of the sequence defined by SEQ ID NO: 1.
This invention further provides human cell lines which have been stably transformed by a recombinant vector comprising a reporter gene operatively linked to a human germline &egr; transcript promoter, and recombinant vectors that can be used to make such stably transformed cell lines.
This invention still further provides methods for detecting human IL-4 agonists in samples comprising:
(a) providing a human cell line which has been stably transformed by a recombinant vector comprising a reporter gene operatively linked to a promoter comprising a human Fc
&egr;
RII IL-4-responsive element, which element has a nucleotide sequence defined by a subsequence of the sequence of SEQ ID NO: 1 and is delimited at the 5′ end by one of bases 1 to 298 and at the 3′ end by one of bases 507 to 678 of the sequence defined by SEQ ID NO: 1;
(b) contacting the transformed cell line with a sample suspected to contain a human IL-4 agonist, under conditions in which human IL-4 would cause increased expression of the reporter gene; and
(c) measuring the level of expression of the reporter gene,
whereby the presence of a human IL-4 agonist in the sample is detected by measurement of an increased level of expression of the reporter gene, compared to the level produced
de Vries Jan E.
Jenh Chung-Her
Narula Satwant K.
Zavodny Paul J.
Carlson Karen Cochrane
Schering Corporation
Thampoe Immac J.
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