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WO2000005403A1 - POLYMORPHISME GENETIQUE DANS LE RECEPTEUR DE IgA - Google Patents

POLYMORPHISME GENETIQUE DANS LE RECEPTEUR DE IgA Download PDF

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Publication number
WO2000005403A1
WO2000005403A1 PCT/US1999/016762 US9916762W WO0005403A1 WO 2000005403 A1 WO2000005403 A1 WO 2000005403A1 US 9916762 W US9916762 W US 9916762W WO 0005403 A1 WO0005403 A1 WO 0005403A1
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cell
genotype
receptor
iga
disease
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PCT/US1999/016762
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English (en)
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Robert P. Kimberly
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Uab Research Foundation
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Priority to US09/744,373 priority Critical patent/US6986987B1/en
Priority to AU52269/99A priority patent/AU5226999A/en
Publication of WO2000005403A1 publication Critical patent/WO2000005403A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to compounds and methods for identifying polymorphism in a cellular receptor, and more particularly, to compounds and methods for identifying and typing single nucleotide polymo ⁇ hisms that code for IgA receptors and applying these polymorphisms to delineation of disease susceptibility and severity.
  • hnmunoglobulins are responsible for responding to antigenic biochemicals encountered by a host eukaryotic organism. Immunoglobulin binding by a host receptor is important in activating immunological responses to an antigen. As a result, polymorphic receptor variations between individual hosts is expected to have implications in the relative susceptibility of individuals to similar antigenic challenge.
  • allelic polymo ⁇ hisms 4-9.
  • the various Fc ⁇ R isoforms have different functional capacities based on different functional domains and/or sequence motifs.
  • allelic variants of Fc ⁇ RIIa, Fc ⁇ RIIIa and Fc ⁇ RIIIb also have distinct functional capacities (Preliminary Results; 4,9-13).
  • the alleles of Fc ⁇ RIIa which differ at amino acid position 131, differ substantially in their capacity to ligate human IgG2 (9,10,12,13) while the F/V- 176 alleles of Fc ⁇ RIIIa differ in their capacity to ligate human IgGl and IgG3 (4).
  • Fc ⁇ RIIa Human neutrophils constitutively express Fc ⁇ RIIa and Fc ⁇ RIIIb, both of which have functionally significant allelic variants.
  • Fc ⁇ RIIa has an ITAM signaling motif in the cytoplasmic domain (14), mediates signaling events that are dependent on both phosphorylation and dephosphorylation, and interacts with actin filaments suggesting direct involvement in phagocytosis (15).
  • Fc ⁇ RIIIb Although the functional capacity of Fc ⁇ RIIIb has been more controversial, in a myeloid environment Fc ⁇ RIIIb initiates a range of cell programs (9,15-17). Importantly, the expression of Fc ⁇ RIIIb is restricted to neutrophils (and eosinophils). Fc ⁇ RI expression is inducible on neutrophils by IFN ⁇ or IL-10 (18-20).
  • Fc ⁇ RIa and Fc ⁇ RIIa Human monocytes constitutively express Fc ⁇ RIa and Fc ⁇ RIIa; and with differentiation into monocyte-macrophages, they express Fc ⁇ RIIIa and Fc ⁇ RIIb.
  • Fc ⁇ RIa and Fc ⁇ RIIIa associate with FceRI ⁇ -chain which has an ITAM signaling motif, and both receptors can initiate degranulation, a respiratory burst and phagocytosis.
  • Fc ⁇ RIIb is distinct in having an inhibitory ITIM motif (21,22). Most carefully studied on B lymphocytes, Fc ⁇ RIIb recruits phosphatases (SHP1 and SHIP) to its signaling complex and downregulates net signaling function (23- 26).
  • SHP1 and SHIP phosphatases
  • Fc receptors are comprised of a single family of molecules most probably derived by alternative splicing from a single gene (27-32).
  • the dominant protein product, Fc ⁇ RIa (CD89), is a transmembrane protein which is heavily and variably glycosylated (33,34).
  • Fc ⁇ RIa lacks recognized signaling motifs in its cytoplasmic domain.
  • Fc ⁇ RIa It associates with FceRI ⁇ -chain which has an ITAM (35), but unlike Fc ⁇ RIIIa, Fc ⁇ RIa does not require the FceRI ⁇ -chain for expression. Similar to Fc ⁇ RIa and Fc ⁇ RT ⁇ a, Fc ⁇ RIa can initiate degranulation, a respiratory burst and phagocytosis at least in some experimental systems (36-38). Fc ⁇ RIa is constitutively expressed on human neutrophils and monocytes with about 6,000-7,000 copies per cell. Its expression is upregulated on activated neutrophils, including exudative neutrophils obtained from the gingival crevice and kidney glomeruli. Among cytokines, TNF ⁇ , IL-8 and GM-CSF increase surface expression of Fc ⁇ RIa while IFN ⁇ and TGF ⁇ downregulate it (39). Alleles of Fc ⁇ RIa are unexplored.
  • Immunoglobulin A plays a prominent role in host defense at mucosal surfaces and in secretions such as tears, saliva and sweat where secretory IgA is the predominant immunoglobulin isotype. IgA appears to serve as a noninflammatory regulator of the local immune response through the production of ILlra and the absence of production of typical pro-inflammatory cytokines (40- 44). These effects contrast with the well known anti-microbial defense functions of IgM and IgG which activate cell programs for inflammation and microbial destruction which may, in turn, injure surrounding host tissues.
  • IgA through its prominent role in protecting surface tissues against invasion by pathogenic organisms is found throughout the respiratory, gastrointestinal and genitourinary tracts. The progression of diseases associated with these tracts is expected to relate to Fc ⁇ R function.
  • Periodontal disease serves as a model for other diseases involving IgA activity. These other diseases include systemic lupus erythematosus (SLE), systemic vasculitis, and IgA nephropathy.
  • Periodontal disease is a chronic, recurrent inflammatory condition initiated and sustained by subgingival plaque bacteria but defined by the host immune system's inflammatory response which results in destruction of structures supporting the teeth (45-50). While bacterial pathogens and their products can damage host supporting tissues, disease can also arise from an exuberant inflammatory immune response by the host. Normally, this response is self- limited and the organism is eliminated. Some individuals, however, seem more susceptible to persistent or recurrent gingival disease, and this may relate to inherent differences in their immune system response. Indeed, some mechanisms of PD may resemble other chronic inflammatory conditions even though PD's distinctive features depend on the unique characteristics of the gingiva, teeth, supporting structures and microorganisms that reside in the oral cavity.
  • Porphyromonas gingivalis has been strongly implicated in adult PD (51,52).
  • P. gingivalis-de ⁇ ved proteases damage tissue directly, as well as indirectly through the induction of collagenase secretion and interruption of complement-mediated defenses (53-56).
  • LPS lipopolysaccharide
  • P. gingivalis LPS induces mononuclear phagocytes to release inflammatory cytokines including TNF ⁇ , IL-1, and IL-6 (57-60).
  • Fimbriae and other surface components of P. gingivalis also stimulate macrophages to produce IL-1 (61,62).
  • IL-l ⁇ is prominent in inflamed gingiva (63).
  • IL-lra secretion of IL-lra by monocytes is a critical anti-inflammatory counterbalance (64).
  • IL-lra is an analog of IL-1 that blocks the effect of IL-1 by competitively binding to IL-1 receptors without transducing an activation signal.
  • IL-lra production is induced in monocytes and macrophages by multiple stimuli, IFN ⁇ , IL-4, IL-6, IL-10 and bacterial LPS.
  • IL-lra production is also stimulated by IgG immune complexes through stimulation of Fc ⁇ RIIa (CD32) and Fc ⁇ RIIIa (CD 16) (65-67), but the addition of LPS to Fc ⁇ R stimulated cells augments IL-l ⁇ and suppresses IL-lra production (68).
  • Fc ⁇ RI (CD89) leads to marked enhancement of IL-lra production without IL-1 and TNF ⁇ (44). Since the ratio of IL-lra:IL-l has been implicated in adult periodontitis as well as several other inflammatory diseases including inflammatory bowel disease and systemic lupus erythematosus (69-71), the relative contributions of Fc ⁇ R and Fc ⁇ RI in stimulating local neutrophils and monocyte-macrophages is clearly of critical importance.
  • Fc ⁇ RI has several different splice isoforms (27-31) and is extensively, yet variably, glycosylated (33-34).
  • Fc ⁇ RI is also genetically polymo ⁇ hic, and whether single nucleotide polymo ⁇ hisms (SNPs) lead to coding changes in both the extracellular and cytoplasmic domains. Characterization of the respective biologies of these SNPs is dependent on an understanding of the functional domains and molecular docking motifs of Fc ⁇ RI. It would also be desirable to assay genetically determined and post- translationally modified variations in Fc ⁇ RI structure and to define the role of both extracellular and cytoplasmic domains essential for functions which may have significant impact on the progression of PD.
  • Fc ⁇ RI splice variants and of both Fc ⁇ RI and Fc ⁇ R polymo ⁇ hisms in determining the ability of a host to fight certain bacterial and viral infections, the susceptibility to and/or severity of autoimmune diseases, as a prognosis indicator or to identify suitable vaccinations or treatments.
  • the present invention is a system and method for correlating the ability of a cell to bind immunoglobulin (IgA) and cellular susceptibility to a disease by identifying a Fc ⁇ RI genotype of a cell and quantifying IgA binding by the cell expressing the Fc ⁇ RI genotype. Thereafter, the IgA binding by the cell and IgA binding by a second cell expressing a second Fc ⁇ RI genotype is compared.
  • IgA immunoglobulin
  • the present invention uses a single nucleotide polymo ⁇ hism or combinations thereof within a Fc ⁇ RI genotype to identify individual susceptibility to a disease.
  • the methods of the present invention also extend to correlating the ability of a cell to bind IgA and cellular susceptibility to a disease through identifying a Fc ⁇ RI phenotype of a given cell and quantifying IgA binding by said cell. Thereafter, IgA binding by the cell is compared to a second cell having a second phenotype of Fc ⁇ RI.
  • single nucleotide polymo ⁇ hisms are responsible for genotypical and phenotypical differences in Fc ⁇ RI herein.
  • the present invention further includes a commercial packaging including reagents for identifying single nucleotide polymo ⁇ hisms in the Fc ⁇ RI genotype or phenotype of an individual as a test to identify individual susceptibility to a disease.
  • the reagents further include instructions for the use thereof.
  • Brief Description of the Drawings Figure 1 - TGF ⁇ causes a significant (p ⁇ 0.01) decrease in Fc ⁇ RI specific phagocytosis in 18-hr cultured human monocytes. Phagocytosis by Fc ⁇ RII and Fc ⁇ RIII are unaffected by TGF ⁇ but there is a modest increase in Fc ⁇ RIIIa expression. Data are reported relative to untreated cells.
  • Phagocytosis normalized to expression is 0.5, 1.2 and 0.9 for Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII respectively.
  • FIG. 2B Pre-incubation of transfected P388D1 cells with saturating levels of murine IgG2a and/or okadaic acid before addition of E-22.2 did not significantly alter Fc ⁇ RIa phagocytosis. Binding of the E-22.2 is not affected by either pre-incubation.
  • FIG. 3 Differential affects of the calcium chelator BAPTA, the protein tyrosine kinase inhibitor genistein and the ser/thr phosphatase inhibitor okadaic acid on receptor specific phagocytosis mediated by transfected WT and MT Fc ⁇ RI. Data are presented relative to control untreated cells.
  • FIG. 4 Cross-linking of transfected WT Fc ⁇ RIa with receptor-specific mAb 22 induces production of both IL-l ⁇ and IL-6 while MT Fc ⁇ RI only induces
  • FIG. 5 Sequence of the cytoplasmic domain of Fc ⁇ RI (CD89) showing potential kinase phosphorylation sites.
  • Figure 7 Blockade of binding of the anti-Fc ⁇ RIII ligand binding site mAb 3G8 by increasing concentrations of human IgG.
  • the level of mAb 3G8- FITC binding to Fc ⁇ RIII after incubation with human IgG is shown.
  • the present invention provides methods for determining the allelic pattern for Fc ⁇ RI genes in human patients.
  • the methods encompass the use of allele specific oligonucleotides as hybridization probes and/or as primers for DNA amplification, as well as the use of direct DNA sequencing. Identification of receptor alleles may also be achieved immunologically, by contacting mucosal cells that express Fc ⁇ RI receptors on their cell surface with antibodies that distinguish between different polymo ⁇ hic forms of the receptor.
  • the present invention is based on the finding that allelic polymo ⁇ hism in a gene encoding Fc ⁇ RI receptor isoform results in functionally distinct gene products.
  • the expression of a polymo ⁇ hic receptor in an individual may have important consequences for the physiological activity of the receptor and thus for the functioning of the cell types that carry the receptor.
  • the present invention Because of the important immunological role of IgA in mucosal immunity, the present invention has utility as a diagnostic to identify high risk patients that warrant early and aggressive treatment.
  • As a diagnostic for infectious disease the present invention has utility in predicting neonatal sepsis and thereby guiding the use of therapeutic gamma globulin.
  • the present invention has utility in diseases illustratively including systemic lupus erythematosus, systemic vasculitis, IgA nephropathy, rheumatoid arthritis, systemic sclerosis, dermatomyositis, Hashimoto's thyroiditis, inflammatory bowel disease and Sjogren's syndrome.
  • diseases illustratively including systemic lupus erythematosus, systemic vasculitis, IgA nephropathy, rheumatoid arthritis, systemic sclerosis, dermatomyositis, Hashimoto's thyroiditis, inflammatory bowel disease and Sjogren's syndrome.
  • the present invention has utility as an immune surveillance test for monitoring protective and nonprotective antibody levels in response to cancer therapies.
  • the present invention provides a method for identifying the Fc ⁇ RI allelic pattern in human patients which comprises testing mucosal tissue cells or DNA from individual patients for the presence of different Fc ⁇ RI allelic variants, using antibody based and/or nucleic based methods which are described in more detail below.
  • the identification of receptor allelic patterns of the present invention further finds utility in identifying gene therapy as a means for providing more effective receptor alleles to a patient.
  • the present invention also encompasses the identification analysis of new allelic forms of Fc ⁇ RI genes, the analysis achieved using methods well known in the art, such as for example, DNA sequencing; single strand conformational polymo ⁇ hism analysis (SSCP) (84); "HOT” cleavage (85); denaturing gradient gel electrophoresis (DVGE) (86) and combinations thereof.
  • SSCP single strand conformational polymo ⁇ hism analysis
  • DVGE denaturing gradient gel electrophoresis
  • a new polymo ⁇ hism has been identified, immunological and/or molecular biological tests are used to genotype patients for the presence or absence of the polymo ⁇ hism.
  • monoclonal antibodies specific to the protein encoding for a newly identified allele are prepared by well known methods; these antibodies can be used for genotyping the patient populations as described above.
  • allele specific oligonucleotides are designed for use as probes and/or as primers in hybridization or PCR-based detection methods, respectively.
  • Fc ⁇ RI and their role in determining unique Fc ⁇ RI signaling events Using the yeast two-hybrid system with CD 89 alpha chain cytoplasmic domain (CY) as "bait,” novel molecular associations with the CY, the residues meeting these associations, and the potential dependence on serine/threonine phosphorylation are determined.
  • CD89 alpha chain signaling alone, the regulation of ⁇ -chain pairing with CD89 alpha chain, and the signaling elements engaged by CD89, as opposed to CD 16 and CD64, are defined.
  • a further method of characterizing the signal elements associated with Fc ⁇ RI includes using stably transfected constructions with specific deletions, truncations and point mutations of CD89. The effect of these constructs on associations, the role of these interactions in modulating signaling and initiation of cell programs is thereby determined.
  • the present invention also encompasses identification of novel single nucleotide polymo ⁇ hisms (SNPs) affecting Fc ⁇ RI structure and function.
  • SNPs are identified using direct cycle sequencing optimized for heterozygote detection, genomic DNA and cDNA obtained by RT-PCR from mRNA so as to provide a template to identify novel SNPs in normal donors, in PD, rheumatoid arthritis patients and in other patients suffering from diseases illustratively including connective tissue, autoimmune, bacterial and viral infection, and cancer.
  • the role of naturally occurring point mutations in modulating signaling and initiation of cell programs is identified in homozygous genotype donors and in stable transfectants. These correlations are utilized to provide the diagnostic utilities of the present invention.
  • the correlations sought are those between particular Fc ⁇ RI allelic polymo ⁇ hs and, for example: the risk for developing the aforementioned diseases; the effectiveness of antibodies protective against biologic- or tumor neo-antigens; and the risk of immune complex disease.
  • Fc ⁇ RI splice variants In order to establish the impact of both pre- and post-translational variations and modifications of Fc ⁇ RI on Fc ⁇ RI function, the variation and expression of Fc ⁇ RI splice variants is determined in circulating and in crevicular fluid myeloid cells from patients suffering from an aforementioned disease in relation to a disease free control group.
  • An RT-PCR method is used to identify translational variations and modifications of the receptor.
  • the Fc ⁇ RI "b" form is investigated for correlations through the use of different TM/CY. Gel electrophoresis of immunoprecipitated Fc ⁇ RI from cell lines and ex vivo myeloid cells is used to identify different receptor molecular weights, as previously done with Fc ⁇ RIIIa (17).
  • Gel electrophoresis serves to characterize the glycosylation state in relationship to the ligand binding ability of the receptor polymo ⁇ h.
  • Translational variations and modifications of receptor glycosylation in patients suffering from an IgA related immune response disease can be correlated with SNPs of the receptor, as well as with the glycosylation status of both IgA and IgG.
  • Statistical methods illustratively including a 2x3 chi-square test is used to determine allele frequencies in disease and control groups. In this manner, it is possible to obtain statistically significant correlations between a given disease and Fc ⁇ RI alleles and thereby a diagnostic as to disease susceptibility and clinical outcome.
  • the Fc ⁇ RI allelic pattern in an individual patient is determined by either: 1) immunological detection of one or more allelic forms of Fc ⁇ RI polypeptides present on the surface of appropriate immune cells ("phenotypic characterization”); or 2) molecular detection of the DNA or RNA encoding one or more Fc ⁇ RI allelic forms using nucleic acid probes, with or without nucleic acid amplification or sequencing ("genotypic characterization") .
  • myeloid cells, mucosal cells, or those of any tissue expressing CD89 or subsets thereof are isolated from a patient to be tested using methods that are well known in the art, such as, for example, gradient centrifugation and or immunoadso ⁇ tion.
  • Antibodies that are capable of distinguishing between different allelic forms of Fc ⁇ RI are then applied to the isolated cells to determine the presence and relative amount of each allelic form.
  • the antibodies may be polyclonal or monoclonal, preferably monoclonal. Measurement of specific antibody binding to cells may be accomplished by any known method, including without limitation quantitative flow cytometry, or enzyme-linked or fluorescence-linked immunoassay. The presence or absence of a particular allele, as well as the allelic pattern (i.e. homozygosity vs. heterozygosity) is determined by comparing the values obtained from the patient with norms established from populations of patients of known genotypes. In an alternate embodiment, DNA is obtained from a patient, and the presence of DNA sequences corresponding to particular Fc ⁇ receptor alleles is determined. The DNA may be obtained from any cell source or body fluid.
  • Non- limiting examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.
  • Body fluids include blood, urine, cerebrospinal fluid, and tissue exudates at the site of infection or inflammation.
  • DNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It is understood that the particular method used to extract DNA will depend on the nature of the source.
  • the DNA may be employed in the present invention without further manipulation.
  • the DNA region corresponding to all or part of the Fc ⁇ RI may be amplified by PCR or other amplification methods known in the art.
  • the amplified regions are specified by the choice of particular flanking sequences for use as primers.
  • Amplification at this step provides the advantage of increasing the concentration of Fc ⁇ RI DNA sequences.
  • the length of DNA sequence that can be amplified ranges from 80 bp to up to 30 kbp.
  • primers are used that define a relatively short segment containing sequences that differ between different allelic forms of the receptor.
  • Fc ⁇ receptor allele-specific DNA sequences may be determined by any known method, including without limitation direct DNA sequencing, hybridization with allele-specific oligonucleotides, and single- stranded conformational polymo ⁇ hism (SSCP).
  • Direct sequencing may be accomplished by chemical sequencing, using the Maxam-Gilbert method (87), or by enzymatic sequencing, using the Sanger method (88). In the latter case, specific oligonucleotides are synthesized using standard methods and used as primers for the dideoxynucleotide sequencing reaction.
  • DNA from a patient is subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers, followed by hybridization with allele-specific oligonucleotides.
  • PCR polymerase chain reaction
  • SSCP analysis of the amplified DNA regions may be used to determine the allelic pattern.
  • allele-specific PCR is used, in which allele-specific oligonucleotides are used as primers and the presence or absence of an amplification product indicates the presence or absence of a particular allele.
  • cells expressing Fc ⁇ RI are isolated by immunoadso ⁇ tion, and RNA is isolated from the immunopurified cells using well-known methods such as guanidium thiocyanate-phenol-chloroform extraction (89).
  • the isolated RNA is then subjected to coupled reverse transcription and amplification by polymerase chain reaction (RT-PCR), using allele-specific oligonucleotide primers.
  • RT-PCR polymerase chain reaction
  • Conditions for primer annealing are chosen to ensure specific reverse transcription and amplification; thus, the appearance of an amplification product is diagnostic of the presence of the allele specified by the particular primer employed.
  • RNA encoding Fc ⁇ RI is reverse-transcribed and amplified in an allele-independent manner, after which the amplified Fc ⁇ receptor-encoding cDNA is identifiable by hybridization to allele- specific oligonucleotides or by direct DNA sequencing.
  • allelic form is intended to mean an alternative version of a gene encoding the same functional protein but containing differences in nucleotide sequence relative to another version of the same gene.
  • allelic polymo ⁇ hism or "allelic variant” is intended to mean a variation in the nucleotide sequence within a gene, wherein different individuals in a general population express different variants of the gene.
  • predetermining polymo ⁇ hic DNA sequence is intended to mean a known allelic variant.
  • allelic pattern is intended to mean the identity of each of the two copies of a particular gene in a patient i.e., homozygosity or heterozygosity.
  • allelic patterns as used herein as being the process of determining the allelic patterns of a human individual.
  • point mutation as used herein is intended to mean a mutation involving a single nucleotide.
  • silent mutation as used herein is intended to mean a change of a nucleotide within a gene sequence that does not result in change in the coded amino acid sequence.
  • missense mutation as used herein is intended to mean a change of a nucleotide within a gene sequence that results in a change in the meaning of a codon, thereby changing the coded amino acid.
  • frame shift mutation as used herein is intended to mean a mutation involving the insertion or deletion of a single nucleotide that results in the remaining downstream sequence being transcribed or translated out of phase.
  • amplification in regard to DNA is intended to mean the use of polymerase chain reaction (PCR) to increase the concentration of a particular
  • DNA sequencing in regard to DNA is intended to mean a method in which DNA is randomly cleaved using individual base specific reactions, illustratively including Maxam-Gilbert sequencing.
  • enzyme sequencing in regard to DNA is intended to mean a method in which single stranded DNA is copied and randomly terminated using DNA polymerase, illustratively including the method of Sanger (88).
  • the term "effective Fc ⁇ RI mediated immune response” is intended to mean immune response that results in the lessening of at least one symptom of a disease to which the host immune response is directed.
  • the receptor mediated immune response is similar in that of other human diseases of tissues protected by neutrophils and monocytes.
  • the diseases illustratively including systemic lupus erythematosus, systemic vasculitis, IgA nephropathy, rheumatoid arthritis, systemic sclerosis, dermatomyositis, Hashimoto's thyroiditis, inflammatory bowel disease and Sjogren's syndrome.
  • FcR-mediated triggering of neutrophils and M ⁇ is a central convergence point in PD. From this perspective, Fc ⁇ R initiate synthesis and secretion of pro-inflammatory cytokines, release of degradative enzymes and release of reactive oxygen intermediates capable of tissue damage (94-96). As counte ⁇ oint, IgA immune complex stimulation of Fc ⁇ RI (CD89) leads to marked enhancement of IL-lra production without IL-1 and TNF ⁇ (44).
  • Fc ⁇ RI CD89
  • Fc ⁇ RIa CD64
  • Fc ⁇ RIIIa CD 16a
  • FceRI ⁇ -chain contains an immunoreceptor tyrosine activation motif (ITAM), is actively tyrosine- and serine/threonine- phosphorylated, and plays a critical role in function (2,14,98,99).
  • ITAM immunoreceptor tyrosine activation motif
  • FceRI ⁇ - chain is both necessary and sufficient for signal transduction (3,100). Indeed, within the field of Fc receptors, there is the common presumption that FceRI ⁇ - chain is the exclusive signaling pathway for those receptors which associate with it.
  • FceRI ⁇ -chain is not the sole actor in FceRI ⁇ -chain-associated Fc receptors.
  • Hogarth and colleagues demonstrated that the cytoplasmic domain (CY) of murine Fc ⁇ RI ligand binding chain (CD64; ⁇ -chain) is actively phosphorylated after treatment of J774 cells with phorbol myristate acetate (101).
  • Transfection studies suggest that Fc ⁇ RI, expressed without ⁇ -chain, appears to be able to mediate some functions like endocytosis ( 102- 104) .
  • the present invention demonstrates that the ⁇ -chain C Y, despite the absence of recognized signaling motifs, participates in initiating cell programs.
  • the Fc ⁇ RI ⁇ -chain CY does interact with actin binding protein (105) and a naturally occurring Fc ⁇ RI ⁇ -chain tail-minus mutant in the NOD mouse has some functional differences (106). While human blood monocytes incubated for 18 hrs with rIFN ⁇ show a modest increase in both Fc ⁇ RI-specific and Fc ⁇ RIII- specific phagocytosis, TGF ⁇ causes a significant decrease in Fc ⁇ RI mediated phagocytosis yet no effect on Fc ⁇ RIII ( Figure 1).
  • P388D1 cells stably transfected with human wild-type (WT) and human CY-minus (MT) Fc ⁇ RI are studied. Both cells lines express comparable levels of receptor. Like the truncated receptor in the NOD mouse, albeit more subtly, the MT in P388D1 endocytoses more slowly than WT.
  • MT phagocytoses less (Fig 2A), an effect which is independent of engagement of the ligand binding site (Fig 2B); it shows a greater sensitivity to chelation of [Ca 2+ ] ; and to the PTK inhibitor genistein and is resistant to the effects of okadaic acid (Fig 3), suggesting a differential engagement of signaling elements compared to WT.
  • Fig 4 shows a model is suggested by the recognition that cytoplasmic domains of different associated chains can interact and modulate function (14,107,108).
  • both the src family tyrosine kinase Lyn and the PKC isoform delta are constitutively associated with ⁇ which in turn facilitates tyrosine phosphorylation and threonine phosphorylation on ⁇ , respectively (98,109,110).
  • Fc ⁇ RIa complex does not have a corresponding ⁇ chain
  • the CY of the ⁇ -chain is involved in the interaction between the ⁇ -chain and may serve to recruit (or perhaps impede) signaling element(s); the okadaic acid data suggests that this element(s) may at least involve ser/thr phosphorylation.
  • the primary sequence of the ⁇ -chain CY domains for Fc ⁇ RI, Fc ⁇ RIII, and Fc ⁇ RIa are different (Table 1), the contributions of each CY domain may provide the basis for the unique receptor function.
  • yeast two-hybrid system is used in the present invention with the GAL4 transcriptional activator, with the CY domain as "bait" cloned in-frame into the pGBT9 vector containing the DNA binding domain of the GAL4 transcriptional activator and with three distinct cDNA libraries cloned into pACT with the GAL4 activation domain.
  • human cDNA libraries an EBV- transformed PBMC library, a PHA-stimulated PBMC library and an unstimulated
  • PBMC library have been used. Recognizing that achieving the native conformation of the CY domain is the goal in the two hybrid system, three different binding domain constructs: (1) binding domain - CY, (2) binding domain - 6 glycines - CY, and (3) binding domain- 6gly-CY-6gly-CY have been constructed. With this approach, a novel member of the band 4.1 family which associates with Fc ⁇ RI CY but not with Fc ⁇ RIII CY, CR1 CY and several other control baits has been identified.
  • Fc ⁇ RIa CY sequence reveals that it is quite distinct from the other FceRI ⁇ -chain associated receptors (Table I) and that it contains several potential serine phosphorylation sites ( Figure 5).
  • the present invention uses the native CY (with or without the 6 glycine linker), the CY with S to D/E to "mimic" serine phosphorylation, and then a variety of truncations and point mutations to establish the role of docking regions and sites with phosphorylation motifs.
  • the present invention Based on the precedent of biologically important SNPs in the coding regions of Fc ⁇ RIIA, Fc ⁇ RIIIA and Fc ⁇ RIIIB, on the recognition of the influence of the Fc ⁇ RIIA and Fc ⁇ RIIIB polymo ⁇ hisms on the risk for PD, and on the knowledge that PD lesions are rich in both IgG and IgA, the present invention identifies novel SNPs in Fc ⁇ RIA. Morton and colleagues have reported a silent SNP in codon 87 (AGA ⁇ AGG, remains Arg), an Asp ⁇ Asn in codon 92 (GAC ⁇ AAC) of extracellular domain 1 (EC1) and a Phe ⁇ Leu at codon 132 (TTT ⁇ CTT) of EC2 (112).
  • Gamma ( ⁇ ) chain plays a critical role in the function of Fc ⁇ RIa, Fc ⁇ RIIIa and Fc ⁇ RI (2).
  • ⁇ -chain provides a necessary signaling element(s) including an ITAM (immunoregulatory tyrosine-based activation motif) (14).
  • ITAM immunoglobulin-associated tyrosine-based activation motif
  • pY phosphorylated tyrosine residues
  • Fc ⁇ RIa is known to be differentially glycosylated in different cell types (33,34) and it has been shown that an altered glycosylation pattern of receptor in certain diseases such as IgA nephropathy.
  • Work with Fc ⁇ RIIIa expressed on NK cells and on monocytes-macrophages provides a framework which suggests that these glycosylation differences in Fc ⁇ RIa are important.
  • Fc ⁇ RIIIa expressed on NK cells and on monocyte-macrophages is also differentially glycosylated despite identical protein cores (Table 3) and these different glycoforms have different affinities for ligand (Figure 15).
  • the macrophage form has a lower apparent Mr, is lower in sialic acid content, and has a lower affinity for IgG (113).
  • Fc receptor polymo ⁇ hisms are important in the development of disease phenotype in chronic inflammatory diseases.
  • the present invention shows that alleles for both Fc ⁇ RIIa and Fc ⁇ RIIIa are altered in their representation in SLE with low binding alleles associated with the SLE phenotype and high binding alleles (176V) protecting against glomerulonephritis (Table 2) (4,114). It is also shown that alleles of Fc ⁇ RIIIB predict the severity of glomerulonephritis in ANCA-positive vasculitis such as Wegener's Granulomatosis (Figure 8). The data presented herein shows that the novel Ser ⁇ Gly in the CKl site of the Fc ⁇ RI CY domain is markedly enriched in chronic inflammatory disease (SLE) (Table 3).
  • Fc receptor polymo ⁇ hisms are also important in host defense against infectious disease (115-117).
  • Fc ⁇ RIIa, Fc ⁇ RIIIa, Fc ⁇ RIIIb and Fc ⁇ RIa are expressed on neutrophils and mononuclear phagocytes, and myeloid cells essential for host defense. Since Fc ⁇ RIIa-R131 binds human IgG2 poorly and IgG2 is a component of the host immune response to encapsulated bacteria, individuals homozygous for Fc ⁇ RIIa-R131 may be at greater risk for bacterial infection. Similarly, individuals homozygous for the low activity Fc ⁇ RIIIb-NA2 may be at greater risk for infection (118).
  • Fc ⁇ RIIa and Fc ⁇ RIIIb polymo ⁇ hisms with PD phenotype suggest that Fc ⁇ R-related function may be important in the pathogenesis of PD.
  • Fc ⁇ RIa polymo ⁇ hisms described above neither has considered the role of the novel Fc ⁇ RIa polymo ⁇ hisms described above.
  • the present invention characterizes the signaling elements associated with Fc ⁇ RI and their role in determining unique Fc ⁇ RI signaling events.
  • Current evidence has been inte ⁇ reted as demonstrating that the ⁇ -chain ITAM is both necessary and sufficient for the function of the FceRI ⁇ -chain associated Fc ⁇ receptors (CD 16 and CD64).
  • the present invention shows that the cytoplasmic domains of the ligand binding ⁇ -chains in these ⁇ -chain receptor complexes are functionally important and provide the basis for diversity in signaling among these FceRI ⁇ -chain associated receptors.
  • the Fc receptor for IgA also forms a receptor complex with the ⁇ -chain, and there is clear precedent that this Fc ⁇ receptor complex is functionally distinct from the Fc ⁇ receptor complexes.
  • the present invention shows that the cytoplasmic domain (CY) of the ligand binding ⁇ -chain of CD89 can modulate the functional responses of the receptor complex and/or possibly elicit functional responses independently of the ⁇ -chain.
  • Fc ⁇ RI has some homology with Fc ⁇ R encoded on human chromosome 1 and with the KLR family of receptors found proximate to Fc ⁇ RI on human chromosome 19
  • Suitable cell lines illustratively include: the murine monocyte-macrophage-like cell lines J774, P388D1 and myeloid cells capable of eliciting appropriate functional responses.
  • Suitable cell lines such as these are transfectable with plasmids and can express transfected human gene products (2,122,123); endogenously express the ⁇ -chain (2).
  • P388D1 has been used extensively to analyze the structural requirements for initiation of functional responses by human Fc ⁇ receptors (especially Fc ⁇ RIa and Fc ⁇ RIIa) and is the preferred choice of host cell for Fc ⁇ RI transfections.
  • Activation of both early signaling events and cell programs are examined in stably transfected lines expressing the wild type and mutant non- ⁇ -chain associated forms of Fc ⁇ RI.
  • Early signals include the phosphorylation of the src- family kinase lyn and of syk, association of these kinases with the receptor, and activation of kinase activity.
  • Fc ⁇ RIa another FceRI ⁇ -chain associated receptor
  • the present invention examines the capacity for CD89 to activate similar or dissimilar pathway(s). To this end, examinations occur of CD89 receptor specific [Ca 2+ ], fluxes and both endocytosis and phagocytosis at low and high levels of receptor cross-linking.
  • Fc ⁇ RI that does not associate with ⁇ -chain
  • a variant of Fc ⁇ RI is constructed from which the TM domain is removed and replaced it with CD8.
  • CD8 does not associate with ⁇ -chain.
  • TCR and BCR 125
  • the magnitude of cross- linking of the receptor complex alters the functional consequences of receptor engagement.
  • a higher level of receptor cross-linking induced by IgA + F(ab') 2 anti-IgA and or anti-receptor mAb + F(ab') 2 GAM
  • IgA + F(ab') 2 anti-IgA and or anti-receptor mAb + F(ab') 2 GAM can result in a distinct pattern of cell activation.
  • a biotin-avidin cross-linking system one can titrate the stoichiometry of receptor aggregations (126).
  • Fc ⁇ RI (carrying specific deletions, truncations, and point mutations) to initiate leukocyte cell functions is initially evaluated in cell lines that have been stably transfected with plasmids that express different forms of Fc ⁇ RI in association with ⁇ -chain. Mutations in the CY domain of Fc ⁇ RI are guided by for instance functionally important regions and/or motifs in Fc ⁇ RI for novel associated molecules identified in the two hybrid system. Comparisons are made between cells that stably express on their membrane equivalent levels of native Fc ⁇ RI (full-length isoform), an Fc ⁇ RI deletion mutants lacking the CY domain and Fc ⁇ RI mutants having truncations or point mutations within the CY domain.
  • Controls include cells not transfected with the CD89-expressing plasmid, or with the parent plasmid that does not contain CD89 DNA.
  • An aspect of present invention is the quantification of polymo ⁇ hic differences in the induction of both rapid signals, — endocytosis, receptor specific [Ca 2+ ] j fluxes, activation of tyrosine kinase activity [lyn and syk], ⁇ and of Fc ⁇ RI- specific effector functions.
  • Receptor specific phagocytosis is evaluated using latex microspheres or erythrocytes opsonized with human IgA or anti-Fc ⁇ RI mAb Fab/F(ab') 2 fragments to avoid co-ligation of endogenous Fc ⁇ receptors.
  • Receptor specific stimulation of oxidative metabolism are quantitated using the same probes as will cytokine production, especially IL-lra, IL-1, IL-6, TNF ⁇ , which are relevant to macrophage function as mediators of inflammatory responses.
  • Fc ⁇ RI expressing transfectants of the present invention are stimulated with surface bound IgG alone or in combination with surface absorbed IgA. Controls include stimulation with LPS in the presence and absence of surface absorbed IgA. Alternatively, mAb is used to engage distinct receptors on the cell surface.
  • mAb 2.4G2 is used to engage murine Fc ⁇ RII/Fc ⁇ RIII and/or murine IgG2a to engage murine Fc ⁇ RIa with and without anti-Fc ⁇ RI mAb Fab/F(ab') 2 fragments.
  • Heterotypic and homotypic cross-linking is provided by either surface absorbed or solution phase F(ab') 2 goat anti-mouse IgG (GAM) or a combination of GAM and anti-human IgA.
  • Peripheral blood neutrophils and monocytes both of which express CD89, are obtained from genotyped donors and studied ex vivo. Peripheral blood monocytes are also be differentiated in vitro into macrophages resembling those found in tissues and having greater abilities to perform phagocytic functions.
  • the approach to the functional consequences of the CD89 SNPs varies with the location of the polymo ⁇ hism in the protein. For polymo ⁇ hisms in the extracellular region, differences in quantitative binding of IgA ligand to mononuclear and polymo ⁇ honuclear cells from genotyped homozygous donors are studied. Binding of both serum IgA and secretory IgA is determined by flow cytometry (127, 128).
  • polymo ⁇ hisms The functional significance of the polymo ⁇ hisms in the CY domain is determined by examination of Fc ⁇ RI induced functions. For example, the polymo ⁇ hisms at positions 245 and 248 alter a casein kinase I phosphorylation site ( Figure 7).
  • genotyped homozygous donors and mAb cross- linking are utilized to rule out any impact of other extracellular polymo ⁇ hisms.
  • neutrophils and monocytes from genotyped homozygous disease free donors a comparison is made between activation of tyrosine kinase activity (lyn and syk) and the induction of cell programs such as receptor specific phagocytosis, oxidative burst, and cytokine production.
  • Fc ⁇ RI has several splice variants and some investigators have hypothesized that these variants may have different functional capacities (29,30,32,112,113). No systematic evaluation of these splice variants in disease states has been previously undertaken to establish their potential relevance. Similarly, Fc ⁇ RI is know to be differentially glycosylated in different cell types and in different disease states (128,132). Using the glycoforms of Fc ⁇ RIIIa as a model (113), the present invention shows that these alterations of glycosylation of Fc ⁇ RI have different functional properties.
  • Example 1 Isolation of DNA/RNA and cDNA preparation: Genomic DNA is extracted from leukocytes (in EDTA anti-coagulated whole blood) using with the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). RNA is isolated from whole blood leukocytes (or Ficoll-Hypaque separated leukocyte fractions) using Triazol RNA isolation solution (Gibco BRL, Gaithersburg, MD). cDNA is synthesized from 5-10 ⁇ g RNA with the Superscript Preamplification System (Gibco).
  • Example 2 - cDNA sequencing To facilitate heterozygote detection, a dye primer strategy is used for fluorescence-based automated cycle sequencing of PCR products on an ABI 377.
  • All sequencing primers are designed with an 18bp M13 sequence tag (ABI PRISMTM Dye Primer Cycle Sequencing -21M13 FS and Ml 3REN FS Ready Reaction Kits (ABI, Foster City, CA)).
  • the PCR products are purified with the QIAquick Gel Extraction Kit (Chatsworth, CA).
  • the BigDye terminator sequencing reaction chemistry (ABI) is also used to detect heterozygosity in multiple Fc receptors genes including CD89. This chemistry has the advantage of allowing longer sequence reads and does not require the inco ⁇ oration of additional M13-based sequence in the primers. It is appreciated that all potentially polymo ⁇ hic residues must ultimately be confirmed by sequencing in both the forward and reverse directions using the dye-primer based strategy.
  • Example 3 Determination of Fc ⁇ receptor alleles (12-14): Allele- specific PCR assays are used to genotype donors for the Fc ⁇ RI alleles illustratively including: Fc ⁇ RIa (ECI) - 87R 87R, Fc ⁇ RIA (ECI) - 92D/92 ⁇ , Fc ⁇ RIa (EC2) - 132F/132L, F ⁇ RI (CY) - 245P/245L and Fc ⁇ RI (CY) - 248S/248G alleles. Since there is a finite error rate in any genotyping assay, each assay is corroborated by direct dye-primer based cycle sequencing of at least 40 homozygous donors and an equal number of heterozygous donors.
  • each assay includes blinded but known genotyped controls to verify the fidelity of the assay.
  • Ambiguities in the assay are resolved by first repeating the allele- specific PCR reaction and then by direct dye-primer based cycle sequencing of genomic DNA samples.
  • Example 4 - Amplification of Fc ⁇ RI cDNA Two sets of overlapping primers inco ⁇ orating either the Ml 3 universal or reverse primer sequences at the appropriate 5' ends are used to amplify the entire coding region of the Fc ⁇ RI cDNA.
  • PCR is performed in a GeneAmp 9600 PCR system with 2.5U of Taq DNA polymerase (Gibco BRL) using the following cycling conditions: 95 °C for 5 min followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 45 sec and extension at 72 °C for one min, followed by a final extension at 72 °C for ten min.
  • the PCR products are gel purified with the QIAquick GEL
  • the first set of primers including the underlined Ml 3 sequence amplify from nt 34 in SI to nt 563 in EC2 (forward primer: 5'-TGT A A A ACG ACG GCC AGT AGC ACG ATG GAC CCC AAA CAG-3' (SEQ ID NO. 1); reverse primer: 5'-CAG GAA ACA GCT ATG ACC GGT GTT CCC CAC TTT GGT GC-3 ' (SEQ LD NO. 2).
  • the second set of primers amplify from nt 458 in ECI to nt 934 in the 3 '-untranslated region (forward primer: 5'-TGT AAA ACG ACG GCC AGT AGA ATA TTT CCC TCA CGT GC-3' (SEQ ID NO. 3); reverse primer: 5'-CAG GAA ACA GCT ATG ACC CTG GCT CCT CTC TGC CTT CAC-3' (SEQ ID NO. 4)).
  • Example 5 - Yeast 2-Hybrid System The yeast two-hybrid system based on the Gal4 transcriptional activator will use our cloned 41 amino acid CD89 cytoplasmic domain in the pGBT9 vector containing the DNA binding domain of the GAL4 transcriptional activator (BD-CD89). Screening the BD-CD89 is performed against a cDNA expression library isolated from total unstimulated human PBL, a cDNA library from EBN infected pooled human PBL and a cD ⁇ A library from PHA-stimulated PBL all of which are cloned into the pACT vector with the Gal 4 activation domain.
  • BD-CD89 GAL4 transcriptional activator
  • a U937 myeloid cD ⁇ A library in pACT is also constructed for this screening pu ⁇ ose.
  • double transformants (BD-CD89 + AD construct) are screened on T ⁇ " ,Leu, His " dropout plates containing 10 mM 3-amino-triazole to select for positive interactions between the CD 89 cytoplasmic domain and the fusion protein encoded by AD-cD ⁇ A.
  • Growth positive colonies are screened secondarily for LacZ activity, the second reporter gene under the control of the Gal 4 promoter.
  • the plasmids containing the AD-cD ⁇ A construct is recovered from growth (+), LacZ (+) colonies by transforming into E. coli HBIOI, harvested, sequenced, and compared to information currently in GenBank.
  • Likely candidates, identified by multiple isolates and by putative biology, are directly rescreened (BD-CD89 + AD-candidate cD ⁇ A), and, if positive, are then tested for protein-protein interactions in co-immunoprecipitations from whole cell lysates and in GST- fusion protein (Pharmacia) affinity matrix/whole cell lysate interactions. Positive candidates in the two hybrid screen which are also positive for bonafide protein- protein interactions are deemed to be true associations.
  • An unstimulated PBL library in the DupLEX-ATM system (Origene) also successfully screened with a
  • Example 6 Transfection: Using the pRC/CMN (Invitrogen) expression vector, COS-7 cells (CaPO4, glycerol shock and chloroquine; and lipofectamine), UC729.6 cells (electroporated using 280N and 960 ⁇ F across 0.4 cm) and P388D1 (CaPO4, glycerol shock and chloroquine and lipofectamine) have been transiently, and transfected P3881 D 1 cells using both CaPO4, glycerol shock and chloroquine and lipofectamine have been stably transfected.
  • COS-7 cells CaPO4, glycerol shock and chloroquine; and lipofectamine
  • UC729.6 cells electroporated using 280N and 960 ⁇ F across 0.4 cm
  • P388D1 CaPO4, glycerol shock and chloroquine and lipofectamine
  • the FACS Nantage is used for flow cytometry with receptor-specific mAbs to cell sort for subpopulations expressing quantitatively different levels of receptor. It is appreciated that comparable levels of transfectant expression are required in studying differences in function by different receptor constructs.
  • Example 7- Receptor and Isoform cloning Products generated by PCR are separated by electrophoresis through low melting point agarose. To generate clones for sequencing the TA Topoisomerase Cloning system (Invitrogen) is used. For mammalian expression, blunt end ligated PCR product is inserted into vector pRC/CMN (Invitrogen), transformed competent E. coli with ampicillin based selection and confirmed plasmid integration with plasmid isolation and restriction fragment pattern analysis. Newer strategies for eukaryotic expression including the use of undirectional TA cloning strategies and the expression vector pCR3-Uni which uses the immediate-early CMV promoter (Invitrogen) are also suitable.
  • Example 8 Construction of GST fusion proteins: To generate glutathione-S-transferase (GST) fusion constructs for expression in E. coli, PCR amplified cDNA or cDNA fragments are subcloned into the BamHI-EcoRI site of the pGEX-2T vector (Pharmacia Biotech). Following transformation into E. coli DH5a, fusion protein is induced in log phase cultures by the addition of 0.1 mM
  • oligonucleotide primers containing the desired mutation(s) are used for
  • DNA glycosylase and used to transform competent E. coli. Mutations are verified by sequencing and then cloned into an appropriate eukaryotic expression vector such as pRC/CMV to generate stable transfectants of P388D1 cells.
  • genotyping is also operative, such as the oligonucleotide ligation assay (OLA).
  • OLA oligonucleotide ligation assay
  • the two labeled primers need to be of different lengths (different by >2 nucleotides) and can be labeled with the same or different fluorescent probes (such as 6-fam and tet for detection on the ABI377). This technique is now widely used for analysis of mutations in the cystic fibrosis gene (63).
  • Example 12 Determination of cytokine promoter alleles: While some microsatellites associated with various cytokine genes have been identified, a systematic search of the 5' promoter region reference sequences for novel SNPs is not available (4,45-51,113). However, SNPs occur with reasonable frequency (approximately 1 per 500-1000 bp) and that at least some of these SNPs are of biological significance. Identifying such SNPs and developing both microsatellite and SNP assays for characterization of a clinical population use the techniques of Examples 1-5, as well as other techniques conventional to the art. Examples of cytokine genes are given:
  • Example 13 General Protein Techniques: Techniques for immunoprecipitation, SDS-PAGE, Western blotting, and related methods are known to the art.
  • MAb to Fc ⁇ R and Fc ⁇ R (CD89) are commercially available (Medarex, Biodesign International, Pharmingen).
  • MAb and polyclonal Ab to src- family tyrosine kinases and syk are also available (UBI, Santa Cruz Biochemicals, Signal Transduction Labs).
  • Example 14 - ⁇ -chain co-immunoprecipitation Using detergent lysing conditions that have been shown to preserve the non-covalent interaction between ⁇ -chain and Fc receptor (35,97), cells are lysed in buffer containing 1% digitonin (Wako) and protease inhibitors. CD89 is immunoprecipitated with anti-receptor mAb using standard techniques. Following SDS-PAGE separation and blotting, ⁇ -chain will be detected with a rabbit polyclonal anti- ⁇ -chain Ab that recognizes both human and murine ⁇ -chain.
  • Wako digitonin
  • CD89 is immunoprecipitated with anti-receptor mAb using standard techniques. Following SDS-PAGE separation and blotting, ⁇ -chain will be detected with a rabbit polyclonal anti- ⁇ -chain Ab that recognizes both human and murine ⁇ -chain.
  • Example 15 - Phosphotyrosine Analysis Stimulated or control neutrophils are lysed as previously described in Example 8 with the addition of the phosphatase inhibitor sodium orthovanadate (0.4 mM). Anti-phosphotyrosine or anti -target immunoprecipitates are separated by SDS-PAGE and transferred to nitrocellulose membranes essentially as described (126). Blots are detected with either anti-target Ab or anti-phosphotyrosine (mAb 4G10 or PY20) and detected with a HRP conjugated sheep-anti-mouse IgG followed by detection with an enhanced chemiluminescence reagent set (Amersham).
  • mAb 4G10 or PY20 anti-target Ab
  • HRP conjugated sheep-anti-mouse IgG followed by detection with an enhanced chemiluminescence reagent set (Amersham).
  • Example 16 - CoUagenase Assay The release of collagenase (MMP-1) from human neutrophils of monocytes is analyzed with the use of a standard sandwich ELISA (129), using plates coated with mAb COMY4A2. After incubating with samples of monocyte supernatant or standards, bound collagenase is detected by means of monoclonal antibody an HRP-conjugated polyclonal anti- collagenase antibody 647.
  • Example 17 - Cytokine assays Levels of cytokines IL-1, IL-lra, IL-6, and TNF ⁇ secreted in culture media are assayed by ELISA, using polyclonal (goat) and monoclonal anti-human cytokine reagents (R&D Systems, Minneapolis, MN). For murine cytokines secreted by transfected cells, anti-mouse cytokine reagents is obtained from Pharmingen (San Diego), or R&D Systems. The assays are calibrated against standards obtained from the same sources.
  • Example 18 - Purification of Igs Human serum Igs are purified by chromatography using anion exchange (Mono Q), molecular sieve (Superose 6 or 12, or Sephacryl S-300), affinity (jacalin-HiTrap for IgAl; protein G-HiTrap for
  • IgG immunoadsorbent (anti-IgM-HiTrap) columns in an FPLC apparatus (Pharmacia Biotech, Piscataway, NJ) or conventional columns.
  • IgA subclasses are separated by means of a jacalin-HiTrap column which retains IgAl while IgA2 passes; IgAl is then recovered by elution with 0.1 M melibiose.
  • Monomeric and polymeric IgA are separated by HPLC on a Biosep Sec-S3000 column
  • IgA myeloma proteins of both subclasses and in different molecular forms are available from a collection kept by Dr. Jiri Mestecky. These are purified by essentially the same procedures as described for normal serum IgA.
  • Example 19 - Assay of Ig and antibodies ELISA: To assay Igs, the plates are coated with anti-Ig antibodies of the desired specificity (Dako Co ⁇ ., Ca ⁇ interia, CA), at optimal levels (1-10 ug/ml). The plates are blocked with 0.15% Tween 20 in PBS, and serially diluted samples, starting from a dilution appropriate to the sample and the expected analyte concentration, are applied overnight. Bound Ig is detected by means of peroxidase-conjugated antibody to the analyte, diluted to the previously determined optimal level in each case (usually 1:1,000-1:5,000), and applied for 4 h. When IgA subclasses are assayed, the bound Ig is detected with monoclonal anti-IgA 1 or anti-Ig A2 antibodies
  • the assay is calibrated by means of a serially diluted standard (Human Ig Reference Preparation; The Binding Site, San Diego, CA), and a standard curve is generated by computer program based on the 4-parameter logistic method (143).
  • PMN, myeloid cells and monocytes are prepared as described (144) using discontinuous ficoll/hypaque density gradient centrifugation. Monocytes are purified by adherence to 100 mm tissue culture plates or directly in 96 well plates for use in functional assays for 90 min at 37°C/5% CO 2 (144). The non-adherent lymphocytes are recovered by rinsing in medium. When appropriate, adherent monocytes are collected by treatment for 2 min.
  • monocytes are allowed to form homotypic clumps by incubation 4°C (145). Clumped monocytes are then sedimented through a FCS cushion at 1 xg, washed and gently dispersed. Purities >90% can routinely be achieved with these techniques (144). The purity of monocytes is assessed by staining for non-specific esterase or with fluorochrome-labeled antibody to CD14.
  • Eosinophils are isolated from the granulocyte fraction of citrated normal human blood (obtained by centrifugation through Percoll SG 1.082). Erythrocytes are removed by hypotonic lysis in water. CD 16+ cells (mainly neutrophils, also NK cells) are removed by magnetic sorting (MACS column, Miltenyi Biotec) using anti-CD 16 antibody coupled to magnetic beads. The yield is -98% pure esosinophils (146).
  • Example 21 - Cell culture Cells are cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, L-glutamine, non-essential amino acids, and antibiotics (complete medium) at 37 ° C in humidified 5 % CO 2 /air ( 123 , 144) .
  • Example 22 - Flow Cytometry Standard analytical flow cytometry (one to four colors) is performed on a FACScan flow cytometer (Becton-Dickinson). Sorting of transfected cell lines is performed on a FACS-Nantage (Becton- Dickinson). The instrument is capable of routine sterile sorts into tubes or tissue culture plates.
  • Example 23 - Phagocytosis Assay The phagocytic response of transfected murine cells or human peripheral blood monocytes are assessed by flow cytometry (41). Fluorescent 1 urn microspheres (Polysciences, Warrington, PA) are coated with human IgA or IgG, or with mAb to CD89 at 25 ug/10 9 microspheres in PBS at 4°C for 16 h and blocked with 1% human serum albumin
  • HSA HSA in PBS.
  • Control microspheres are coated with HSA only.
  • microspheres are coated with anti-human light chain F(ab)'2 to capture human Ig in a more physiological presentation of the Fc region for receptor engagement.
  • the coated microspheres are added to the cultured monocytes at a ratio of 10 microspheres/monocytes for 4 h at 37°C in 5% C02, and the cells are collected for analysis by flow cytometry (FACScan, Becton Dickinson).
  • biotinylated anti-receptor mAb or Ig are bound to streptavidin saturated biotinylated PKH26-labeled E as we have previously reported (147).
  • the opsonized E are mixed with phagocytes at a ratio of 50:1, incubated for varying periods of time, and the non-internalized E are removed by hypotonic lysis. Phagocytosed E are then quantitated by flow cytometry.
  • Example 24 Superoxide Assay: The production of superoxide (O2) by transfected cells of human monocytes is determined by superoxide dismutase
  • Example 26 Novel molecular associations with the cytoplasmic domain of Fc ⁇ RI: Using the yeast two-hybrid system based on the GAL4 transcriptional activator, the entire 41 amino acid cytoplasmic domain of CD89 in-frame with a 6-gly linker is cloned into the pGBT9 vector containing the DNA binding domain of the GAL4 transcriptional activator. Using this binding domain construct expressing the cytoplasmic domains of Fc ⁇ RI, screening against cDNA expression libraries that have been cloned into the pACT vector with the GAL4 activation domain (AD) occurs.
  • AD GAL4 activation domain
  • AD-cD ⁇ A construct plasmid is recovered from growth (+), LacZ (+) colonies by transforming into E. coli, harvested, sequenced, and compared to information currently in GenBank.
  • the yeast two-hybrid system is best suited for higher affinity protein- protein interactions which are not phosphorylation dependent. Therefore, while true positives are highly informative, they do not comprise the entire repertoire of interacting proteins. The ability to identify true positives also depends on the suitability of the screening library. These libraries are particularly useful in identifying proteins that interact with Fc ⁇ R expressed in myeloid and lymphoid cells. When difficulties are encountered with the GAL4-based system, the DupLex-A TM system has several potential advantages including a reduction in the number of false positives and ease of co-immunoprecipitation with the HA tag downstream of the B42 activation protein.
  • Example 27 Identification of novel SNPs in Fc ⁇ RI: Through the examination of cDNA derived from disease free-individuals, the present invention identifies multiple single nucleotide polymo ⁇ hisms in the coding region of the CD89 gene. Indeed, there are SNPs in each of the three exons encoding the ECI, EC2 and the TM CY domains ( Figure 4).
  • Fc ⁇ RI 29,30,32,112,131. Many of these transcripts are co-expressed in the same cell type. Accordingly, the present invention amplifies the Fc ⁇ RI cDNA in two overlapping segments and gel purifies fragments corresponding to the full length Fc ⁇ RI transcript (expressing SI, S2, ECI, EC2 and TM/CY). The 5' segment is amplified with a pair of primers located in the SI region and the EC2 region (nt
  • the 3' segment of the Fc ⁇ RI cDNA is amplified with a pair of primers located in ECI and the 3 '-untranslated region (nt458 to 934).
  • the gel- purified PCR amplified segments are cycle-sequenced from both directions.
  • the sequencing of a specific PCR-amplified Fc ⁇ RI cDNA may be complicated by the presence of multiple splice variants that cannot be adequately resolved by agarose gel electrophoresis. For example, if there is a splice variant that does not express exon S2 (36 nt), which allows co-purification with wild type Fc ⁇ RI. Sequencing of the 5' segment in the forward direction is then not possible. However, in this situation, reverse direction sequencing up to the S2-EC1 splice site yields the necessary information.
  • Example 28 Expression of Fc ⁇ RIb and Fc ⁇ RI splice variants in patients with PD: A protein product of a sequence is studied by biosynthetically labeling cells expressing the sequence with 35 S-methionine, the cells are lysed and anti-CD89 mAb is used for immunoprecipitation from both cell supematants and from cell lysates. Following deglycosylation with endoglycosidase F (N- Glycanase®), the putative product is resolvable from the larger a isoforms on a higher percentage polyacrylamide gel.
  • N- Glycanase® endoglycosidase F
  • the mature proteins in 125 I-labeled anti- CD89 immunoprecipitates from PMN and eosinophils are each heterogeneous but very distinct in apparent Mr, 55-75kD vs. 70-lOOkD respectively (128).
  • these forms Upon treatment with endoglycosidase F, these forms reduce to a common major band of 32kD with a minor 36 kD band evident from PMN.
  • the mol wt of Fc ⁇ RI from human monocytes and U937 cells are analyzed in comparison to PMN and eosinophils with and without endoglycosidase F (N-glycanase ® ) treatment as we have previously done for human Fc ⁇ RIII (144).
  • the present invention demonstrates the CD89 polymo ⁇ hism can be exploited as a diagnostic assay for determining susceptibility to and or severity of various diseases involving IgA.
  • the present invention further demonstrates a process through which the use of genotyping and/or phenotyping to assist in defining disease prognosis.
  • Clark MR Clarkson SB
  • Ory PA Stollman ⁇ , Goldstein IM. Molecular basis for a polymo ⁇ hism for human Fc ⁇ receptor II on human monocytes. J Immunol 143: 1731, 1989. 6. Warmerdam PAM, van de Winkel JGJ, Gosselin EJ, Capel PJA.
  • Clark MR Stuart SG, Kimberly RP, Ory PA, Goldstein LM.
  • a single amino acid distinguishes the high-responder from low-responder form of Fc receptor II on human monocytes. Eur J Immunol 21: 1911-1916, 1991.
  • Fc gamma receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fc gamma receptor II. J Immunol 146: 997- 1004, 1991.
  • IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10. J Immunol 149: 4048-4052, 1992. 21. Daeron M, Latour S, Malbec O, Espinosa E, Pina P, Pasmans P, Fridman WH.
  • TGF-bl Transforming growth factor-beta 1 (TGF-bl) down-regulates IgA Fc- receptor (CD89) expression on human monocytes. Clin. Exp. Immunol. 103: 161-166, 1996. 40. Russell MW, Reinholdt J, Kilian M. Anti-inflammatory activity of human IgA antibodies and their Fab fragments: inhibition of IgG-mediated complement activation. Eur. J. Immunol. 19: 2243-2249. 1989.
  • Wilson ME Kalmar JR. Fc ⁇ RIIa (CD32): a potential marker defining susceptibility to localized juvenile periodontitis. J Periodontol. 67: 323- 331, 1996.
  • GM-CSF induces human neutrophil IgA-mediated phagocytosis by an IgA Fc receptor activation mechanism. Nature 332:647-648, 1988.

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Abstract

L'invention concerne une méthode et une combinaison permettant d'identifier des polymorphismes nucléotidiques simples dans FcαRI, cette méthode et cette combinaison pouvant être utilisées pour identifier la sensibilité d'un sujet à une maladie. FcαRI agit comme récepteur cellulaire de l'immunoglobuline (IgA), la sensibilité à une maladie liée à IgA, ou la sévérité de cette maladie, étant définie par génotypage ou phénotypage d'un sujet selon FcαRI.
PCT/US1999/016762 1998-07-24 1999-07-23 POLYMORPHISME GENETIQUE DANS LE RECEPTEUR DE IgA WO2000005403A1 (fr)

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US09/744,373 US6986987B1 (en) 1998-07-24 1999-07-23 Genetic polymorphism in the receptor for IgA
AU52269/99A AU5226999A (en) 1998-07-24 1999-07-23 Genetic polymorphism in the receptor for iga

Applications Claiming Priority (2)

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US9409698P 1998-07-24 1998-07-24
US60/094,096 1998-07-24

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WO2000005403A1 true WO2000005403A1 (fr) 2000-02-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7947463B2 (en) * 2000-05-10 2011-05-24 Signe Biopharma, Inc. Compositions and methods for the diagnosis, treatment and prevention of steroid hormone responsive cancers

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DE WIT T.P.M. ET AL: "Structure of the Gene for the Human Myeloid IgA Fc Receptor (CD89)", J. IMMUNOL., vol. 155, 1995, pages 1203 - 1209, XP002921996 *
PATRY C. ET AL: "Identification of Fc.alpha. Receptor (CD89) Isoforms Generated by Alternative Splicing that are Differentially Expressed Between Blood Monocytes and Alveolar Macrophages", J. IMMUNOL., vol. 156, 1996, pages 4442 - 4448, XP002921995 *
RETERINK T.J.F. ET AL: "Alternative Splicing of IgA Fc Receptor (CD89) Transcripts", GENE, vol. 175, no. 1-2, February 1996 (1996-02-01), pages 279 - 280, XP002921998 *
VAN DIJK T.B. ET AL: "Cloning and Characterization of Fc.alpha.Rb, a Novel Fc.alpha. Receptor (CD89) Isoforms Expressed in Eosinophils and Neutrophils", BLOOD, vol. 88, no. 11, 1 December 1996 (1996-12-01), pages 4229 - 4238, XP002921997 *
WU J. ET AL: "A Novel Polymorphism of Fc. gamma RIIIa (CD16) Alters Receptor Function and Predisposes to Autoimmune Disease", J. CLIN. INVEST., vol. 100, no. 5, September 1997 (1997-09-01), pages 1059 - 1070, XP002921994 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7947463B2 (en) * 2000-05-10 2011-05-24 Signe Biopharma, Inc. Compositions and methods for the diagnosis, treatment and prevention of steroid hormone responsive cancers
US8563249B2 (en) 2000-05-10 2013-10-22 Signe Biopharma Inc. Receptor gene screening for detecting or diagnosing cancer

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