CK2α - protein phosphatase 2A molecular complex: Possible interaction with the MAP kinase pathway

Despite its wide range of known substrates, the signaling function of protein kinase CK2 is still enigmatic. Mounting evidence suggests that CK2, the catalytic subunit of holoenzymic CK2, may exist free of its usual regulatory partner CK2, raising the possibility that free CK2 has regulation and function distinct from those of the holoenzyme. We previously reported that CK2 could bind to the core dimer of protein phosphatase 2A, and indirectly cause down-regulation of the PP2A substrate MEK1, possibly via activation of PP2A and/or targeting of PP2A to some element of the Ras/Raf/MEK pathway. Here, these results are confirmed and extended. By using transfection experiments and immune kinase assays, we show that endogenous PP2Ac and CK2 are the only major substrates associating with epitope-tagged CK2, and that expression of activated Raf results in disruption of the CK2- PP2A association. Such disruption might be a necessary step for maximal activation of the MAP kinase pathway by Raf. In keeping with this idea, overexpression of CK2 dose-dependently inhibits the mitogen-induced activation of cotransfected, epitope-tagged MAP kinase. We suggest that the CK2 free form of CK2 is both a target and a regulator of Raf/MAPK signaling.     

Localization of individual subunits of protein kinase CK2 to the endoplasmic reticulum and to the Golgi apparatus

The protein kinase CK2 is composed of two catalytic - or - and two regulatory -subunits. In mammalian cells there is ample evidence for the presence of individual CK2 subunits beside the holoenzyme. By immunofluorescence studies using peptide antibodies which allow us to detect the CK2-, - and -subunits we found all three subunits to be co-localized with a 58 KDa Golgi protein which is specific for the Golgi complex. Subfractionation studies using dog pancreas cells revealed the presence of all three subunits of CK2 at the smooth endoplasmic reticulum (sER)/Golgi fraction whereas the rough endoplasmic reticulum (rER) harboured only the catalytic - and -subunits. We found that the microsomal preparation from dog pancreas cells contained CK2 which phosphorylated a CK2 specific synthetic peptide and which was heparin sensitive. Furthermore, we could immunoprecipitate the CK2-subunit that exhibited a kinase activity which phosphorylated a CK2 specific substrate and which was heparin sensitive. Protease digestion experiments revealed that the CK2 subunits were located on the cytosolic side of the rER and the sER/Golgi complex. Thus, we could demonstrate an asymmetric distribution of the CK2 subunits at the rER and sER/Golgi complex. Since the CK2- and -subunits exhibit a substrate specificity which is different from the CK2 holoenzyme one might speculate that the asymmetric distribution of the CK2 holoenzyme and the CK2 catalytic subunits may have regulatory functions.     

Functional specialization of CK2 isoforms and characterization of isoform-specific binding partners

In mammals, protein kinase CK2 has two isozymic forms of its catalytic subunit, designated CK2gr; and CK2. CK2 and CK2 exhibit extensive similarity within their catalytic domains but have completely unrelated C-terminal sequences. To systematically examine the cellular functions of each CK2 isoform in mammalian cells, we have generated human osteosarcoma U2-OS cell lines with the expression of active or inactive versions of each CK2 isoform under the control of an inducible promoter [22]. Examination of these cell lines provides evidence for functional specialization of CK2 isoforms at the cellular level in mammals with indications that CK2 is involved in the control of proliferation and/or cell survival. To understand the molecular basis for functional differences between CK2 and CK2, we have undertaken studies to identify proteins that interact specifically with each isoform of CK2 and could contribute to the regulation of their independent functions. A novel pleckstrin-homology domain containing protein, designated CK2-interacting protein 1 (i.e. CKIP-1) was isolated using the yeast two hybrid system as a protein that interacts with CK2 but not CK2 [23]. When expressed in cells as a fusion with green fluorescent protein, CKIP-1 localizes to the cell membrane and to the nucleus. In this study, we present evidence from deletion analysis of CKIP-1 suggesting that a C-terminal region containing a putative leucine zipper has a role in regulating its nuclear localization. Collectively, our data supports a model whereby CKIP-1 is a non-enzymatic regulator of CK2 that regulates the cellular functions of CK2 by targeting or anchoring CK2 to specific cellular localization or by functioning as an adapter to integrate CK2-mediated signaling events with components of other signal transduction pathways.     

Multiple forms of protein kinase CK2 present in leukemic cells: In vitro study of its origin by proteolysis

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2 was more resistant to these proteases than CK2. When these proteases were assayed on the reconstituted (22 holoenzyme, a 37 kDa -band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2 deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells.     

Expression and regulation of protein kinase CK2 during the cell cycle

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2, CK2 and CK2 on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2 and CK2 following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2 to CK2 during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2 relative to CK2 in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2 increase in mitotic cells, that CK2 and CK2 are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.     

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic ( and ) and regulatory () subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of free CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemaggutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with and subunits of Xenopus CK2, with the subunit of D. rerio, and with Xl CK2A¹⁵⁶, which although inactive can bind tightly to CK2, and with Xl CK2E⁷⁵E⁷⁶, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10–20% of treated cells.Expression of CK2 or CK2E⁷⁵E⁷⁶ in COS-7 cells caused an increase of 5–7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2, the activity increased further to 15–20-fold of the controls. Transfection of CK2 alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2A¹⁵⁶ yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, cotransfection of CK2 or CK2E⁷⁵E⁷⁶ with CK2A¹⁵⁶ caused a 60–70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2 subunits. These results can be interpreted as meaning that CK2A¹⁵⁶ is a dominant negative mutant that can compete with the other catalytic subunits for the CK2 subunit. Addition of recombinant CK2 to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2 subunit is limiting in the extracts and that an excess of free CK2 has been produced in the transfected cells. Transfection of cells with CK2E⁷⁵E⁷⁶ results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2E⁷⁵E⁷⁶ to heparin increases considerably when it forms part of the holoenzyme CK2₂₂.     

Characterization,subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

We have isolated and characterized the genomic clone of maize casein kinase 2 (CK2) subunit using the previously described CK2-1 cDNA clone as a probe. The genomic clone is 7.5 kb long and contains 10 exons, separated by 9 introns of different size, two larger than 1.5 kb and the others around 100–150 bp. The sequence of the exons is 100% homologous to the sequence of the CK2-1 cDNA. Southern hybridization of total genomic DNA from maize embryos with CK2 cDNA indicated that the CK2-1 gene is part of a multigenic family. We also isolated a new embryo cDNA clone coding for an CK2-2 subunit. We studied the regulation of the enzyme in embryos at the mRNA level, at the protein level and by activity testing. By using immunocytochemistry the CK2 protein was localized in several types of cells of mature embryos. Particularly strong signals were visible in the cytoplasm of epidermis and meristematic cells. Decoration of nuclei of root cortex and scutellum cells was also observed suggesting that CK2 can shift from the cytoplasm into nuclei in specific cell types. We examined whether CK2 contained specific protein domains which actively target the protein to the nucleus by using in-frame fusions of the maize CK2 subunit to the reporter gene encoding -glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved in other CK2 proteins.     

Response of cancer cells to molecular interruption of the CK2 signal

Protein kinase CK2 is one of the key cellular signals for cell survival, growth, and proliferation. It is has been observed to be elevated in various cancers that have been examined. Various observations suggest that moderate dysregulation of CK2 may profoundly influence the cell response. We have examined the effects of interfering with the CK2 signal in various cancer cell lines by employing antisense oligodeoxynucleotides (ODN) against the and subunits of CK2. Our results demonstrate that antisense CK2- and antisense CK2- ODNs markedly influence cell viability of these cancer cells in a dose and time-dependent manner. Antisense CK2- was slightly more effective than antisense CK2- in most of the cells tested. The efficacy of the antisense ODN seemed to vary with the cell type; however, in all cases potent induction of apoptosis was observed. Significantly, the effects of the antisense ODN on the CK2 activity in the nuclear matrix were relatively small compared to the much stronger induction of apoptosis in cells. This suggests that modest down-regulation of CK2 can evoke a much greater apoptotic response in cancer cells.     

Intermolecular contact sites in protein kinase CK2

Chemical crosslinking and analysis of CNBr-digested fusion products by immunoblotting with sequence-specific antibodies identifies an interaction between positions 55-70 of subunit (55-70) and 65-80 of subunit (65-80). This has been supported by crosslinking of subunits with peptides 65-80 and 55-70, by binding of subunits to immobilized peptides, and by the hindrance of coprecipitation with peptide-raised antibodies (anti-65-80; anti-55-70). Functionally, 55-70 is a negative regulatory region for the kinase activity of subunit . The opposite, stimulatory property of subunit has been assigned to its C-terminal part. Subdivision of peptide 155-181, that has stimulatory effect, into overlapping peptides and assaying for a binding and binding competition revealed a tight physical contact at 162-175. This region, however, is non-stimulatory indicating binding a necessary but not sufficient quality for stimulation. A contact might exist to regions surrounding C147 and/or C220 at subunit a as indicated by crosslinking and peptide competition. The crosslinking data also confirm a - contact in CK2 holoenzyme. Effects by non-ionic detergents show hydrophobic interactions to play an important role in catalytic activity adjustment.     

Novel Type of Signaling Molecules: Protein Kinases Covalently Linked with Ion Channels

Recently we identified a new class of protein kinases with a novel type of catalytic domain structurally and evolutionarily unrelated to the conventional eukaryotic protein kinases. This new class, which we named alpha-kinases, is represented by eukaryotic elongation factor-2 kinase and the Dictyosteliummyosin heavy chain kinases. Here we cloned, sequenced, and analyzed the tissue distribution of five new putative mammalian -kinases: melanoma -kinase, kidney -kinase, heart -kinase, skeletal muscle -kinase, and lymphocyte -kinase. All five are large proteins of more than 1000 amino acids with an -kinase catalytic domain located in the carboxyterminal part. We expressed the catalytic domain of melanoma -kinase in Escherichia coli, and found that it autophosphorylates at threonine residues, demonstrating that it is a genuine protein kinase. Unexpectedly, we found that long aminoterminal portions of melanoma and kidney -kinases represent new members of the TRP ion channel family, which are thought to mediate the capacitative Ca²⁺entry in nonexcitable mammalian cells. This suggests that melanoma and kidney -kinases, which represent a novel type of signaling molecule, are involved in the regulation of Ca²⁺influx in mammalian cells.     

Identification of proteins that associate with protein kinase CK2

In order to aid in an understanding of the cellular functions of protein kinase CK2, a search for interacting proteins was carried out using a 32P-labeled CK2 overlay method. Several proteins were found to associate with CK2 by this assay; among them, one protein of 110 kDa appeared to be the most prominent one. The possible association of CK2 with p110 was suggested by experiments involving the co-immunoprecipitation using anti-CK2 antibodies. Further analysis using GST-CK2 fusion proteins demonstrated that the CK2-p110 interaction occurred through the CK2/ subunits. To identify p110, it was purified using a GST-CK2 affinity column, and internal amino acid sequencing was then performed. p110 was found to be nucleolin, a nucleolar protein that may be important for rRNA synthesis; a possible role of CK2 in the control of this process is suggested. Using the same CK2 overlay technique, another interacting protein, insulin receptor substrate 1 (IRS-1), was also identified. By applying a modified overlay method using individual 35S-labeled CK2 subunits, obtained by in vitro translation in rabbit reticulate lysates, it was determined that CK2 associates with IRS-1 through its / subunits; i.e. in keeping with the fact that IRS-1 is a known substrate for CK2. However, further work is needed to examine the association of CK2 with IRS-1 in vivo in order to fully understand the significance of the interaction.     

Sequence analysis of 22 kDa-like α-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements

Several genomic and cDNA clones encoding the 22 kDa-like -coixin, the -prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like -coixin genes designated -3A, -3B and -3C were found in the 15 kb -3 genomic clone. The -3A and -3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the -3B gene, suggesting that the three -coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication.Comparison of the deduced amino acid sequences of -coixin clones with the published sequences of 22 kDa -zein and 22 kDa-like -kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15–20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like -prolamins and the 19 kDa -zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins.Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5 and 3 flanking regions of -3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. –300 prolamin box are present at conserved positions in -3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in -3B that occupies approximately the same positions as those identified for the 22 kDa -zein and -kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes.The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like -prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Depletion of catalytic and regulatory subunits of protein kinase CK2 by antisense oligonucleotide treatment of neuroblastoma cells

Summary 1. The use of antisense oligonucleotides to inhibit expression of the genes coding for the catalytic (/') and regulatory () subunits of protein kinase casein kinase 2 (CK2) has allowed study of the role of this enzyme in mouse neuroblastoma cells.2. Selective depletion of catalytic (/') subunits results in the blocking of neuritogenesis. The depletion of catalytic subunits also affects the sorting of the regulatory () subunit of CK2, as the absence of catalytic subunits prevents the translocation of the regulatory subunit to the nuclei. These results emphasize the existence of a control mechanism linking the expression and sorting of CK2 catalytic and regulatory subunits.3. Selective depletion of the regulatory () subunit of protein kinase CK2 by an specific antisense oligonucleotide causes partial inhibition of neurite extension.     

Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2/ antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2.We had also observed that rat liver membrane glycoproteins obtained through chromatography on wheat-germ lectin-Sepharose contain CK2 activity which copurifies with grp94/endoplasmin. We have now confirmed that this activity was due to the presence of protein kinase CK2 as evidenced by immunodetection with antibodies against CK2/. The fractions enriched in grp94/endoplasmin and CK2 also contained another 55-kDa polypeptide (p55) phosphorylated by CK2 which has been identified as calreticulin by N-terminal sequencing. Calreticulin and grp94/endoplasmin could be partially resolved from CK2 by chromatography on heparin-agarose and almost completely on ConA-Sepharose. However, phosphorylation of immunoprecipitated grp94/endoplasmin was enhanced by its preincubation with purified CK2 prior to immunoprecipitation, what confirms the easy reassociation between these proteins.The association of protein kinase CK2 with eIF-2 and with grp94/endoplasmin may serve to locate the enzyme in the cellular machinery involved in protein synthesis and folding, and reinforces the possible involvement of CK2 in these processes.     

Generation of mutants of CK2α which are dependent on the β-subunit for catalytic activity

To shed light on the structural features underlying high constitutive activity of protein kinase CK2 a number of mutants of the human CK2 subunit altered in the interactions between the N-terminal segment and the activation loop have been generated and shown to be defective in catalytic activity. In particular the truncated mutant 2-12 displays under standard conditions an almost complete loss of catalytic activity accounted for by a dramatic rise in its Km for ATP (from 10 to 206 M) and a reduced Kcat. Such a drop in efficiency is paralleled by conformational disorganization, as judged from Superdex 75 gel filtration profile. Both catalytic properties and gel filtration behaviour similar to those of wild type CK2 were restored upon association with the regulatory -subunit, suggesting that constitutive activity is conferred to CK2 and to CK2 holoenzyme through different molecular mechanisms. In the holoenzyme an assumable release of tension at the backbone of Ala-193 (as seems to be indicated by a comparison of the crystal structures of maize CK2 alone vs. a CK2– peptide complex) may result in the ability of the activation loop to adopt its proper conformation independently of interactions with the N-terminal segment.     

α-Globin gene deletion causes α-thalassemia syndromes in two German families

Summary Restriction endonuclease mapping of chromosomal DNA has been used to determine whether the -globin gene deletion or non-deletion form of -thalassemia is the underlying molecular defect in individuals of two unrelated German families with -thalassemia syndromes. The obtained DNA pattern in all cases indicated loss of -globin genes resulting in-/,--/, and--/- genotypes in thalassemia-2, -thalassemia-1, and Hb H individuals respectively. The chromosomes showing loss of one -globin gene in -thalassemia-2 and Hb H disease were characterized by the so-called rightward deletion form exhibiting loss of a 3.7 kb DNA fragment in the -gene cluster.     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Modulation of cytokine responses of murine CD8+ αβ intestinal intraepithelial lymphocytes by IL-4 and IL-12

The immune responses of the intestine mucosa feature the noninflammatory type, such as IgA production and oral tolerance. Th2 type cytokines have been implicated in the induction of these noninflammatory responses. In the present study, cytokine responses of CD8+ and CD4+ TCR+ intestinal intraepithelial lymphocyte ( iIEL) subsets to TCR stimulation under the influence of IL-12, IL-4, or CD28 costimulation were examined. IL-12 enhanced production of IL-10 and IFN- by the CD8+ iIEL significantly but only marginally affected the CD8+ subset, whereas IL-4 induced IL-4, IL-5, and IL-10 production and augmented TGF- production by both subsets. CD28 costimulation induced production of Th2 cytokines by CD4+ iIEL in the absence of exogenous IL-4. Unlike lymph node CD4+ cells, the CD28 costimulation-induced Th2 differentiation of CD4+ iIEL was not inhibited by IFN-. These results demonstrate active cytokine production by CD4+, CD8+, as well as CD8+ iIEL. The Th2-skewed cytokine profile of CD8+ iIEL and the IFN--resistance of Th2 differentiation of the CD4+ iIEL suggest that both iIEL subsets contribute to the induction of noninflammatory mucosal immune responses.