1 Food Science and Human Nutrition Department, Center for Nutritional Sciences, and 2 Department of Medicine, College of Medicine, University of Florida, Gainesville 32611; and 3 Gainesville Veterans Affairs Medical Center, Gainesville, Florida 32608
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ABSTRACT |
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Cysteine-rich intestinal protein (CRIP),
which contains a double zinc finger motif, is a member of the Group 2 LIM protein family. Our results showed that the developmental
regulation of CRIP in neonates was not influenced by conventional vs.
specific pathogen-free housing conditions. Thymic and splenic CRIP
expression was not developmentally regulated. A line of transgenic (Tg)
mice that overexpress the rat CRIP gene was created. When challenged with lipopolysaccharide, the Tg mice lost more weight, exhibited increased mortality, experienced greater diarrhea incidence, and had
less serum interferon- (IFN-
) and more interleukin (IL)-6 and
IL-10. Similarly, splenocytes from the Tg mice produced less IFN-
and IL-2 and more IL-10 and IL-6 upon mitogen stimulation. Delayed-type
hypersensitivity response was less in the Tg mice. Influenza virus
infection produced greater weight loss in the Tg mice, which also
showed delayed viral clearance. The observed responses to
overexpression of the CRIP gene are consistent with a role for this LIM
protein in a cellular pathway that produces an imbalance in cytokine
pattern favoring Th2 cytokines.
interferon-; interleukin-1; interleukin-6; endotoxin; zinc
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INTRODUCTION |
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CYSTEINE-RICH INTESTINAL PROTEIN (CRIP) is a zinc-binding protein that was initially identified as a developmentally regulated intestinal gene and has subsequently been identified in several other tissues and cells (3, 15, 16). The zinc-binding property of CRIP has been attributed to the LIM domain, which consists of a specific two zinc finger motif that has a highly conserved Cys2HisCys plus Cys4 sequence (9, 13). The LIM protein family is large. CRIP is the most elementary member of the Group 2 LIM protein family, because it contains only one LIM domain. This group within the LIM protein family does not contain a homeodomain as found in other LIM proteins. In general, it is proposed that the LIM protein family plays a regulatory role that influences differentiation and growth of eukaryotic cells. However, the exact function of any of the Group 2 LIM proteins is not established. LIM proteins may increase in relative abundance with increasing genomic complexity of the organism. For example, in Saccharomyces cerevisiae and Caenorhabditis elegans, the LIM domain genes represent 2 and 6% of all zinc finger domain genes in the respective genomes (9). This increasing abundance points to key regulatory functions for all LIM proteins.
CRIP expression is upregulated in the intestine of young rats just before weaning (3, 24). We proposed that CRIP expression in neonates is induced by glucocorticoid hormones (24). Indeed, the CRIP promoter has consensus sequences for glucocorticoid response elements and imparts glucocorticoid responsiveness in reporter constructs, and glucocorticoid hormone will induce precocious CRIP synthesis in newborn rats (23, 24). Consequently, glucocorticoid hormones most likely contribute in part to the postnatal increase in expression.
Our experiments have demonstrated that CRIP is expressed in immune cells and tissues, specifically in peritoneal macrophage and peripheral blood mononuclear cells, at levels similar to the high levels of expression in the small intestine of weanling and adult rats (15, 24). Moderate expression has been observed in the lung, spleen, heart, and thymus, with very low expression in liver, brain, and kidney (12, 15). The tissue specificity of CRIP expression plus its upregulation during early postnatal development suggest that this LIM protein may have a function in cells involved in host defense.
To further study the function of CRIP, we developed a line of transgenic mice that overexpress the rat CRIP (rCRIP) gene in a tissue-specific fashion (12). Because CRIP is highly expressed in immune tissues, we challenged these mice with both influenza virus (intracellular pathogen) and lipopolysaccharide (LPS; extracellular pathogen) to evaluate the effect of CRIP overexpression on two different types of host immunity. In this study, we report that mice that produce more CRIP are more sensitive to an endotoxic challenge and influenza virus and exhibit an altered cytokine production pattern. Both findings are consistent with a regulatory function for CRIP, particularly in immune cells.
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MATERIALS AND METHODS |
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Animals and experimental design. A 15-kb rat genomic clone was isolated and subjected to multiple restriction enzyme digests. Subsequently, a 5-kb fragment containing the rCRIP gene was ligated into sites of a pGEM cloning vector to transform competent cells. Appropriate colonies were grown and, after rounds of nested deletions and sequencing, PCR primers were designed and used to amplify an ~5-kb fragment. This PCR product contained the rCRIP gene, including 2,644 bp of the 5' flanking regulatory region and 1.8 kb of the structural portion of the rCRIP gene including all five exons, and was used to produce the transgene construct as described in detail previously (12). Because the rat and mouse CRIP cDNAs, and thus presumably the genes, are highly homologous, a 91-bp of pGEM vector sequence was included in the construct for the purpose of transgene identification in transgenic (Tg) mice. The responsiveness of the rCRIP promoter in a reporter construct in IEC-6 transfected cells has been previously described (23).
Homozygous Tg mice were developed in the B6SJL hybrid strain from the founder line exhibiting the greatest CRIP expression (12). After the mating of F1 animals, which carried the transgene (both female and male), the expected ratio of offspring (1:2:1; noncarriers-heterozygous-homozygous) was obtained. Determination of transgene copy number by the slot blot technique was described previously (12). Heterozygous mice had ~5 copies of the transgene, whereas the homozygous animals had ~8 copies. Homozygous littermates were then bred to maintain the transgenic line. Some of the mice used in these studies, both Tg and wild-type (NTg), were born in a specific pathogen-free (SPF) mouse facility. They were allowed free access to autoclaved, deionized water and an irradiated commercial diet (Teklad Mouse Breeder Sterilizable Diet 7904; Harlan, Madison, WI). Tg and NTg mice were also bred and maintained in conventional housing conditions and were provided tap water and a commercial rodent diet (Teklad 8604). The mice were age-matched (4-12 wk of age, except the neonatal study) and, where possible, were sex-matched for experiments. There were no significant differences in body weights or organ weights between the Tg and NTg mice from either environment (data not shown). In some experiments, Tg and NTg mice from the conventional environment were injected with LPS [Eschericia coli Serotype 0127:B8; Sigma Chemical (St. Louis, MO); 20 mg/kg body wt ip] or saline at various times before being killed. In separate experiments, mice were infected intranasally with virus (Influenza A/Port Chalmers/1/73[H3N2]) (39). Delayed-type hypersensitivity (DTH) reactions of mice were evaluated in another series of experiments by application of 2,4-dinitro-1-fluorobenzene (DNFB, 50 µl; 0.5% in 4:1 acetone-olive oil) to the shaved abdomen on two successive days (25). Four days after the second application, the mice were rechallenged with a 0.2% DNFB application (20 µl to the left pinna and 20 µl of acetone-olive oil vehicle to the right). Twenty-four hours later, swelling of pinnae was measured with a micrometer. The DTH response was expressed as the percent change in pinna thickness after DNFB challenge. The mice were anesthetized with methoxyflurane, halothane, or pentobarbital in various experiments. All animal procedures were approved by the University of Florida Institutional Animal Care and Use Committee.Tissue processing. Zinc concentrations of serum derived from blood obtained by cardiac puncture were measured by atomic absorption (15). The proximal duodenum of the small intestine was excised and flushed with ice-cold 0.9% saline. Mucosal cells were removed by scraping and homogenized with a Potter-Elvehjem homogenizer in 4 volumes of 20 mM HEPES, pH 7.4, 1 mM EDTA, and 300 mM mannitol containing 5% protease inhibitor cocktail (Sigma) added just before use. A 40,000-g cytosol fraction from these homogenates and those from spleen and thymus were similarly prepared, as described previously (16, 24). Samples of small intestine, spleen, and thymus were also immediately homogenized in TriPure Isolation Reagent (Roche Molecular Biochemicals, Indianapolis, IN) with a polytron (Brinkman, Westbury, NY) to isolate total RNA. Protein concentration of the cytosol was determined colorimetrically (27).
Lung tissue was collected from the influenza-infected mice and treated as described below. Phytohemagglutinin (PHA)-stimulated cytokine production used splenocytes isolated by passage of minced spleen fragments through nylon mesh. Cells were washed with RPMI (Mediatech, Cellgro, Herndon, VA)-complete medium (RPMI-1640 supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 25 mM HEPES buffer, and 50 µM 2-mercaptoethanol). After the cells had been washed for a total of three washes in RPMI-complete, they were suspended in RPMI-complete with 10% heat-inactivated fetal calf serum at 2 × 106 cells/ml. An aliquot of cells was incubated with 10 µg/ml of PHA in 48-well cell culture plates for 24 h at 37°C, 95% humidity, and 5% CO2. The supernatants were removed and frozen atQuantitative PCR and Western analysis. cDNA was synthesized from 1-3 µg of total RNA using reverse transcriptase (RT) and amplified with DNA polymerase in a 25-µl reaction mixture. Measurement of rat or mouse (r/m)CRIP mRNA levels by quantitiative PCR (Q-PCR) used primers (forward) 5'-GCGACAAGGAGGTATTTCA-3' and (reverse) 5'-TTCCACATTTCTCGCACTTC-3', and a fluorescence resonance energy transfer probe 5'-6FAM-TGACGTCACTAGGCAAGGACTGGCAT-TAMRA, selected with Primer Express software (Applied Biosystems, Foster City, CA). These were based on sequence information for rCRIP (12). The primer and probe set for 18S ribosomal RNA used for normalization were from Applied Biosystems. Relative quantitation was calculated from a standard curve generated by 1:10 dilutions of total RNA to produce a 4- to 5-log range as described recently in detail (28). Amplification of PCR products was measured fluorimetrically (ABI model 5700 Sequence Detection System). Routinely, RT reactions were for 30 min at 48°C, and PCR was for 40 cycles.
Proteins in tissue cytosol preparation were separated by 15% Tris-Tricine SDS-PAGE (16). After transfer to Immobilon-P membrane (Millipore, Bedford, MA), immunodetection was with ammonium sulfate precipitate and protein A-purified rabbit IgG prepared against a synthetic peptide (CGFGRGGAESHTFK) representing amino acids 65-77 of the COOH terminus of r/mCRIP with a cysteine added to facilitate conjugation to keyhole limpet hemocyanin. These methods have been described previously (14, 20). A secondary antibody-alkaline phosphatase conjugate and an extracellular fluid reagent (Amersham Pharmacia Biotech, Piscataway, NJ) were used for fluorescence imaging (Molecular Dynamic Storm 840 Imager). Recombinant human CRIP, prepared with an expression system described previously (20), was used as the standard.Lung viral titers/cytokine assays.
Viral titers were determined by inoculation of ground lung samples into
Madin Darby canine kidney cells, as described previously (39). Splenocyte IFN-, IL-6, IL-10, and tumor necrosis
factor-
(TNF-
) after PHA challenge, as well as serum IFN-
,
IL-6, IL-10, and TNF-
after LPS challenge, were measured using
commercial ELISA kits (Cytimmune, College Park, MD). IL-2 production
was based on [3H]thymidine incorporation by murine HT-2
lymphocytes measured using a bioassay, as described previously
(22).
Statistical analysis. Tests for significance were performed using the Student's t-test, the Mann-Whitney test, or 2 × 2 factorial ANOVA with GraphPad Software (San Diego, CA) or the SAS System (SAS Institute, Cary, NC). When necessary, data were subjected to log10 transformation to lessen variance heterogeneity.
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RESULTS |
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Response to the environment.
Relative quantitative analysis of CRIP mRNA quantities (abundance) by
Q-PCR shows the wide range of CRIP expression that occurs in neonates
from birth to 41 days of age (Fig. 1). Of
note is that SPF-maintained NTg mice exhibited essentially the same
CRIP upregulation, starting at ~15 days of age, as did conventionally maintained mice. Also of considerable interest is that CRIP mRNA levels
in thymus and spleen are negligible during these early weeks of life
(Fig. 1).
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Response to LPS challenge.
Because CRIP expression is highly expressed in immune cells, which
respond to microbial factors, we subjected both the NTg and Tg mice to
LPS as a model extracellular pathogen challenge. The induction of CRIP
expression by LPS is shown in Fig. 2.
CRIP mRNA was measured by real-time PCR, and data are presented as relative quantities (Fig. 2A). LPS produced increases in
CRIP mRNA ranging from 1.4 to 2.2 in intestine, spleen, and thymus. These were statistically significant (P 0.02) for each
comparison pair (±LPS). Expression was markedly greater in the Tg
mice. Similarly, Western analysis showed that LPS induced changes in
CRIP protein that were present in cytosol extracts of these tissues.
However, the sensitivity was such that only the data from the Tg mice
were analyzed statistically (Fig. 2B). As shown, LPS
produced a significant increase for each comparison pair (P
0.05).
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Response to influenza virus.
Infection with influenza virus served as a model to test the effect of
CRIP overexpression on the response to an intracellular pathogen. Serum
zinc concentrations at day 2 were comparable in the two
genotypes, but there was a significant decline in the Tg mice by
day 8 (Fig. 5A).
The ability to clear influenza virus from the lung was significantly
compromised (P < 0.05) in the Tg genotype (Fig.
5B). By day 8, all of the NTg mice, but only one
of the Tg mice, had cleared the virus from the lungs (P < 0.05). All mice began to lose weight during the first 24 h
after infection (Fig. 5C). After day 3, the NTg
mice started to regain weight. The Tg mice continued to lose weight
through day 4 and had significantly (P < 0.05) lower body weight throughout the 8-day observation period. The
reduced response to viral challenge in the Tg mice points to an altered
host defense due to CRIP overexpression.
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DISCUSSION |
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The normal physiological role of CRIP is not known. However, the high level of expression in immune cells compared with other cells is evidence that this protein may have a role in host defense (14, 15, 20). In support of this notion, high levels of CRIP expression in peritoneal macrophages, thymus, and spleen are increased upon induction by LPS (14, 15). Intestinal CRIP abundance varies among intestinal cell types, being highest in Paneth cells, followed by eosinophils within the lamina propria (14). Both cell types are well defined immune cells. CRIP is also localized, albeit to a lesser extent, in intestinal epithelial cells (14). Recently, the gut epithelium has been shown to produce inflammatory cytokines in response to pathogenic bacteria (33). Consequently, enterocytes must also be considered to be immune cells, perhaps by aiding in tolerance of the gut to pathogens. To further test the possibility of an immune function for CRIP, in these studies we examined the result of CRIP overexpression to models of extracellular (LPS) and intracellular (viral infection) pathogen exposure.
The intestine provides an important part of the mucosal immune system (6, 8, 18); consequently, factors regulating this system should respond to conditions within the intestinal lumen. This time period corresponds to the point at which the gut is adapting to both colonization with enteric microflora and marked changes in diet (17, 18). As shown in Fig. 1, the developmental increase in CRIP expression is not a response to environmental factors, because CRIP concentrations increase to the same extent in neonates maintained in SPF condition as in those raised in a conventional environment. This suggests that glucocorticoid hormone, which is able to produce a precocious increase in CRIP expression in neonatal rat pups (24, 32), is the primary stimulus for the developmental increase in CRIP in rodent intestine. An immunological response to enteric pathogens may be a factor in CRIP expression, but that possibility was not tested in these experiments.
Our Tg mice maintained in an adult breeding colony have not shown any abnormality in reproduction, growth, or gross intestinal morphology; however, as shown in this report, these mice are more sensitive to LPS and influenza virus. This suggests to us that the function of CRIP is as a modifier of a response rather than as a direct initiator of a response, which, if overexpressed, would result in obvious morbidity or morphology.
LPS is known to cause a complex reaction of the immune system
(36) and initially has strong stimulatory effects on
macrophages and other immune cells, including release of cytokines,
such as IL-1, IL-6, and TNF-. The time period chosen to examine
these cytokines was one when some, e.g., IL-2, exhibit maximal levels after stimulation. CRIP overexpression appears to alter cytokine patterns in LPS-stimulated mice with a shift to an increase in IL-6 and
IL-10 and a decrease in IFN-
and IL-2. Production of TNF-
, a
macrophage-derived cytokine, is not changed with CRIP overexpression
within the time period examined, suggesting that differences in
production of these cytokines is somewhat specific and that CRIP
overexpression affects lymphocyte function more than monocyte function.
Because IL-10 suppresses lymphocyte and macrophage function, the marked
(threefold) increase in its production may explain, in part, the
increased sensitivity to LPS that the Tg mice display.
Evaluation of splenocyte proliferation and cytokine production of
mitogen-challenged splenocytes provided additional support for the
hypothesis that CRIP overexpression influences cytokine balance. This
hypothesis is further supported by the decreased ability of the Tg mice
to clear virus. As with aging mice, there appears to be a decrease in
IFN- and IL-2 and an increase in IL-6 and IL-10 in response to this
immune challenge in Tg mice (39). The decrease in
Th1-associated cytokines, including IFN-
and IL-2, in aged mice is
related to a decreased ability to clear influenza virus from the lung
(39). Additionally, Th2 cytokines such as IL-6 and IL-10
may cause delayed virus clearance in influenza-infected mice
(29).
To balance the immune response, there is cross-regulation between the
cytokines that affect immune cells, such as Th1/Th2 cells (10,
30). For example, IL-4 drives Th2 T cell differentiation, and
IFN- and IL-12 drive Th1 T cell differentiation. In addition, IL-4
and IFN-
exert antagonistic effects on Th1 and Th2 processes, respectively (35). Thus, from the data collected in this
group of experiments, two questions arise: 1) is CRIP
overexpression affecting macrophage or T-helper cell function, i.e.,
upregulating Th2 cell functions and downregulating Th1 cell functions;
or 2) is CRIP overexpression affecting the differentiation
pathways of these two cell types? CRIP expression may also be related
to a combination of these two events. Using the data presented here, we
have developed a working model, briefly described earlier
(11), based on CRIP-related cytokine dysregulation that
will be tested in future experiments. There is precedence in the
literature for cytokine expression regulation by a zinc finger protein.
Specifically, TNF-
production is regulated by tristetraprolin, a
CCCH zinc finger protein (7), through a feedback
inhibitory mechanism influencing TNF-
mRNA.
Our model of CRIP function currently focuses on the decreased levels of
IFN- and IL-2 secreted by CRIP-Tg mice described in this report.
This emphasis is supported by other evidence, including: 1)
presence of consensus response elements in the CRIP promoter
(23) necessary for IFN-
-induced gene expression;
2) protein-protein interaction observed in vitro (J. Nicewonger and R. Cousins, unpublished data) between CRIP and the
IFN-
chaperone calreticulin (21); and 3)
zinc inducibility of calreticulin through metal response elements in
the calreticulin promoter (34). This model will be applied
to test the response of CRIP function in other situations, e.g., zinc
deficiency. Th2 cytokine production is decreased in zinc-deficient mice
(37). The same response has been shown in zinc-deficient
humans (2). Because CRIP is a zinc finger protein that may
be influenced by cellular zinc pools, a link between zinc-related
immune dysfunction and the proteins CRIP and calreticulin will be a
focus of our further research.
Like other LIM proteins, CRIP may be involved in control of cellular determination and differentiation or the regulation of specific processes. The LIM protein MLP is involved in muscle cell differentiation (1), and LMO1 (previously Rbtn1 or Ttg-1) is a transcription factor involved in T cell proliferation (5). Similarly, the protein LMO2 (previously Rbtn2 or Ttg-2) was found to be part of a DNA-binding complex important for mouse hematopoietic development (31, 40, 41). The function of CRIP may involve protein-protein interactions that include interaction with cytoplasmic factors and/or nuclear factors such as the nuclear LIM interactor protein (19) or the Clim/Ldb/Nli coregulator family (38).
In summary, we have shown in these experiments that CRIP overproduction
decreases IFN- and IL-2 levels and increases IL-6 and IL-10 levels.
The imbalance favoring Th2 cytokines suggests that CRIP regulates
expression/secretion of these cytokines from cells where this zinc
finger protein is very highly expressed. Mice producing larger than
normal amounts of CRIP are, therefore, more susceptible to toxins (as
shown here with LPS) produced by pathogens and to viral infection. The
response of CRIP expression to LPS and viral challenges further
strengthens a role for this LIM protein as an adapter molecule for
modifying the action of factors associated with cellular mechanisms
leading to dysregulation of cytokines required for cellular host defense.
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ACKNOWLEDGEMENTS |
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We express our appreciation to Warren R. Clark, Robert J. Cottey, Leah M. Coy, and Kelli A. Herrlinger-Garcia for help with some analyses.
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FOOTNOTES |
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This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-31127 (R. J. Cousins), Boston Family Endowment Funds (R. J. Cousins), and Department of Veterans Affairs (B. S. Bender).
Current addresses: L. Lanningham-Foster, Department of Pathology, Wake Forest University School of Medicine, Winston-Salem NC 27157; B. A. Davis, Department of Nutrition Research, Wyeth Nutritionals International, Collegeville, PA 19426.
Address for reprint requests and other correspondence: R. J. Cousins, Food Science and Human Nutrition Dept., Univ. of Florida, 201 FSHN, Gainesville FL 32611-0370 (E-mail: RJCousins{at}mail.ifas.ufl.edu or cousins{at}ufl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 11, 2002;10.1152/ajpendo.00508.2001
Received 12 November 2001; accepted in final form 7 February 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arber, S,
Halder G,
and
Caroni P.
Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation.
Cell
79:
221-231,
1994[ISI][Medline].
2.
Beck, FW,
Prasad AS,
Kaplan J,
Fitzgerald JT,
and
Brewer GJ.
Changes in cytokine production and T cell subpopulations in experimentally induced zinc-deficient humans.
Am J Physiol Endocrinol Metab
272:
E1002-E1007,
1997
3.
Birkenmeier, EH,
and
Gordon JI.
Developmental regulation of a gene that encodes a cysteine-rich intestinal protein and maps near the murine immunoglobulin heavy chain locus.
Proc Natl Acad Sci USA
48:
2516-2520,
1986.
4.
Blanchard, RK,
and
Cousins RJ.
Differential display of intestinal mRNAs regulated by dietary zinc.
Proc Natl Acad Sci USA
93:
6863-6868,
1996
5.
Boehm, T,
Foroni L,
Kaneko Y,
Perutz MF,
and
Rabbitts TH.
The rhombotin family of cysteine-rich LIM-domain oncogenes: distinct members are involved in T-cell translocations to human chromosomes 11p15 and 11p13.
Proc Natl Acad Sci USA
88:
4367-4371,
1991[Abstract].
6.
Brandtzaeg, P.
Development and basic mechanisms of human gut immunity.
Nutr Rev
56:
S5-S18,
1998[ISI][Medline].
7.
Carballo, E,
Lai WS,
and
Blackshear PJ.
Feedback inhibition of macrophage tumor necrosis factor- production by tristetraprolin.
Science
281:
1001-1005,
1998
8.
Castro, GA,
and
Arntzen CJ.
Immunophysiology of the gut: a research frontier for integrative studies of the common mucosal immune system.
Am J Physiol Gastrointest Liver Physiol
265:
G599-G610,
1993
9.
Clarke, ND,
and
Berg JM.
Zinc fingers in Caenorhabditis elegans: finding families and probing pathways.
Science
282:
2018-2022,
1998
10.
Constant, SL,
and
Bottomly K.
Induction of TH1 and TH2 CD4+ T cell responses: the alternative approaches.
Ann Rev Immunol
15:
297-322,
1997[ISI][Medline].
11.
Cousins, RJ,
and
Lanningham-Foster L.
Regulation of cysteine-rich intestinal protein, a zinc finger protein, by mediators of the immune response.
J Infect Dis
182:
S81-S84,
2000[ISI][Medline].
12.
Davis, BA,
Blanchard RK,
Lanningham-Foster L,
and
Cousins RJ.
Structural characterization of the rat cysteine-rich intestinal protein gene and overexpression of this LIM-only protein in transgenic mice.
DNA Cell Biol
17:
1057-1064,
1998[ISI][Medline].
13.
Dawid, IB,
Breen JJ,
and
Toyama R.
LIM domains: multiple roles as adapters and functional modifiers in protein interactions.
Trends Genet
14:
156-162,
1998[ISI][Medline].
14.
Fernandes, PR,
Samuelson DA,
Clark WR,
and
Cousins RJ.
Immunohistochemical localization of cysteine-rich intestinal protein in rat small intestine.
Am J Physiol Gastrointest Liver Physiol
272:
G751-G759,
1997
15.
Hallquist, NA,
Khoo C,
and
Cousins RJ.
Lipopolysaccharide regulates cysteine-rich intestinal protein, a zinc finger protein, in immune cells and plasma.
J Leukocyte Biol
59:
172-177,
1996[Abstract].
16.
Hempe, JM,
and
Cousins RJ.
Cysteine-rich intestinal protein binds zinc during transmucosal zinc transport.
Proc Natl Acad Sci USA
88:
9671-9674,
1991[Abstract].
17.
Hooper, LV,
Wong MH,
Thelin A,
Hansson L,
Falk PG,
and
Gordon JI.
Molecular analysis of commensal host-microbial relationships in the intestine.
Science
291:
881-884,
2001
18.
Insoft, RM,
Sanderson IR,
and
Walker WA.
Development of immune function in the intestine and its role in neonatal disease.
Pediatr Clin North Am
43:
551-571,
1996[ISI][Medline].
19.
Jurata, LW,
Kenny DA,
and
Gill GN.
Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.
Proc Natl Acad Sci USA
93:
11693-11698,
1996
20.
Khoo, C,
Blanchard RK,
Sullivan VK,
and
Cousins RJ.
Human cysteine-rich intestinal protein: cDNA cloning and expression of recombinant protein and identification in human peripheral blood mononuclear cells.
Protein Expr Purif
9:
379-387,
1997[ISI][Medline].
21.
Kosuge, T,
and
Toyoshima S.
Increased degradation of newly synthesized interferon-gamma (INF-gamma) in anti CD3-stimulated lymphocytes treated with glycoprotein processing inhibitors.
Biol Pharm Bull
23:
545-548,
2000[ISI][Medline].
22.
Langkamp-Henken, B,
Johnson LR,
Viar MJ,
Geller AM,
and
Kotb M.
Differential effect on polyamine metabolism in mitogen- and superantigen-activated human T-cells.
Biochim Biophys Acta
1425:
337-347,
1998[ISI][Medline].
23.
Levenson, CW,
Shay NF,
and
Cousins RJ.
Cloning and initial characterization of the promoter region of the rat cysteine-rich intestinal protein gene.
Biochem J
303:
731-736,
1994[ISI][Medline].
24.
Levenson, CW,
Shay NF,
Lee-Ambrose LM,
and
Cousins RJ.
Regulation of cysteine-rich intestinal protein by dexamethasone in the neonatal rat.
Proc Natl Acad Sci USA
90:
712-715,
1993[Abstract].
25.
Lewis, B,
and
Langkamp-Henken B.
Arginine enhances in vivo immune responses in young, adult and aged mice.
J Nutr
130:
1827-1830,
2000
26.
Lowell, CA,
and
Berton G.
Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr.
Proc Natl Acad Sci USA
95:
7580-7584,
1998
27.
Markwell, MA,
Haas SA,
Bieber LL,
and
Tolbert NE.
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.
Anal Biochem
87:
206-210,
1978[ISI][Medline].
28.
Moore, JB,
Blanchard RK,
McCormack WT,
and
Cousins RJ.
cDNA array analysis identifies thymic LCK as upregulated in moderate murine zinc deficiency before T-lymphocyte population changes.
J Nutr
131:
3189-3196,
2001
29.
Moran, TM,
Isobe H,
Fernandez-Sesma A,
and
Schulman JL.
Interleukin-4 causes delayed virus clearance in influenza virus-infected mice.
J Virol
70:
5230-5235,
1996[Abstract].
30.
Muraille, E,
and
Leo O.
Revisiting the Th1/Th2 paradigm.
Scand J Immunol
47:
1-9,
1998[ISI][Medline].
31.
Neale, GA,
Rehg JE,
and
Goorha RM.
Ectopic expression of rhombotin-2 causes selective expansion of CD4-CD8 lymphocytes in the thymus and T-cell tumors in transgenic mice.
Blood
86:
3060-3071,
1995
32.
Needleman, DS,
Leeper LL,
Nanthakumar NN,
and
Henning SJ.
Hormonal regulation of the mRNA for cysteine-rich intestinal protein in rat jejunum during maturation.
J Pediatr Gastroenterol Nutr
16:
15-22,
1993[ISI][Medline].
33.
Neish, AS,
Gewirtz AT,
Zeng H,
Young AN,
Hobert ME,
Karmali V,
Rao AS,
and
Madara JL.
Prokaryotic regulation of epithelial responses by inhibition of IB-
ubiquitination.
Science
289:
1560-1563,
2000
34.
Nguyen, TQ,
Capra JD,
and
Sontheimer RD.
Calreticulin is transcriptionally upregulated by heat shock, calcium and heavy metals.
Mol Immunol
33:
379-386,
1996[ISI][Medline].
35.
Paludan, SR.
Interleukin-4 and interferon-: the quintessence of a mutual antagonistic relationship.
Scand J Immunol
48:
459-468,
1998[ISI][Medline].
36.
Rietschel, ET,
and
Brade H.
Bacterial endotoxins.
Sci Am
267:
54-61,
1992[ISI][Medline].
37.
Shi, HN,
Scott ME,
Stevenson MM,
and
Koski KG.
Energy restriction and zinc deficiency impair the functions of murine T cells and antigen presenting cells during gastrointestinal nematode infection.
J Nutr
128:
20-27,
1998
38.
Sugihara, TM,
Bach I,
Kioussi C,
Rosenfeld MG,
and
Andersen B.
Mouse deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4 & CLIM coregulators.
Proc Natl Acad Sci USA
95:
15418-15423,
1998
39.
Taylor, SF,
Cottey RJ,
Zander DS,
and
Bender BS.
Influenza infection of beta 2-microglobulin-deficient (beta 2m/
) mice reveals a loss of CD4+ T cell functions with aging.
J Immunol
159:
3453-3459,
1997[Abstract].
40.
Wadman, IA,
Osada H,
Grütz GG,
Agulnick AD,
Westphal H,
Forster A,
and
Rabbitts TH.
The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins.
EMBO J
16:
3145-3157,
1997
41.
Yamada, Y,
Warren AJ,
Dobson C,
Forster A,
Pannell R,
and
Rabbitts TH.
The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis.
Proc Natl Acad Sci USA
95:
3890-3895,
1998