Overexpression of CRIP in transgenic mice alters cytokine patterns and the immune response

Lorraine Lanningham-Foster1, Calvert L. Green1, Bobbi Langkamp-Henken1, Barbara A. Davis1, Khanh T. Nguyen1, Bradley S. Bender2,3, and Robert J. Cousins1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma (IFN-gamma ) and more interleukin (IL)-6 and IL-10. Similarly, splenocytes from the Tg mice produced less IFN-gamma 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-gamma ; interleukin-1; interleukin-6; endotoxin; zinc


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -80°C until the cytokines were measured, as we will describe.

Quantitative 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-gamma , IL-6, IL-10, and tumor necrosis factor-alpha (TNF-alpha ) after PHA challenge, as well as serum IFN-gamma , IL-6, IL-10, and TNF-alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Cysteine-rich intestinal protein (CRIP) mRNA in intestine of nontransgenic neonatal mice raised in specific pathogen-free (SPF) or conventional (CONV) environments. CRIP mRNA quantities were measured by quantitative PCR (Q-PCR). Mice were weaned at 21 days of age. Values are from intestines pooled from 3 mice (males and females) at each time point. Levels of thymic and splenic CRIP mRNA from mice at 19 and 30 days of age are shown for comparison.

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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Fig. 2.   Expression of CRIP in intestine, spleen, and thymus of CRIP transgenic (Tg) and nontransgenic (NTg) mice after lipopolysaccharide (LPS). Mice were treated with LPS or saline and killed 24 h later. Tissues were processed for analysis of CRIP mRNA quantities by Q-PCR and cytosolic CRIP protein abundance by Western analysis. Values are means ± SE (n = 3-5) from individual tissues. For each comparison pair (±LPS), the values for the LPS-treated mice were significantly greater (CRIP mRNA, P<= 0.02; CRIP protein, P <=  0.05). A: CRIP mRNA normalized to 18S ribosomal RNA; B: CRIP in cytosol from Tg mice. Recombinant CRIP was used as the standard.

Reduced serum zinc concentrations are a well recognized consequence of acute endotoxemia and demonstrate here the comparable systemic response of NTg and Tg mice to LPS (Fig. 3A). Within 48 h after LPS challenge, over 50% of Tg mice died, in contrast to only 10% of the NTg mice (Fig. 3B). During the course of the challenge, all Tg mice developed diarrhea (Fig. 3C). These mice were lethargic and did not groom themselves, both features of endotoxic shock (26). In comparison, morbidity was less in the NTg mice. Tg mice also lost significantly more weight than NTg mice by day 2 after LPS (Fig. 3D). After this point, NTg mice began to recover from the LPS, as observed by increased weight. At the end of the 8-day comparison period, the Tg mice still had a lower body weight. Saline-treated NTg and Tg mice did not lose weight, and there was no mortality (Fig. 3, B and D). Because intestinal CRIP expression was greater in Tg mice than in NTg mice, the higher CRIP level is correlated to the higher incidence of LPS-induced diarrhea in the Tg mice after LPS challenge.


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Fig. 3.   Measurable responses of CRIP Tg and NTg mice to LPS. Male mice were treated with LPS or saline. Some mice were killed at 24 h for serum zinc measurements. The remainder were monitored for 8 days after the injection. A: serum zinc concentration. Values shown are means ± SE; n = 3-5. Differing numbers of asterisks indicate that means are significantly different (P < 0.001). B: percent survival (n = 7-8). C: percent diarrhea incidence (n = 7-8). D: change from initial body weight. Values shown are means ± SE; n = 7-8. Saline-treated NTg and Tg weights are not significantly different. Asterisks indicate that means from LPS-treated Tg mice are significantly different from the corresponding mean for LPS-treated NTg mice (P < 0.01).

The increased sensitivity to LPS observed in the Tg mice suggests that the response could be the result of cytokine dysregulation. To investigate this possibility, serum cytokines were measured after LPS challenge (Table 1). Serum IFN-gamma concentrations in Tg mice were about one-third of those found in the NTg group. Serum TNF-alpha was also depressed in the two genotypes, but not significantly. Serum IL-10 was approximately threefold higher in Tg mice, and serum IL-6 was twofold higher in these mice. These results are consistent with an increase in cytokine dysregulation in the CRIP-overexpressing mice after a stimulus of an extracellular pathogen. To further investigate the possibility of cytokine dysregulation in CRIP overexpression, splenocytes obtained from both genotypes were stimulated in culture with the mitogen PHA (Table 1). There was significantly less IFN-gamma and IL-2 production by the cultured Tg splenocytes, with correspondingly greater IL-6 and IL-10 production. There was no difference in TNF-alpha production between splenocytes from the two genotypes. Splenocytes from Tg mice also exhibited significantly (P < 0.008) decreased proliferation (24% of the NTg cells) using the [3H]thymidine incorporation assay (data not shown).

                              
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Table 1.   Serum cytokines after LPS challenge and PHA-stimulated cytokine release by splenocytes from nontransgenic and CRIP transgenic mice

The observed increases in Th2 cytokines IL-6 and IL-10 observed in the CRIP Tg mice suggest that they would show an altered Th1 response, e.g., DTH. As shown in Fig. 4, the DTH response to DNFB was significantly depressed in the Tg mice. This supports the finding of cytokine imbalance favoring Th2 over Th1 cytokines in the Tg mice.


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Fig. 4.   Delayed-type hypersensitivity response of CRIP Tg and NTg controls. Male and female mice were challenged with DNFB, as described in MATERIALS AND METHODS. The right pinna was treated with the vehicle; the left pinna was challenged with DNFB. Data are expressed as the %change in thickness of the DNFB vs. vehicle-treated pinnae from NTg and Tg mice. Values are means ± SE (n = 26). Asterisk indicates that the value is significantly different (P < 0.05) from that of NTg mice.

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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Fig. 5.   Response of CRIP Tg and NTg control mice to influenza virus. Mice (males) were challenged intranasally with influenza virus, as described in MATERIALS AND METHODS. Values shown are means ± SE; n = 3/group. A: serum zinc concentration; B: lung viral titers on days 2, 4, and 8 postinfection; C: %change from initial body weight after influenza virus infection. Asterisks indicate that values are significantly different (P < 0.05) from NTg mice.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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-alpha . 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-gamma and IL-2. Production of TNF-alpha , 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-gamma 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-gamma 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-gamma and IL-12 drive Th1 T cell differentiation. In addition, IL-4 and IFN-gamma 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-alpha production is regulated by tristetraprolin, a CCCH zinc finger protein (7), through a feedback inhibitory mechanism influencing TNF-alpha mRNA.

Our model of CRIP function currently focuses on the decreased levels of IFN-gamma 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-gamma -induced gene expression; 2) protein-protein interaction observed in vitro (J. Nicewonger and R. Cousins, unpublished data) between CRIP and the IFN-gamma 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-gamma 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.


    ACKNOWLEDGEMENTS

We express our appreciation to Warren R. Clark, Robert J. Cottey, Leah M. Coy, and Kelli A. Herrlinger-Garcia for help with some analyses.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 282(6):E1197-E1203