Epidermal growth factor stimulates the tyrosine phosphorylation of SHPS-1 and association of SHPS-1 with SHP-2, a SH2 domain-containing protein tyrosine phosphatase

SHPS-1 is a 120 kDa glycosylated receptor-like protein that contains immunoglobulin-like domains in its extracellular region and four potential tyrosine phosphorylation for SH2 domain binding sites in its cytoplasmic region. Epidermal growth factor (EGF) stimulated the rapid tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2, a protein tyrosine phosphatase containing SH2 domains, in Chinese hamster ovary cells overexpressing human EGF receptors. In the cells overexpressing SHPS-1, the tyrosine phosphorylation of SHPS-1 was more evident than that observed in parent cells. However, overexpression of SHPS-1 alone did not affect the activation of MAP kinase in response to EGF. These results suggest that SHPS-1 may be involved in the recruitment of SHP-2 from the cytosol to the plasma membrane in response to EGF.     

A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities

The vascular endothelial growth factor (VEGF) and the VEGF-C promote growth of blood vessels and lymphatic vessels, respectively. VEGF activates the endothelial VEGF receptors (VEGFR) 1 and 2, and VEGF-C activates VEGFR-3 and VEGFR-2. Both VEGF and VEGF-C are also potent vascular permeability factors. Here we have analyzed the receptor binding and activating properties of several cysteine mutants of VEGF-C including those (Cys156 and Cys165), which in other platelet-derived growth factor/VEGF family members mediate interchain disulfide bonding. Surprisingly, we found that the recombinant mature VEGF-C in which Cys156 was replaced by a Ser residue is a selective agonist of VEGFR-3. This mutant, designated DeltaNDeltaC156S, binds and activates VEGFR-3 but neither binds VEGFR-2 nor activates its autophosphorylation or downstream signaling to the ERK/MAPK pathway. Unlike VEGF-C, DeltaNDeltaC156S neither induces vascular permeability in vivo nor stimulates migration of bovine capillary endothelial cells in culture. These data point out the critical role of VEGFR-2-mediated signal transduction for the vascular permeability activity of VEGF-C and strongly suggest that the redundant biological effects of VEGF and VEGF-C depend on binding and activation of VEGFR-2. The DeltaNDeltaC156S mutant may provide a valuable tool for the analysis of VEGF-C effects mediated selectively via VEGFR-3. The ability of DeltaNDeltaC156S to form homodimers also emphasizes differences in the structural requirements for VEGF and VEGF-C dimerization.     

Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase)

Flt-1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor-1, binds VEGF and a new VEGF-related ligand, placenta growth factor, but KDR/Flk-1 (VEGF receptor-2) binds only VEGF. To characterize the functional regions in the Flt-1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt-1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N-terminal 1st to 3rd immunoglobulin (Ig)-like domains of Flt-1 binds both ligands with high affinity. However, for dimer formation of soluble Flt-1, a region further downstream in the Flt-1 extracellular domain was required. Mutant Flt-1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt-1 tyrosine kinase. Soluble Flt-1 carrying the N-terminal 1st to 3rd Ig region suppressed VEGF-dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt-1, suggesting that the binding of one soluble Flt-1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.     

Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

Enhanced phosphorylation of Src family kinase substrates containing SH2 domain binding sites

Src family protein-tyrosine kinases possess several modular domains important for regulation of catalytic activity and interaction with potential substrates. Here, we explore interactions between the SH2 domain of Hck, a Src family kinase, and substrates containing SH2 domain-binding sites. We have synthesized a series of peptide substrates containing a high affinity SH2 domain binding site, (phospho)Tyr-Glu-Glu-Ile. We show that the presence of this sequence in a peptide results in a dramatic increase in the phosphorylation rate of a second tyrosine located at the N terminus. Enhanced phosphorylation is not a consequence of stimulation of enzymatic activity by C-terminal tail displacement but is imparted instead by a 10-fold reduction in the Km of the phosphotyrosine-containing peptide when compared with a control. The isolated catalytic domain of the non-receptor tyrosine kinase Abl does not show a preference for the pYEEI motif-containing peptide; however, the preference is restored when the SH2 domain of Src is introduced into Abl. Furthermore, enhanced phosphorylation is dependent on the distance between SH2 domain-binding site and phosphorylatable tyrosine, with the minimum distance requirement being seven amino acids. Reversing the orientation of the pYEEI motif with respect to the substrate sequence decreases phosphorylation by down-regulated Hck, but both orientations are utilized equally well by activated Hck. We discuss the possible implications of these results for processive phosphorylation of substrates in vivo by Src family kinases.     

Immunolocalization of basic fibroblast growth factor and fibroblast growth factor receptor-1 and receptor-2 in rat cranial sutures

Craniosynostosis is a common disorder with an unknown etiology. Recent genetic mapping studies have demonstrated a strong linkage between several familial craniosynostotic syndromes and mutations in fibroblast growth factor receptor 1 (FGF-R1) and 2 (FGF-R2). The purpose of this experiment was to investigate by immunohistochemistry the protein production of these receptors as well as of their most prevalent ligand, basic fibroblast growth factor (bFGF), before, during, and after sutural fusion in rat cranial sutures. The posterior frontal (normally fuses between postnatal days 12 and 22) and sagittal (remains patent) sutures of embryonic day 20 and neonatal days 6, 12, 17, 22, and 62 (n = 3 per group) were harvested, fixed, and decalcified. Five-micrometer sections were stained with polyclonal antibodies against bFGF, FGF-R1, and FGF-R2, and patterns of immunohistochemical staining were assessed by independent reviewers. Our results indicate that increased bFGF production correlates temporally with suture fusion, with increased staining of the dura underneath the fusing suture prior to fusion followed by increased staining within osteoblasts and sutural cells during fusion. FGF-R1 and, to a lesser extent FGF-R2 immunostaining revealed a different pattern of localization with increased immunostaining within the patent sagittal suture at these time points. These results implicate bFGF in the regulation of sutural fusion and may imply autoregulatory mechanisms in fibroblast growth factor receptor expression.     

Crystallization of the receptor binding domain of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells. In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups. In order to facilitate the production of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet-derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space group P2(1), with cell parameters a = 55.6 A, b = 60.4 A, c = 77.7 A, beta = 90.0 degrees, and four monomers in the asymmetric unit. Native and derivative data were collected for two of the cysteine mutants as well as for wild-type VEGF.     

Mapping of the sites for ligand binding and receptor dimerization at the extracellular domain of the vascular endothelial growth factor receptor FLT-1

The vascular endothelial growth factor (VEGF) receptor FLT-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven Ig-like loops within the extracellular domain. Upon VEGF binding FLT-1 becomes phosphorylated, which has been thought to be preceded by receptor dimerization. To further investigate high affinity binding of VEGF to FLT-1 and ligand-induced receptor dimerization, we expressed in Sf9 cells the entire extracellular domain comprising all seven Ig-like loops: sFLT-1(7) and several truncated mutants consisting of loop one, one and two, one to three, one to four, and one to five. The corresponding proteins, named sFLT-1(1), (2), (3), (4), and (5) were purified. Only mutants sFLT-1(3) to (7) were able to bind 125I-VEGF with high affinity. No binding of VEGF was observed with sFLT-1(1) and sFLT-1(2), indicating that the first three Ig-like loops are involved in high affinity binding of VEGF. The binding of VEGF to sFLT-1(3) could be competed with placenta growth factor (PlGF), a VEGF-related ligand, suggesting that high affinity binding of VEGF and PlGF is mediated by the same or closely related contact sites on sFLT-1. Deglycosylation of the sFLT-1(3), (4), (5), and (7) did not abolish VEGF binding. Furthermore, unglycosylated sFLT-1(3), expressed in Escherichia coli, was able to bind VEGF with similar affinity as sFLT-1(3) or sFLT-1(7), both expressed in Sf9 cells. This indicates that receptor glycosylation is not essential for high affinity binding. Dimerization of the extracellular domains of FLT-1 upon addition of VEGF was detected with all mutants containing the Ig-like loop four. Although sFLT-1(3) was able to bind VEGF, dimerization of this mutant was inefficient, indicating that sites on Ig-like loop four are essential to stabilize receptor dimers.     

青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子及其受体表达的影响

目的 研究青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子(VEGF)及其受体( VEGFR)的影响。方法酶联免疫吸附法检测非细胞毒性浓度(5、10、20 ng/ml)青蒿琥酯作用SHI-1细胞后培养上清液VEGF浓度,流式细胞术检测有或无青蒿琥酯作用时,SHI-1细胞表面VEGFR-1及VEGFR-2阳性表达率。结果培养24、48 h后,无青蒿琥酯作用的SHI-1细胞培养上清液VEGF质量浓度分别为( 980.3±2.2)、(982.4±2.3) pg/ml,VEGFR-1表达率分别为(5.40±3.11)％和(4.45±2.85)％,VEGFR-2表达率分别为(13.90.± 2.26)％和（13.95±1.96）％。5、10、20 ng/ml青蒿琥酯作用24h后,SHI-1细胞培养上清液VEGF质量浓度分别为(234.6±1.8)、(114.9±1.6)、(108.8±1.5) pg/ml,作用48 h后分别为(62.3±1.7)、(60.9±1.6)、(32.7±1.7) pg/ml,与培养相同时间无青蒿琥酯组相比,VEGF浓度明显下降（均P＜ 0.05）,且相同浓度青蒿琥酯作用24 h与48 h间差异亦有统计学意义(均P＜ 0.05)。5、10、20 ng/ml青蒿琥酯作用24 h,VEGFR-1阳性率分别为(4.30±2.21)％、(4.20±1.37)％和(3.90±1.86)％,作用48 h后分别为(3.80±2.87)％、(3.60±1.73)％和(3.00±1.82)％,相同作用时间不同浓度青蒿琥酯组间及相同浓度作用不同时间组间VEGFR-1阳性率差异均无统计学意义(均P＞ 0.05);作用24h后,SHI-1细胞VEGFR-2阳性率分别为(4.40±1.15)％、(3.10±0.68)％和(1.10±0.72)％,作用48 h后分别为(3.00±1.68)％、(2.20±0.93)％和(0.60±0.92)％,3个不同浓度青蒿琥酯作用相同时间后VEGFR-2表达率降低（均P＜ 0.05）,相同浓度作用24与48 h间差异均无统计学意义（均P＞ 0.05）。结论SHI-1细胞株高分泌VEGF,青蒿琥酯可下调VEGF分泌及VEGFR-2的表达,而对VEGFR-1表达的调节作用不显著。     

Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor

The kinase insert domain-containing receptor (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed various deletion mutants of the extracellular region fused with the crystallizable fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH2-terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule. The first Ig-like domain may regulate the ligand binding.     

Arachidonate-induced tyrosine phosphorylation of epidermal growth factor receptor and Shc-Grb2-Sos association

PURPOSE: Subdural grid arrays are used when seizure activity cannot be located by ictal scalp recordings and when functional cortical mapping is required before surgery. This study was performed to determine and compare the CT and MR imaging appearance of subdural EEG grids and to identify the types and frequency of associated complications. METHODS: We retrospectively reviewed the medical records and imaging studies of 51 consecutive patients who underwent 54 craniotomies for subdural EEG grid implantation with either stainless steel or platinum alloy contacts between June 1988 and September 1993. Twenty-two patients had both CT and MR examinations, 27 patients had CT only, and five patients had MR imaging only. All studies were assessed for image quality and degradation by the implanted EEG grids, for intra- and extraaxial collections, and for mass effect, with differences of opinion resolved by consensus. RESULTS: Subdural EEG grids caused extensive streak artifacts on all CT scans (corresponding directly to grid composition) and mild to moderate magnetic susceptibility artifacts on MR images. Sixteen associated complications were detected among the 54 patients imaged, including four significant extraaxial hematomas, four subfalcine or transtentorial herniations, two tension pneumocephali, two extraaxial CSF collections, two intraparenchymal hemorrhages, and one case each of cerebritis and brain abscess. In all but four cases, the detected complications were not clinically apparent and did not require specific treatment. There were no residual sequelae. CONCLUSION: Because of extensive streak artifacts, CT showed only gross complications, such as herniation and grid displacement by extraaxial collections. MR imaging artifacts were more localized, allowing superior evaluation of subdural EEG grid placement and associated complications.     

Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis

Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.     

Platelet-derived growth factor (PDGF) stimulates the association of SH2-Bbeta with PDGF receptor and phosphorylation of SH2-Bbeta

We recently identified SH2-Bbeta as a JAK2-binding protein and substrate involved in the signaling of receptors for growth hormone and interferon-gamma. In this work, we report that SH2-Bbeta also functions as a signaling molecule for platelet-derived growth factor (PDGF). SH2-Bbeta fused to glutathione S-transferase (GST) bound PDGF receptor (PDGFR) from PDGF-treated but not control cells. GST fusion protein containing only the SH2 domain of SH2-Bbeta also bound PDGFR from PDGF-treated cells. An Arg to Glu mutation within the FLVRQS motif in the SH2 domain of SH2-Bbeta inhibited GST-SH2-Bbeta binding to tyrosyl-phosphorylated PDGFR. The N-terminal truncated SH2-Bbeta containing the entire SH2 domain interacted directly with tyrosyl-phosphorylated PDGFR from PDGF-treated cells but not unphosphorylated PDGFR from control cells in a Far Western assay. These results suggest that the SH2 domain of SH2-Bbeta is necessary and sufficient to mediate the interaction between SH2-Bbeta and PDGFR. PDGF stimulated coimmunoprecipitation of endogenous SH2-Bbeta with endogenous PDGFR in both 3T3-F442A and NIH3T3 cells. PDGF stimulated the rapid and transient phosphorylation of SH2-Bbeta on tyrosines and most likely on serines and/or threonines. Similarly, epidermal growth factor stimulated the phosphorylation of SH2-Bbeta; however, phosphorylation appears to be predominantly on serines and/or threonines. In response to PDGF, SH2-Bbeta associated with multiple tyrosyl-phosphorylated proteins, at least one of which (designated p84) does not bind to PDGFR. Taken together, these data strongly argue that, in response to PDGF, SH2-Bbeta directly interacts with PDGFR and is phosphorylated on tyrosine and most likely on serines and/or threonines, and acts as a signaling protein for PDGFR.     

Common proteins bind mRNAs encoding erythropoietin, tyrosine hydroxylase, and vascular endothelial growth factor

Lysophosphatidic acid (LPA) is a growth factor-like mediator for fibroblasts or smooth muscle cells produced and released by activated platelets. Platelet activation occurs with hepatic necrosis and subsequent liver regeneration and fibrosis. In the fibrosis, hepatic stellate cells proliferate with phenotypic transformation to myofibroblasts. Thus, effects of LPA on proliferation of hepatocytes and stellate cells were investigated. In cultured rat stellate cells, LPA increased DNA synthesis with enhanced MAP kinase activity. Pertussis toxin (PTX) attenuated this mitogenic action. In contrast, LPA decreased DNA synthesis by cultured rat hepatocytes induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting protein synthesis. Enhanced MAP kinase activity by HGF or EGF was not changed by LPA. This anti-mitogenic action was attenuated by PTX. TGFbeta level in the medium was less than the level effective for inhibiting the DNA synthesis in the presence of LPA. Our results suggest that LPA might affect proliferation of hepatocytes and stellate cells in liver diseases complicating platelet activation.     

Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3

Lymphatic vessels have been difficult to study in detail in normal and tumor tissues because of the lack of molecular markers. Here, monoclonal antibodies against the extracellular domain of the vascular endothelial growth factor-C receptor that we have named VEGFR-3 were found to specifically stain endothelial cells of lymphatic vessels and vessels around tumors such as lymphoma and in situ breast carcinoma. Interestingly, the spindle cells of several cutaneous nodular AIDS-associated Kaposi's sarcomas and the endothelium around the nodules were also VEGFR-3 positive. The first specific molecular marker for the lymphatic endothelium should provide a useful tool for the analysis of lymphatic vessels in malignant tumors and their metastases and the cellular origin and differentiation of Kaposi's sarcomas.     

Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind approximately 100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.     

Expression of vascular endothelial growth factor and placenta growth factor in human placenta

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.     

Different tyrosine autophosphorylation requirements in fibroblast growth factor receptor-1 mediate urokinase-type plasminogen activator induction and mitogenesis

BACKGROUND: Although rapid fluid resuscitation continues to be an important component of the initial therapy for trauma patients, it remains unknown whether the rate of fluid administration after trauma-hemorrhage has any deleterious or beneficial effects on immunity. METHODS: Male C3H/HeN mice were subjected to sham operation or soft-tissue trauma (midline laparotomy) and hemorrhagic shock (mean arterial blood pressure of 35+/-5 mm Hg for 90 minutes) followed by resuscitation with four times the volume of shed blood in the form of lactated Ringer's solution over 30 minutes (rapid rate), 60 minutes (moderate rate), or 120 minutes (slow rate). The animals were killed at either 4 hours or 4 days after the end of trauma-hemorrhage. Spleens were harvested and splenocyte interleukin (IL)-3 and interferon-gamma (IFN-gamma) release as well as splenic macrophage IL-1beta and IL-6 release were determined. RESULTS: The results indicate that at 4 hours after trauma-hemorrhage, splenocyte IL-3 and IFN-gamma release were significantly depressed in all animals subjected to trauma-hemorrhage compared with sham-operated animals. At 4 days after trauma-hemorrhage, splenocyte IL-3 and IFN-gamma release were restored in mice resuscitated with the slow rate of resuscitation; however, the release of these cytokines remained significantly depressed in animals resuscitated with the moderate or rapid rates. Splenic macrophage IL-1beta and IL-6 release were significantly depressed at 4 hours after trauma-hemorrhage. At 4 days after trauma-hemorrhage, the release of these proinflammatory cytokines was still depressed in animals resuscitated with the rapid rate. In contrast, splenic macrophage IL-1beta and IL-6 release were restored in mice receiving the slow rate of resuscitation. CONCLUSION: These results suggest that a slower rate of fluid resuscitation after trauma-hemorrhage leads to a faster restoration of the depressed cell-mediated immunity, whereas rapid fluid resuscitation produces a prolonged depression of immune responses. In view of this, we propose that a prospective clinical study of this type must be performed in a select group of trauma patients to determine whether or not a slower rate of fluid resuscitation also improves immune responses in trauma patients.