Regulation of Human Decidual Cell Macrophage Inflammatory Protein-1α (MIP-1α) Production by Inflammatory Cytokines

PROBLEM : Inflammation of human gestational tissues is a key pathophysiologic event in the genesis of infection-associated preterm labor. Human gestational tissues produce several inflammatory cytokines after stimulation with bacterial products. These include interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), and IL-6. Another class of cytokines includes chemokines of the “C-C” subclassification such as macrophage inflammatory protein-1α (MlP-1α). The purpose of this study was to determine whether cultured human decidual cells produce MIP-1α in response to other inflammatory cytokines. METHODS : Various concentrations of IL-1β, TNFα, IL-6, and IL-4 were incubated with confluent monolayer cultures of decidual cells isolated from normal term placentae for 16 h at 37°C, and MlP-1α concentrations in culture supernatants were measured by ELISA. RESULTS : We found that incubation of decidual cells with IL-1β, TNFα, and IL-4 resulted in significant concentration-dependent increases in M1P-1α production. IL-6 had no effect on MlP-1α production. CONCLUSIONS : Our data are the first to show that human decidual cells in culture produce MlP-1α in response to other inflammatory cytokines. We suggest that decidual cell production of MIP-1α is an important early event in the pathophysiology of infection-associated preterm labor.     

Inhibition of M-tropic HIV-1 infection by the fd phage-gene 3 protein with MIP-1α-binding activity

CCR5 is a chemokine receptor with seven transmembrane-domains. It is expressed on T cells and macrophages and functions as the principal co-receptor for macrophage (M)-tropic strains of HIV-1. The anti-CCR5 monoclonal antibody (mAb) 2D7 inhibits the binding and chemotaxis of the three natural β-chemokine ligands of CCR5, macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES, to CCR5⁺ cells. The mAb also efficiently blocks the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro.In this study, we attempted to determine the peptide motif recognized with the 2D7 mAb. We isolated phage clones by panning a phage display library using 2D7 and identified three peptide motifs. One of these phage clones (M23) showed a marked inhibitory activity on HIV-1 infection. The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the g3p of the M23 phage clone (M23-g3p). The M23-g3p was purified by fast-performance liquid chromatography (FPLC). We show here that (1) M23-g3p was specifically recognized with anti-CCR5 mAb; (2) M23-g3p showed inhibitory activity on the infectivity of M-tropic but not T-tropic HIV-1 strains; (3) M23-g3p bound to MIP-1α, MIP-1β, and RANTES but not MCP-1. These results suggested that the M23-g3p might mimic the CCR5-binding domain shared by β-chemokines, MIP-1α, MIP-1β, and RANTES as well as the HIV-1 infection.     

Molecular characterization and clinical presentation of HKαα and anti‐HKαα alleles in southern Chinese subjects

The HKαα allele is a rearrangement occurring in the α‐globin gene cluster containing both the ‐α^3.7 and ααα^anti4.2 unequal crossover junctions. The anti‐HKαα allele is the reciprocal product containing both the ‐α^4.2 and ααα^anti3.7 unequal crossover junctions, which had been predicted but had not been detected previously. The phenotypic feature and population frequency of these two unusual alleles were not described. We report the identification of nine individuals carrying the HKαα allele and two individuals carrying the anti‐HKαα allele in southern China and describe their phenotype and haplotype data. The molecular structures of HKαα allele and anti‐HKαα allele were confirmed by two‐round nested polymerase chain reaction assay. The mechanism of origin of both alleles is related to probably simultaneous double crossover. Heterozygotes of HKαα or anti‐HKαα allele show a normal hematological phenotype. Finally, we report the carrier rates of these both alleles in the Guangxi Zhuang Autonomous Region of southern China, namely, ∼0.07% for the HKαα allele and ∼0.02% for the anti‐HKαα allele.     

Macrophage inflammatory protein-1α influences eosinophil recruitment in antigen-specific airway inflammation

Allergic airway inflammation is characterized by peribronchial eosinophil accumulation within the submucosa of the airway of the lung. In the present study we have utilized a model of airway inflammation induced by intratracheal challenge with parasite (Schistosoma mansoni) egg antigen (SEA) in presensitized mice. The recruitment of neutrophils and eosinophils into the airway was found to be maximal at 8 and 48 h post challenge, respectively. Since macrophage inflammatory protein-1α (MIP-1α) has previously been found to be chemotactic for eosinophils, in vitro, we postulated that MIP-1α was involved in the airway inflammation and more specifically in eosinophil recruitment into the airway. Initial studies demonstrated an increase in MIP-1α mRNA expression at 8 h post-SEA challenge, as compared to vehicle-treated control mice. We next demonstrated a significant increase in MIP-1α protein in the lungs of SEA-challenged mice at 8 h compared to control challenged mice, correlating to the mRNA data. Immunohistochemical staining of lungs from SEA-challenged mice demonstrated MIP-1α protein expression in airway epithelial cells, alveolar macrophages and in recruited mononuclear cell populations. Immunolocalization of MIP-1α to cells within the bronchoalveolar lavage fluid demonstrated that macrophages and eosinophils stained positive for the protein. To determine the contribution of MIP-1α expression to eosinophil accumulation, SEA-challenged mice were passively immunized with either neutralizing MIP-1α antibodies or normal rabbit IgG, 3–4 h prior to the intratracheal SEA challenge. These studies demonstrated a > 50% decrease in eosinophil recruitment to the lungs and airway in animals receiving neutralizing MIP-1α antibodies with no effect on early neutrophil recruitment. These results suggest that the production of MIP-1α, induced by an antigen-specific response, plays an important role in recruitment of eosinophils in this airway model of inflammation.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Involvement of spinal monocyte chemoattractant protein-1 (MCP-1) in cancer-induced bone pain in rats

In this study, we examined the involvement of chemokine monocyte chemoattractant protein-1 (MCP-1) in the spinal cord of a rat model of cancer-induced bone pain (CIBP). In this model, CIBP was established by an intramedullary injection of Walker 256 cells into the tibia of rats. We observed a significant increase in expression levels of MCP-1 and its receptor CCR2 in the spinal cord of CIBP rats. Furthermore, the intrathecal administration of an anti-MCP-1 neutralizing antibody attenuated the mechanical allodynia established in CIBP rats. Likewise, an intrathecal injection of exogenous recombinant MCP-1 induced a striking mechanical allodynia in naïve rats. These results suggest that increases in spinal MCP-1 and CCR2 expression are involved in the development of mechanical allodynia associated with bone cancer rats.     

Expression of HNF-1α and HNF-1β in various histological differentiations of hepatocellular carcinoma

Hepatic nuclear factor 1 (HNF-1) regulates genes in a hepatocyte-specific manner. It has been previously reported that the ratio of HNF-1α and HNF-1β mRNA is related to histological differentiation hepatocellular carcinoma (HCC). In this study, the expression levels of the HNF-1α and HNF-1β proteins were analysed relatively and quantitatively in various histologically differentiated HCC and surrounding non-cancerous tissues, and HNF-1α binding activity for the AT element of the B domain of the human α-fetoprotein enhancer was examined. Western blot analysis demonstrated that HNF-1α protein was expressed at a higher level in well-differentiated HCC tissues than in the surrounding non-HCC tissues; on the other hand, the HNF-1α protein was expressed at lower levels in moderately and poorly differentiated HCCs than in the surrounding non-HCC tissues. The levels of HNF-1β expression in well-differentiated and poorly differentiated HCCs were similar to and higher than those found in the respective surrounding non-cancerous portions. In binding assays, HNF-1 binding activity was high in well-differentiated HCC and lower in moderately and poorly differentiated HCCs. Most well-differentiated HCC cases showed immunohistochemical expression of HNF-1α. These findings show that poor histological differentiation of HCC correlates with decreases in the level and activity of HNF-1α proteins. © 1998 John Wiley & Sons, Ltd.     

A γδ T cell specific surface receptor (WC1) signaling G0/G1 cell cycle arrest

Three monoclonal antibodies (mAb; SC-6, SC-12, and SC-29) reactive with the γδ T cell-restricted antigen WC1 were obtained immunizing mice with an ovine interleukin (IL)-2-dependent γδ T cell line. These mAb strongly inhibited DNA synthesis in IL-2-dependent γδ T cell lines with cell cycle arrest in G0/G1 phase, but did not induce apoptosis. The mAb-induced growth arrest was reversible, either by removing the mAb or by co-culture with mitogen or anti-CD3 in the presence of IL-2. In contrast, addition of phorbol ester, ionomycin and IL-2 had no effect on the mAb-induced growth arrest. The observations define a biologically important role for the cell surface molecule WC1 in the regulation of γδ T cell proliferation and also provide a suitable system to study the relevant signal transduction events.     

Helper activity of CD4+ αβ T cells is required for the avian γδ T cell response

We have studied the in vitro activation of chicken γδ T cells. Both splenic αβ and γδ T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4⁺ cells or αβ T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of γδ T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4- or αβ TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the γδ T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-γδ TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-γδ TcR monoclonal antibody, a strong proliferation of γδ T cells was observed. In contrast, both Vβ1- and Vβ2-expressing αβ T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either Vβ1⁺ or Vβ2⁺ T cell subset alone had no negative effect on the proliferation or blast formation of γδ T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4⁺ αβ T cells (both Vβl- and Vβ2-expressing) play a role in the activation and response of chicken γδ T cells.     

Postjunctional α1- and α2-adrenoceptors mediating contraction in isolated human groin arteries and veins

By means of selective agonists and antagonists for α₁- and α₂-receptors, the α-receptor subtypes in human groin arteries and veins were characterized and compared. In the arteries the α₁-receptor blocker prazosin caused a concentration-dependent parallel displacement of the noradrenaline (NA) concentration-response (cr) curve without reduction of maximum (pA₂=9.86); the selective α₂-receptor antagonist rauwolscine in the concentration 10^-8 M caused a right-ward shift of the NA cr-curve without reduction of E_max, but 10^-7 M and 10^-6 M caused little or no further shift. In the veins, the two antagonists had the opposite effects. Rauwolscine caused a concentration-dependent right-ward shift of the NA cr-curve without depression of maximum (pA₂=9.03); prazosin 10^-9 M significantly displaced the NA cr-curve, whereas 10^-8 M and 10^-7 M caused little or no further shift. The responses to the α₂-receptor agonist clonidine in the arteries were too small to allow calculations of pEC50 values; in the veins contractions were elicited in all vessel segments investigated (pEC₅₀=6.24). Phenylephrine, selective for α₁-receptors, was significantly more potent in arteries than in veins. NA was significantly more potent in veins than in arteries. It is concluded that in human groin vessels, there is a functional predominance of arreceptors in the arteries and of a2-receptors in the veins.     

Catalase and α-enolase: two novel granulocyte autoantigens in inflammatory bowel disease (IBD)

In IBD, the target antigens of anti-neutrophil cytoplasmic autoantibodies (ANCA) have not been fully identified, which limits the analysis of the diagnostic significance as well as of the possible pathophysiological role of these antibodies. In this study, we identify the target antigens of ANCA in large groups of patients with ulcerative colitis (UC) and Crohn's disease (CD). Apart from antibodies against lactoferrin and bactericidal/permeability-increasing protein (BPI), which have been reported before, antibodies against two novel granulocyte antigens were identified: antibodies against a 57/56-kD doublet were found in 38% of samples from UC patients and in 26% of samples from CD patients, whereas antibodies against a 47-kD protein were found in 10% of samples from UC patients and in 18% of samples from CD patients. Partial purification and amino acid sequence analysis identified the 57-kD protein as catalase and the 47-kD protein as α-enolase. This study is the first to report catalase and α-enolase as granulocyte antigens for autoantibodies in IBD.     

γδ T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ αβT cells

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4⁺ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

Murine T Cell Determination of Pregnancy Outcome: I. Effects of Strain, αβ T Cell Receptor, γδ T Cell Receptor,and γδ T Cell Subsets

PROBLEM: T cells bearing αβ T cell receptor (TcR) and γδ TcR are present at the fetomaternal interface, and the latter, which express surface activation markers, can react with fetal trophoblast cell antigens. What is the role of these cells? METHOD: Using stress-abortion-prone DBA/2-mated CBA/J and abortion-resistant C57/B16 mice, αβ, γδ, and CD8^+/- T cell subsets were measured in spleen and uterine decidua. The effect of immunization against abortion and administration of anti-TcR antibody in vivo was examined. Cytokine synthesis was measured by intracellular staining of Brefeldin A-treated cells. RESULTS: Abortion-prone matings showed an unexpected accumulation of γδ T cells beginning in the peri-implantation period and this was suppressed by immunization against abortion. The immunization deleted γδ T cells producing the abortogenic cytokines, TNF-α and γ-interferon, and increased production of the anti-abortive cytokines, IL-10 and transforming growth factor-β2 (TGF-β2). Immunization also boosted the number of αβ T cells which were present in the decidua as early as 2 days after implantation. In vivo injection of GL4 (anti-δ) depleted γδ T cells producing Th1 cytokines in the peri-implantation period, and prevented abortions, whereas H57 (anti-β) decreased the number of αβ T cells and led to 100% abortions. CD8⁺ T cells present in peri-implant decidua before onset of abortions were mostly αβ TcR⁺, although some were γδ⁺. Changes in γδ and αβ T cells in pregnancy were most dramatic in uterine tissue. CONCLUSION: Although decidual γδ T cells after formation of a distinct placenta and fetus produce anti-abortive TGF-β2-like molecules and IL-10, prior events can lead to abortion. High local production of TNF-α and γ-interferon develop during the peri-implantation phase because of an excessive increase in the Th1 cytokine⁺ subset of γδ cells; these cytokines may be contributed by other tissues in decidua, and the contribution of bioactive factors by γδ T cells may augment the cytokine pool. In contrast, αβ T cells (which may be inactivated by stress that causes abortions) may mediate the anti-abortive effect of alloimmunization. Alloimmunization involves a shift from a Th1 to a Th2 pattern in the γδ T cells in decidua.     

α-thalassaemia due to a single codon deletion in the α-1-globin gene. Computational structural analysis of the new α-chain variant

A new unstable α-globin chain associated with α-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the α-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the α-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 or DK61 results in a structurally abnormal α-globin chain, not previously described, named Hb Clinic, which leads to the α-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this α-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer. Hum Mutat 11:412, 1998. © 1998 Wiley-Liss, Inc.     

The α4β1 integrin in sickle cell disease

The α4β1 integrin is an adhesion receptor expressed on reticulocytes in sickle cell disease (SCD) and mediates the adhesion of these cells to sub-endothelial matrix proteins and the endothelium. In this review, we describe the mechanism of activation of the α4β1 integrin on sickle reticulocytes and discuss novel roles for this integrin in SCD as a result of this activation. We also illustrate novel therapies in SCD that may target the integrin and alleviate vaso-occlusion.     

High frequency of CD8αα homodimer-bearing T cells in human fetal intestine

Immunohistochemistry on frozen sections was used to identify CD8αα cells and CD8αβ cells in human intestine. As observed previously, CD8αβ cells predominate (>95%) in tonsil and post-natal intestine. However in human fetal intestine (16–24 weeks gestation), almost half the CD8⁺ cells in the lamina propria are CD8αα, and many CD8αα cells can be identified in the epithelium. In contrast, in the T cell zones of the Peyer's patches, CD8αβ cells are dominant. The CD8αα cells are virtually all αβ T cell receptor positive. By analogy with the murine system, these CD8αα cells in the fetal gut may be directly derived from the marrow, undergoing thymus-independent differentiation in the gut mucosa.