Evidence for involvement of lymphocyte function-associated antigen 1 in T cell migration to epidermis.

Although it is well known that in various T cell-mediated skin diseases T cells migrate preferentially to epidermis, no direct evidence has been presented in which molecules on T cells are important in directing T cell traffic to epidermis. We have previously established CD4+ autoreactive cloned T cells with a special tropism for epidermis in vitro as well as in vivo. Antibody inhibition studies demonstrated that only anti-lymphocyte function associated Ag 1 (anti-LFA-1) mAb completely inhibited the in vitro migration of the T cells toward the epidermis, whereas mAb against other T cell surface molecules had little or no effect. Monovalent F(ab) fragment of the anti-LFA-1 mAb, although less efficient, also inhibited the T cell migration. The apparent dependency of the inhibition on the anti-alpha-chain mAb suggested a major role for the alpha-chain of LFA-1 in T cell migration to epidermis. The relevance of an LFA-1-dependent mechanism to the epidermotropic migration of T cells was further strengthened by the findings that the T cell migration to epidermis was inhibited by divalent cation depletion, cytochalasin B, and low temperature. These findings indicate that the LFA-1 molecule, which is thought to be primarily involved in cell-to-cell adhesions, also plays an important role in directing T cell migration to epidermis.     

Spectrin involvement in a 40 degrees C structural transition of the red blood cell membrane

Proteins involved in a structural transition detected in red blood cell membranes at 40 degrees C by spin labeling methods have been investigated. Antibodies specific for spectrin, band 3, and protein 4.1 have been used as specific probes to modify membrane thermotropic properties. Spectrin seems to be involved in a 40 degrees C transition detected in ghosts by both a stearic acid spin label (16-doxyl stearic) and a sulfhydryl-specific maleimide analogue spin label. Circular dichroism and maleimide spin labeling studies of purified spectrin show a slow unfolding of the protein structure starting at 25-30 degrees C and a massive transition with an onset temperature of 48 and 40 degrees C, respectively. This thermotropic behavior of spectrin could be the process that modifies membrane physicochemical properties above 40 degrees C that are detected by the stearic acid spin label. The transition detected by the stearic acid spin label was modified both by antispectrin antibodies and anti-4.1 protein antibodies, but not by antibodies specific for the cytoplasmic domain of band 3. These results suggest an involvement of protein 4.1 in regulating spectrin unfolding at the membrane level. A selective inhibition of the transition detected by the maleimide spin label has been obtained with a monoclonal antispectrin antibody at 1:1 molar ratio. The involvement in this transition of a localized spectrin domain(s) containing few exposed sulfhydryl groups is proposed.     

Evidence for a voltage-gated, non-selective cation channel in the human red cell membrane.

Using the patch clamp technique we have identified a voltage-dependent, non-selective cation channel in the human red cell membrane. Basic properties of this channel are reported, and it is proposed that it may be involved in the increased transport of cations which is seen when intact human red cells are suspended in a depolarising media.     

Evidence for the involvement of the thromboxane synthase pathway in human natural cytotoxic cell activity

We and other investigators have recently shown that inhibitors of lipoxygenase reversibly inhibit natural cytotoxic (NC) or natural killer (NK) cell activity, whereas some inhibitors of cyclooxygenase enhance these functions. In addition, exogenous LTB4 augments NC and NK activity, whereas PGE2 depresses it. In the present studies, we sought to investigate the possible role of the TxA2 synthase pathway in NC function. Inhibition of this pathway by OKY-1581 or dazoxiben significantly inhibited NC activity against HSV-infected cells as well as NK function against K562 target cells. The inhibition was dose dependent, reversible, and not due to direct toxicity. NC activity was also significantly inhibited by the addition of PGE2 or PGI2 to the 4-hr assay, whereas addition of 6-keto-PGF1 alpha had no effect. Addition of PGH2, which could be converted to TxA2 or other PG, had no significant effect, but concomitant use of OKY-1581 produced a greater inhibition of NC function than by using OKY-1581 alone. U44069, a TxA2 analog, was inhibitory by itself and could not alter the inhibition caused by OKY-1581 or dazoxiben. In contrast, the TxA2 receptor blocker 13-APA significantly enhanced NC activity and even reversed the inhibitory effect of U44069 at equimolar (10(-7)M) concentrations. Taken together, these data suggest that most of the inhibitory effect of the TxA2 synthase inhibitors on NC and NK cell function derives from their ability to reorient cyclic endoperoxide metabolism toward more inhibitory compounds. In addition, TxA2 itself could exert a negative feedback on NC function through its receptor, as evidenced by the use of a TxA2 analog and a TxA2 blocker.     

Evidence for involvement of HMGB1 protein in human DNA mismatch repair

Defects in human DNA mismatch repair predispose to cancer, but many components of the pathway have not been identified. We report here the identification and characterization of a novel component required for mismatch repair in human cells. A 30-kDa protein was purified to homogeneity by virtue of its ability to complement a depleted HeLa extract in repair of mismatched heteroduplexes. The complementing activity was identified as HMGB1 (the high mobility group box 1 protein), a non-histone chromatin protein that facilitates protein-protein interactions and recognizes DNA damage. Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair.     

Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I

Two and three color flow cytometry of normal human bone marrow was used to identify CD34+ progenitor cells and examine their binding to the plant lectin Ulex europaeus I (Ulex). In normal bone marrow, 48.48 +/- 17.4% of the CD34+ cells bind to Ulex. Two color flow cytometry was used to sort CD34 + cells, and subsets of CD34+ cells, CD34+ Ulex+ and CD34+ Ulex-. These populations were sorted into colony assays to assess myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The CD34+ Ulex+ subset was 84 +/- 14% BFU-E colonies (mean +/- S.D.) and had the highest cloning efficiency of 28 +/- 13%. Three color analysis of CD34+ Ulex+ cells showed staining with other erythroid (CD71, GlyA) antibodies and lack of stain. ing with myeloid (CD13, CD45RA) antibodies. These studies confirmed the erythroid characteristics of this subpopulation.     

Unmasking of a potassium leak in resealed human red blood cell ghosts

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0° C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.     

Antigenic and functional properties of the human red blood cell urea transporter hUT-B1

The Kidd (JK) blood group locus encodes the urea transporter hUT-B1, which is expressed on human red blood cells and other tissues. The common JK*A/JK*B blood group polymorphism is caused by a single nucleotide transition G838A changing Asp-280 to Asn-280 on the polypeptide, and transfection of erythroleukemic K562 cells with hUT-B1 cDNAs carrying either the G838 or the A838 nucleotide substitutions resulted in the isolation of stable clones that expressed the Jk(a) or Jk(b) antigens, respectively, thus providing the first direct demonstration that the hUT-B1 gene encodes the Kidd blood group antigens. In addition, immunochemical analysis of red blood cells demonstrated that hUT-B1 also exhibits ABO determinants attached to the single N-linked sugar chain at Asn-211. Moreover, immunoadsorption studies, using inside-out and right-side-out red cell membrane vesicles as competing antigen, demonstrated that the C- and N-terminal ends of hUT-B1 are oriented intracellularly. Mutagenesis and functional studies by expression in Xenopus oocytes revealed that both cysteines Cys-25 and Cys-30 (but not alone) are essential for plasma membrane addressing. Conversely, the transport function was not affected by the JK*A/JK*B polymorphism, C-terminal deletion (residues 360-389), or mutation of the extracellular N-glycosylation consensus site and remains poorly para-chloromercuribenzene sulfonate (pCMBS)-sensitive. However, transport studies by stopped flow light scattering using Jk-K562 transfectants demonstrated that the hUT-B1-mediated urea transport is pCMBS-sensitive in an erythroid context, as reported previously for the transporter of human red blood cells. Mutagenesis analysis also indicated that Cys-151 and Cys-236, at least alone, are not involved in pCMBS inhibition. Altogether, these antigenic, topologic, and functional properties might have implications into the physiology of hUT-B1 and other members of the urea transporter family.     

Characterization of a red blood cell antigen in donkeys and mules associated with neonatal isoerythrolysis

A red cell antigen of donkeys and mules was identified using antibodies in serum from a mare which produced a mule foal affected with neonatal isoerythrolysis (NI). Subsequently antibodies with similar activity were identified in the sera of other mares which had produced mule foals and were produced by immunization of horses with blood from donkeys. The antigen detected by these antibodies does not correspond to any recognized horse red cell alloantigen. This may be a xenoantigen since all donkeys (and mules) tested have shared this antigen and all horses tested have lacked the antigen. The results suggest that all mule pregnancies (donkey sire x horse dam) are incompatible with regard to this factor and a potential for neonatal isoerythrolysis exists in all cases.     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (Ea). Below 15 or 25 degrees C (for Cl- and Br-, respectively), Ea is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). For the temperature dependence of SO4(2-) transport up to 37 degrees C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO4(2-) transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60 degrees C and to show that, even for the slowly permeating SO4(2-) in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37 degrees C and where Ea changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using 35Cl- -NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472-6480), we also determined the temperature dependence of Cl- -binding. We found no significant change over the entire range from 0 to 57 degrees C, regardless of whether the measurements were performed in the absence or presence of competing SO4(2-). We conclude that the enthalpy changes associated with Cl- - or SO4(2-)-binding are negligible as compared to the Ea values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl-, Br- and SO4(2-) below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (E_a). Below 15 or 25°C (for Cl⁻ and Br⁻, respectively), E_a is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283–306). For the temperature dependence of SO₄²⁻ transport up to 37°C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO₄²⁻ transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60°C and to show that, even for the slowly permeating SO₄²⁻ in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37°C and where E_a changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using ³⁵Cl⁻-NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472–6480), we also determined the temperature dependence of Cl⁻-binding. We found no significant change over the entire range from 0 to 57°C, regardless of whether the measurements were performed in the absence or presence of competing SO₄²⁻. We conclude that the enthalpy changes associated with Cl⁻-or SO₄²⁻-binding are negligible as compared to the E_a values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl⁻, Br⁻ and SO₄²⁻ below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Studies on antigenic competition. V. Evidence for the involvement of a thymic-derived cortisone-sensitive cell in the mediation of antigenic competition

Animals depleted of lymphoid subpopulations by neonatal thymectomy, by adult thymectomy, irradiation, and bone marrow reconstitution, or by hydrocortisone treatment did not exhibit antigenic competition. In contrast, injection of anti-lymphocyte serum, while depressing the magnitude of the immune response, did not effect antigenic competition. It is concluded that a thymic-dependent cortisone-sensitive cell population is involved in the initiation of antigenic competition.     

Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.