Normal reproductive and macrophage function in Pem homeobox gene-deficient mice.

Interaction between germ cells and the supporting somatic cells guides many of the differentiative processes of gametogenesis. The expression pattern of the Pem homeobox gene suggests that it may mediate specific inductive events in murine reproductive tissues. During gestation, Pem is expressed in migrating and early postmigratory primordial germ cells, as well as in all embryo-derived extraembryonic membranes. Pem expression ceases in the germline after Embryonic Day 14 in both sexes and then reappears postnatally in the supporting cells of the gonad. In mature mice, Pem is produced by testicular Sertoli cells during stages VI-VIII of spermatogenesis and transiently by ovarian granulosa cells lining periovulatory follicles. Despite this tightly regulated reproductive expression pattern, mice with a targeted mutation in Pem have normal fecundity, with no detectable alteration in extraembryonic testicular or ovarian development or function. We also show that Pem is expressed throughout embryonic and adult development in a subset of a tissue-specific class of macrophages, Kupffer cells, as well as in a localized fraction of cells in macrophage cell lines. Although the number of Pem-positive Kupffer cells increases in mice treated with lipopolysaccharide, loss of Pem does not detectably interfere with the cells' ability to induce iNOS expression, demonstrating this Kupffer cell function does not require Pem. No differences were observed between Pem-knockout mice in 129, C57BL6/J, or mixed genetic backgrounds. Together, these data show that Pem is dispensable for embryonic and postnatal development, gonadal function, and Kupffer cell activation, perhaps due to compensatory expression of a similar homeobox gene.

Effect of membrane cholesterol depletion on the immunoreactivity of the D antigen and IgG anti-D.

The immunoreactivity of the main Rh antigen (D) and its corresponding antibody, as determined by a ti-IgG to IgG combining ratio, antibody dissociation and antigen accessibility to antibody, was examined in cholesterol depleted human red cells and ghost membranes. The anti-IgG reactivity of IgG anti-D bound to cholesterol depleted red cells and ghosts was demonstrably enhanced in vitro and in electron microscopy studies, particularly in ghosts. Dissociation of cell bound anti-D during buffer incubation was greater after cholesterol depletion, especially in ghosts. There was also reduced binding of anti-D to cholesterol-depleted cells as previously reported. All these effects appeared to be independent of endogenous or exogenous proteolysis in either cholesterol-depleted membranes or controls as judged from membrane electrophoretic analyses. A2C, an agent which increases membrane fluidity, had no effect on anti-D binding or the antiglobulin reactivity of cell bound IgG. A reduction in anti-D binding also was observed in red cells depleted of cholesterol following immobilization of membrane proteins by glutaraldehyde crosslinking. The findings show that cholesterol depletion not only affects the antigen but also Rh antibody reactivity. They also suggest that factors other than vertical antigen movement in a fluid bilayer may influence the behavior of the D antigen in cholesterol-modified erythrocytes.

Proteolysis of band 3 is enhanced by anti-Rho(D) binding to the red cell membrane.

Surface radioiodinated human red cells were incubated with IgG fractions and the radioelectrophoretic profile of the ghost membranes determined. The patterns of RhO(D)-negative membranes exposed to anti-RhO(D) IgG and RhO(D)-positive membranes exposed to non-immune IgG fractions remained intact. Membranes of RhO(D)-positive membranes following incubation with anti-RhO(D) IgG showed a sharp reduction in the quantity of intact band 3, the main glycoprotein of the red cell membrane. This process was significantly abrogated in the presence of protease inhibitors. The results suggest a possible role for IgG binding in promoting the generation of band 3-derived fragments described by others as normal constituents of isolated ghosts.

Anti-D prozone and membrane sulfhydryl modification.

An IgG anti-D prozone is produced by progressive inactivation of the D antigen following red cell exposure to increasing concentrations of thimerosal and phenol present as antibody excess is achieved. Partial inactivation of the D antigen by routinely added thimerosal, an organic mercurial, and phenol is associated with an unstable D antigen-antibody complex resulting in an increased rate of dissociation of anti-D and with a decreased reactivity of the cell-bound anti-D in the antiglobulin reaction. Complete D inactivation occurs with concentrations in excess of 0.43 microM thimerosal. If attributable to inactivation of a surface-exposed sulfhydryl group, it suggests that less than 5 percent of these are involved in D-antigen activity. The data do not exclude the possibility that D inactivation may result from alteration of sulfhydryl groups other than those exposed at the surface.

Differential binding of IgG anti-D and IgG autoantibodies to reticulocytes and red blood cells.

125I IgG anti-D binding to reticulocytes obtained by density fractionation is reduced relative to that bound to all other red cell (RBC) fractions. Maximum D antigen reactivity occurs following reticulocyte maturation with no detectable change in D reactivity of mature RBC throughout their life span. Reticulocytes have in the range of about 60% of the content of mature RBC. Previously reported increased anti-D agglutinability and binding to old RBC it is not due to an intrinsic increase in D antigen with age, but results from an 'apparent' decrease in anti-D binding to young RBC fractions due to reticulocyte enrichment. IgG RBC autoantibodies obtained by elution from the RBC of eight Coombs-positive blood donors, probably associated with alpha-methyldopa (alpha-MD) administration, showed decreased binding to reticulocytes as determined by 125I protein A (PA). Reticulocytes bound about 70% of the IgG bound to mature RBC, indicating that the membrane antigenic determinant defined by these autoantibodies was incompletely expressed in the reticulocyte. This difference in IgG autoantibody binding between reticulocytes and mature RBC is similar to the decreased D antigen content of reticulocytes and consistent with an autoantibody determinant associated with the Rh complex. Direct testing of density fractionated Coombs-positive RBC in four out of five patients with autoimmune haemolytic anaemia (AIHA) showed reduced quantities of IgG on reticulocytes. The distribution of IgG between reticulocytes and mature RBC may be useful in serologically characterizing patients with AIHA and in identifying subpopulations of patients with this disorder.

Effects on blood group antigens from storage at low ionic strength in the presence of neomycin.

Red blood cells (RBC) stored without plasma in a neomycin, low ionic strength medium at 4 degrees C in excess of 24 h show alterations in antigen reactivity. There is a loss of protease-sensitive RBC antigens and a protease-type increased IgG saline agglutinability of Rh antigens that is associated with increased binding of 125I anti-D. Both the serological findings and the alteration in RBC membrane polypeptides are consistent with protease modification of the membrane due to contamination of the RBC by leukocytes. Neomycin, low ionic strength or leukocytes alone or in dual combination do not produce the observed changes in antigen reactivity. The role of neomycin and low ionic strength in this phenomenon and implication for quality control of reagent RBC used for antibody detection and identification are discussed.

Labeling of Rh antibodies on solid-phase protein A.

Rh0(D) antibodies which retain immune specificity after radiolabeling were prepared by a procedure which does not require IgG isolation from serum, requires 10-fold less isotope than conventional techniques and yields antibody solutions of defined composition. The method involves radioiodination of IgG on immobilized protein A, depends on employing human red cells reduced in surface cytophilic IgG, and exploits the inability of goat IgG to interact with Staphylococcus aureus protein A. The technique concentrates IgG by affinity adsorption and should prove useful in preparing radiolabeled alloantibodies from dilute human antisera and for red cell autoantibodies.

Exposure of the Rh0(D) antigen on the surface and cytoplasmic domains of the red cell membrane.

Inside-out (IO) and right-side-out (RO) vesicles derived form human red blood cells were tested for their ability to bind 125I-labelled IgG anti-RHO(D). The binding of anti-RHO(D) to RO vesicles from RHO(D)-positive cells was quantitatively similar to that exhibited by intact cells when compared on a membrane surface area basis. There was no significant binding of labelled antibody to IO vesicles from RhO(D)-positive cells or to either RO or IO vesicles derived from RhO(D)-negative cells. The RhO(D) antigen was immunologically accessible on only the plasma side of the membrane in RhO(D)-positive red cells, as has been shown for blood group antigens defined by carbohydrate determinants. No immunologically reactive RhO(D) antigen was present on either RO or IO vesicles derived from RHO(D)-negative red cells.

Inhibition of histidyl-tRNA-adenosine triphosphate phosphoribosyltransferase complex formation by histidine and by guanosine tetraphosphate.

Formation of the complex between the first enzyme of histidine biosynthesis from Salmonella typhimurium, ATP phosphoribosyltransferase [1-(5'-phosphoribosyl)-ATP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.17], and histidyl-tRNA is shown to be inhibited by L-histidine and by guanosine-5'-diphosphate-3'-diphosphate in the presence of histidine. Higher histodine levels make guanosine tetraphosphate a more effective inhibitor. Relatively high concentrations of guanosine-5'-triphosphate also inhibit complex formation, but this inhibition is not enhanced by histidine. The possible implications of these observations with respect to the gene regulatory activity of this enzyme are discussed.