Fate of surface immunoglobulin during induction of lymphocyte proliferation

The modulation of immunoglobulin on the surface of rabbit B lymphocytes by goat antibodies with specificity for rabbit surface membrane immunoglobulin or by such goat antibodies covalently linked to Sepharose was studied in relation to the proliferative response to these agents. Although the induction of DNA synthesis was greater in the presence of Sepharose-linked antibody than in the presence of free antibody, modulation of surface membrane immunoglobulin was induced with free but not with Sepharose-linked antibody. Thus, in the presence of free antibody the surface membrane immunoglobulin content of cells was rapidly decreased and remained at a low level throughout the culture period, whereas the surface immunoglobulin content of cells incubated with Sepharose antibody was essentially unaltered. The surface immunoglobulin lost from cells incubated with free goat antibodies reappeared slowly upon further incubation in culture medium devoid of antibody, and such reappearance of rabbit surface membrane immunoglobulin was inhibited by puromycin. Upon culture with Sepharose-linked antibody the surface membrane immunoglobulin content of B cells was unaffected by puromycin. This result was interpreted as indicating that surface membrane immunoglobulin loss followed by reappearance does not occur. Lastly, the linkage of surface membrane immunoglobulin to cytoskeletal elements induced by free antibody was not induced by Sepharose-linked antibody as judged from differences in detergent solubilization characteristics. Possible mechanisms to account for these differences in surface membrane immunoglobulin modulation as they relate to the proliferative response are considered.     

Inhibition of lymphocyte activation by cyclosporin A: interference with the early activation of the membrane phospholipid metabolism in rabbit lymphocytes stimulated with concanavalin A, anti-rabbit immunoglobulin or the Ca2+ ionophore A 23187

Rabbit lymphocytes from the mesenteric lymph nodes were stimulated with concanavalin A, goat anti-rabbit immunoglobulin, or the Ca2+ ionophore A 23187. The stimulated incorporation of labeled uridine into RNA as well as of labeled thymidine into DNA was suppressed within a dose range of 40-1000 ng/ml cyclosporin A in both Con A-stimulated T lymphocytes and in anti-immunoglobulin-stimulated B lymphocytes, without affecting the resting cells. A 23187-stimulated rabbit lymphocytes proved to be more sensitive to cyclosporin A. At 40 ng/ml the immunosuppressive drug was effective in inhibiting elevated incorporation of labeled nucleosides into macromolecules in ionophore-stimulated cells. Cyclosporin A, at the same concentrations that were effective in inhibiting stimulated RNA and DNA synthesis, suppressed one of the earliest events occurring in stimulated lymphocytes, i.e., enhanced incorporation of unsaturated fatty acids into membrane phospholipids. Whereas cyclosporin A significantly inhibited the incorporation of arachidonic acid into phosphatidylcholine and phosphatidylethanolamine in concanavalin A-, anti-immunoglobulin-, and A 23187-stimulated cells, it proved to be ineffective in inhibiting the incorporation of arachidonate into phosphatidylinositol. The data indicate that cyclosporin A inhibits both T- and B-cell stimulation by interfering with a common target, e.g., the early activation of membrane phospholipid metabolism of rabbit lymphocytes.     

Anti-immunoglobulin-induced proliferation of B cells : Parallelism in the inhibition by chloroquine, monensin and immunoglobulin

Proliferation of rabbit lymphocytes was induced with goat anti-rabbit immunoglobulin. Chloroquine and monensin, known to inhibit internalization-related events, yielded inhibition of proliferation that paralleled the inhibition by a specific competitive ligand, rabbit immunoglobulin (IgG), whereas inhibition by puromycin did not. Moreover, virtually all of the cells that can be activated in freshly isolated populations adhered to anti-immunoglobulin-coated Petri plates, whereas all of the activatable population was recovered in the non-adherent fraction after a brief incubation of the cells with anti-immunoglobulin to induce internalization of surface membrane immunoglobulin. Using immunofluorescence it was further observed that monensin and Chloroquine inhibit the reappearance of surface immunoglobulins on the cell surface to some extent subsequent to their removal induced by anti-immunoglobulin.     

Surface immunoglobulin on rabbit lymphoid cells. I. Ultrastructural distribution and endocytosis of b4 allotypic determinants on peripheral blood lymphocytes

Immunoglobulin (Ig) b4 allotypic determinants are detected on the surface membrane of rabbit peripheral blood lymphocytes by an indirect immunoferritin labeling technique. Cells coated with antiallotype antibodies are labelled with soluble complexes of ferritin and rabbit antiferritin of a given allotype. At 0 °C a patchy distribution of labeled surface immunoglobulin is visualized on 80% of the lymphocytes examined. Warming of the cells for 1–5 min at 37 °C causes rapid endocytosis of surface label in a perinuclear fashion. Cap formation is not observed. Cross-linking of immunoferritin labelled surface determinants with sheep anti-rabbit Ig (SARG) inhibits endocytosis and promotes aggregation of small surface patches. Indirect evidence suggests that sloughing and/or stripping of labelled surface Ig can occur after this aggregation. These surface changes may be the first step in the induction of lymphocyte activation.     

DNA and immunoglobulin synthesis by rabbit peripheral blood lymphocytes in vitro: complete and incomplete stimulation

Activation of resting (G0) rabbit peripheral blood lymphocytes (PBLs) into DNA synthesis and IgG synthesis was studied using sheep anti-rabbit IgG (SARIgG), protein A, pokeweed mitogen (PWM), and lipopolysaccharide (LPS). DNA synthesis was assayed by [125I]iododeoxyuridine incorporation. IgG synthesis was measured by determination of Ig in culture supernatants by an ELISA assay. Rabbit PBLs cultured with SARIgG or protein A for 48 hr and then without these reagents for 72 hr showed both DNA synthesis and Ig synthesis, whereas PWM and LPS had very little, if any, effect. PBLs stimulated with SARIgG for 6 hr and then without SARIgG for subsequent 114 hr did not become activated into DNA synthesis or IgG synthesis. However, PBLs prestimulated with SARIgG for 6 hr and then with PWM for 114 hr showed prominent DNA and IgG synthesis. LPS also maintained activation of PBLs after prestimulation of these cells with SARIgG, but the effect was much smaller than that of PWM. No evidence was found for production of factors by SARIgG-stimulated PBLs that could, by themselves, either stimulate resting cells or maintain activation of SARIgG-prestimulated cells. These results suggest that anti-IgG and protein A are complete activating mitogens for resting rabbit B cells to proliferate and differentiate into IgG-producing cells, whereas PWM and LPS are not able to activate G0 cells directly, but have a sustaining effect after activation of resting B cells with anti-IgG, either directly or via production of factors by accessory cells.     

Determination of the LDL receptor binding capacity of human lymphocytes by immunocytofluorimetric assay

The determination of the LDL receptor binding capacity of human blood lymphocytes was assessed by indirect immunocytofluorimetric assay. To produce the maximal synthesis of the LDL receptor, the cholesterol efflux was enhanced by incubation of lymphocytes with HDL3 subfractions. The binding capacity of the LDL receptor was measured by incubation at 4 degrees C either with LDL and rabbit anti-LDL immunoglobulins or with peptide receptor antibody (ARP-Ig) raised against the NH2-terminal sequence of the LDL receptor. Thereafter complexes were incubated with fluorescein-labelled anti-rabbit immunoglobulin (FITC-Ig). Fluorescence flow cytometry was used to quantify the number of fluorescent lymphocytes and results were expressed as the percentage of lymphocytes with a fluorescent intensity above the threshold. Using preimmune rabbit immunoglobulin and then FITC-Ig, only 5-10% of cells were fluorescent. Neither LDL nor ARP-Ig could bind to homozygous familial hypercholesterolemia (FH) lymphocytes. Normal lymphocytes preincubated with HDL3 could bind LDL or ARP-Ig, the number of fluorescent cells being 59 and 39.2% respectively. Subjects with confirmed or suspected heterozygous FH demonstrated cell fluorescence at about half the normal level.     

Cell surface mobility of a bacterial R-form glycolipid, mR595, after binding to rat cells, and its effect on the mobility of concanavalin A receptors

Binding of small amounts of glycolipid mR595 to rat cells, followed by sequential incubation of cells at 37 °C with rabbit anti-glycolipid mR595 and fluorescein-conjugated sheep anti-rabbit γ-globulin antisera results in the localization of fluorescence at one pole of the cell surface (capping). Binding of higher amounts of glycolipid mR595 to cells not only inhibits formation of glycolipid caps but those of the ConA receptor-fluorescent ConA complex as well. Glycolipid mR595 binding does not alter [³H]ConA binding to cells but cell agglutination by ConA is inhibited in a competitive fashion. Binding of small amounts of ConA to cells does not affect glycolipid capping. Colchicine and cytochalasin B (CB) treatment of cells inhibits glycolipid cap formation.     

Effects of trifluoperazine, a calmodulin antagonist, on rabbit T- and B-cell responses to mitogens and antigen

Trifluoperazine (TFP), an inhibitor of the calcium-binding protein, calmodulin (CaM), was used to assess the role of calmodulin in the responses of rabbit lymphoid cells to stimulation with mitogen and antigen. After binding goat anti-rabbit Fab antibody, rabbit B cells lose their surface immunoglobulin (Ig) through endocytosis and then reexpress this protein during the next 24 hr. This reexpression was markedly inhibited by TFP. The brief and early addition of TFP markedly inhibited the increased [3H]thymidine (Tdr) uptake by rabbit T cells treated with concanavalin A and B cells exposed to anti-Fab. TFP greatly inhibited the induction by keyhole limpet hemocyanin (KLH) of the in vitro syntheses of antibody, Ig, and protein by KLH-primed lymph node cells (LNC). The earlier the TFP the greater was the inhibition of induction of these syntheses. However, once induced, synthesis and secretion of antibody were not inhibited by TFP. In striking contrast to the inhibition by TFP of the mitogenic and antigenic responses of lymphoid cells was the lack of effect of this drug on resting lymphocytes. Since TFP was not cytotoxic for either resting or mitogen- or antigen-stimulated LNC, it is highly unlikely that the observed inhibitory effects of this drug were due to its cytotoxicity. We postulate that an early signal for the activation of LNC proliferation, differentiation, and the syntheses of antibody, Ig, and protein involves a calcium-CaM-mediated reaction. Based on this work and that of others, the calcium-CaM complex may mediate an interaction between the ligand-occupied surface receptor and the cytoskeleton.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling

Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G–fluorescein isothiocyanate (IgG–FITC) for flow cytometry or with anti-rabbit IgG–peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with ¹²⁵I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1–100 ng/ml LPS; however, it was reduced by approximately 30–40% after 2 h of treatment with 1 μg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.     

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA

A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)⁺RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)⁺RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

Immunoelectron-microscopy localization of abscisic acid with colloidal gold on Lowicryl-embedded tissues of Chnopodium polyspermum L.

Further study on the localization of abscisic acid (ABA) has been undertaken at the ultrastructural level in Chenopodium polyspermum L. Axillary-bud-bearing nodes on the main axis were fixed with soluble 1-(3-dimethylaminopropyl)-3 ethyl carbodiimide, then postfixed with paraformaldehyde and embedded in Lowicryl K4M at-20° C. Ultrathin sections mounted on grids were successively incubated with rabbit anti-ABA antibodies and with gold-labelled goat anti-rabbit anti-bodies (40 nm particle size). Control sections treated with preimmune rabbit serum and ABA-preabsorbed antibodies were devoid of label. The background staining was very low with this technique. Quantitative analysis of the immunolabelling showed that two main sites of ABA accumulation could be defined: first, plastids in cortical cells and vascular parenchyma cells associated with sieve elements and xylem vessels; second, the cell cytoplasm and nucleus in the axillary bud tip and in procambial strands. In vascular bundles, the cambial cells showed no immunoreactivity. These observations support the hypothesis for the cytoplasmic synthesis of ABA which is subsequently trapped in plastids as cells mature.Abbreviations ABA abscisic acid - EDC 1-(3-dimethyl-aminopropyl)-3-ethyl carbodiimide - GAR 40 goat anti-rabbit antibodies labelled with colloidal gold of particle size 40 nm - IgG immunoglobulin G     

Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) and N′-Nitrosonornicotine (NNN) by isolated rabbit lung cells

The genotoxic potentials of N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) were studied in fresh preparations of Clara cells and type II cells isolated by centrifugal elutriation and density gradient centrifugation, and macrophages from rabbit lung. The activation of the compounds to bacterial mutagens was assayed in the Salmonella mutagenicity test using strains of TA 100 and TA 1530 preincubated with test chemicals and cells placed in chambers with nucleopore membranes to separate cells and bacteria. Unscheduled DNA synthesis was measured by incorporation of [³H]-thymidine in the cells after exposure to the compounds. NHMI, NNK and NNN were not activated to bacterial mutagens by Clara cells, type II cells or macrophages, presumably because the reactive metabolites generated were not released into the incubation medium. However, NHMI and NNK increased unscheduled DNA synthesis in Clara cells, and the highest repair activity was found after incubation with NNK. The effect of NNN was only marginal. This indicates that NHHI and NNK are genotoxic in the rabbit lung and that the Clara cells are involved in the metabolic activation of these compounds.Abbreviations NHMI N-nitrosoheptamethyleneimine - NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone - NNN N-nitrosonornicotine Supported by a grant and a fellowship (R.B.) from the Royal Norwegian Council for Scientific and Industrial Research.     

Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: The endocytic activation hypothesis

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity ¹²⁵I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of ³H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6–8 h period as an obligatory step in production of “second messenger” in the action of this hormone.     

Binding of monoclonal antibody to the Epstein Barr virus (EBV)/CR2 receptor induces activation and differentiation of human B lymphocytes

A panel of B cell-specific monoclonal antibodies that identify the CR2/EBV receptor were examined for their ability to mimic the T-independent mitogenic agent, EBV, and thus activate human peripheral blood B lymphocytes. Two of four different anti-CR2/EBV monoclonal antibodies, OKB7 and AB-1, produced a 50-fold to 200-fold dose-dependent stimulation of DNA synthesis of peripheral blood mononuclear cells. One of the other monoclonal antibodies, anti-B2, had slight activity, and the other, HB-5, was completely inactive. One of the mitogenic antibodies, OKB7, which directly inhibits binding and infection of B cells by EBV in the absence of a second anti-immunoglobulin antibody, was examined in further detail. Both the intact antibody in soluble form and its pepsin-derived F(ab')2 fragment stimulated DNA synthesis of unseparated B and T lymphocytes. Peak stimulation of DNA synthesis in peripheral blood mononuclear cells occurred between 4 to 6 days. B cells were responsible for incorporation of [3H]thymidine. However, T cells were required for activation of peripheral blood mononuclear cells by OKB7. OKB7, as well as the other mitogenic monoclonal anti-EBV/CR2 receptor antibody, also induced B cells to differentiate after 6 to 10 days of culture as indicated by polyclonal Ig secretion. IgM was the predominate immunoglobulin secreted. These studies thus indicate that certain epitopes on the EBV/CR2 receptor trigger B cells to divide and differentiate. This pathway of B cell activation, in contrast to that produced by EBV, is T cell dependent.     

Early activation and cell cycle entry of resting B cells after Fab-anti-Ig treatment: role of receptor crosslinking

Comparison of the effect of goat anti-rabbit Ig (GARIg) and its monovalent fragment (Fab-GARIg) demonstrates that surface Ig (sIg) crosslinking is not necessary to effect G0 to G1 transition in rabbit peripheral blood B cells but is required for induction of DNA synthesis. Five micrograms per milliliter or more of GARIg is sufficient to induce DNA synthesis but up to 50 micrograms/ml of Fab-GARIg is not. However, the monovalent reagent induces microscopically observable cytoplasmic and nuclear changes (blast transformation) in a dose-dependent manner. These differ qualitatively and quantitatively from the morphological changes seen with comparable doses of GARIg; Fab anti-Ig produces "small blasts" whereas complete GARIg induces large blasts. The monovalent reagent, in a wide range of concentrations, is as effective as the complete antibody in modulating sIg from rabbit B cells. Fab-GARIg treatment modulates sIg in a biphasic manner. It clears the high-density sIg within 5 min, whereas the remaining low-density receptors disappear after 4 hr. Cytosolic protein kinase C levels decline equally after treatment with either Fab-GARIg or whole anti-Ig. RNA synthesis, as measured by [3H]uridine incorporation, increases for the first 12 hr in cells activated with either reagent. It declines to basal levels in Fab-GARIg stimulated cells, but a continuous increase occurs in cells stimulated with 5 and 50 micrograms/ml of complete antibody. Simultaneous addition of 50 micrograms/ml Fab-GARIg with 5 microgram/ml of GARIg causes greater RNA synthesis for 12 hr after stimulation than is caused by GARIg alone. After 12 hr the monovalent reagent has an inhibitory effect on RNA synthesis. Fluorescence-activated cell sorter analysis of acridine orange-stained cells shows that Fab anti-Ig-stimulated cells have higher RNA content than resting cells, but lower than GARIg-activated cells. These findings suggest that rabbit B cells can be activated from the G0 stage of cell cycle to G1 by monovalent anti-Ig reagents but further cell cycle progression requires maintenance signals provided by receptor crosslinking. The implications of these results for B cell activation signalling are discussed in the context of the floating receptor model.     

An avidin-biotin-peroxidase method for Fc receptors on macrophages isolated from and in sections of rat lung

Receptors for the Fc region of immunoglobulin G (Fc receptors) were detected on pulmonary macrophages by adapting an avidin-biotin-peroxidase technique to isolated cells and sections of rat lung. After incubation with soluble rabbit immunoglobulin G (IgG), surface bound IgG was identified consistently and reproducibly on glass-adherent pulmonary macrophages and on macrophages in tissue sections made from incubated lung slices. Control experiments indicated that binding was specifically mediated by surface Fc receptors. This method may be useful for identifying macrophages in intact tissues.     

Reaction of antibody to normal human hypoxanthine phosphoribosyltransferase with products of mutant genes.

Immunoglobulin produced in rabbits against normal human red cell hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8) was used to study cell lysates of individuals with deficient enzyme activity. The reaction of immunoglobulin with HPRT formed partially active insoluble and fully active soluble complexes. The insoluble complexes were separated from soluble complexes and the free enzyme by centrifugation. The soluble complexes and free enzyme were separated by electrophoresis. Hemolysates from 13 patients with the Lesch-Nyhan syndrome who have virtually total deficiency of HPRT activity and 2 patients with hyperuricemia and 2–5% of normal activity were unable to neutralize immunoglobulin and showed no evidence of cross-reacting material (CRM). In contrast, 2 other partially deficient males with 4.5 and 50% of normal actvity, and a partially deficient heterozygous female with 34% of normal activity, were CRM⁺ in this assay. The amount of CRM present in the cells of these 2 males appeared to be disproportionate to their HPRT activity. The heterozygous female contained about 30% of normal CRM which was consistent with the estimated activity provided by her normal cell population. This indicated that her abnormal cells were CRM^?. Absence of CRM in her abnormal cells was consistent with the observed lack of CRM in hemolysates of her hyperuricemic half-brother. These data indicate the presence of considerable heterogeneity in human mutation at the HPRT locus.     

Somatic hypermutation of immunoglobulin genes: lessons from proliferating cell nuclear antigenK164R mutant mice

Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.e. they can replicate across DNA lesions. The lack of proofreading activity, however, renders TLS highly mutagenic. The advantage is that B cells use mutagenic TLS to introduce somatic mutations in immunoglobulin (Ig) genes to generate high-affinity antibodies. Given the critical role of PCNA-Ub in activating TLS and the role of TLS in establishing somatic mutations in immunoglobulin genes, we analysed the mutation spectrum of somatically mutated immunoglobulin genes in B cells from PCNA^K164R knock-in mice. A 10-fold reduction in A/T mutations is associated with a compensatory increase in G/C mutations—a phenotype similar to Polη and mismatch repair-deficient B cells. Mismatch recognition, PCNA-Ub and Polη probably act within one pathway to establish the majority of mutations at template A/T. Equally relevant, the G/C mutator(s) seems largely independent of PCNA^K164 modification.