C1 inhibitor hinge region mutations produce dysfunction by different mechanisms.

Heterozygosity for a mutant dysfunctional C1 inhibitor protein, a member of the serine proteinase inhibitor (serpin) superfamily, results in type II hereditary angioneurotic oedema. We identified a "hinge" region mutation in C1 inhibitor with a Val to Glu replacement at P14 Val-432. Recombinant C1 inhibitors P10 Ala-->Thr and P14Val-->Glu did not form stable complexes with fluid phase C1s or kallikrein. The P14 Val-->Glu mutant, however, was cleaved to a 96K form by C1s, while the P10 Ala-->Thr mutant was not. The recombinant P10 mutant also did not complex with C1s, kallikrein or beta-factor Xlla-Sepharose. The two mutations, therefore, result in dysfunction by different mechanisms: in one (P14 Val-->Glu), the inhibitor is converted to a substrate, while in the other (P10 Ala-->Thr), interaction with target protease is blocked.

N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility.

The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

Identification of a subpopulation of lymphocytes in human peripheral blood cytotoxic to autologous fibroblasts.

A naturally occurring subpopulation of human peripheral blood lymphocytes is cytotoxic to autologous and/or allogeneic fibroblasts. The autocytotoxic lymphocytes have a receptor for the third component of complement and for aggregated gamma globulin, do not form rosettes with sheep red blood cells, and are not removed by passage through nylon. The autocytotoxic subpopulation is not present in the thymus and tonsils of normal children or in the peripheral blood of individuals with X-linked agammaglobulinemia. Fibroblast absorption experiments demonstrate that the autocytotoxic cells are "sensitized" to antigens expressed on allogeneic fibroblasts in addition to the antigens expressed on autologous cells. Some normal individuals have a second subpopulation of lymphocytes that may "regulate" the autocytotoxic cells. The relevance of these observations to the murine autocytotoxic cells is discussed.

Capping and adenosine metabolism. Genetic and pharmacologic studies.

Capping of membrane Ig was studied in lymphocytes treated with agents that interfere with adenosine metabolism. Treatment of murine or human B cells with combinations of coformycin, an inhibitor of adenosine deaminase, homocysteine, and adenosine impaired Ig capping. Inhibition of capping was also produced by 3-deazaadenosine, a specific inhibitor of adenosylhomocysteine hydrolase. The inhibitors did not affect capping of the Thy-1 antigen or membrane sites reactive with antilymphocyte antibodies. Two patients with a hereditary deficiency in adenosine deaminase had impairment of Ig capping. Such an impairment was not found in lymphocytes of two other patients who had undergone successful bone marrow transplantation. It is known that the addition of a calcium ionophore results in activation of microfilament function and in disruption of Ig caps. The ionophore effect was not inhibited by the agents mentioned above. Our results suggest that the inhibition of Ig capping during aberrant adenosine metabolism may be caused by a methylation defect preceding the contracticle event that produces membrane reorganization.

Induction of human immunoglobulin synthesis and secretion in somatic cell hybrids of mouse myeloma and human B lymphocytes from patients with agammaglobulinemia.

Somatic cell hybrid clones were isolated from the fusion of RPC 5,4 mouse myeloma cells and B lymphocytes from three patients with agammaglobulinemia. One patient had X-linked agammaglobulinemia; the remaining two patients had common varied agammaglobulinemia. All three patients had B lymphocytes which fail to secrete immunoglobulin. The hybrid nature of the clones was established by examination of metaphase chromosome spreads. Most of the clones from all three patients expressed surface immunoglobulin of mouse and human parental origin. Clones from two of the patients had fewer cells with surface Ig than hybrids from normal persons, while clones from the third patient had large numbers of surface Ig fluorescent cells. Most of the clones from all three patients synthesized and secreted human and mouse immunoglobulin. As determined by sodium dodecyl sulfate acrylamide gel electrophoresis of radioactively labeled proteins, clones from each of the patients produced human gamma, alpha, and mu-heavy chains. These studies demonstrate the presence of functional structural genes coding for human immunoglobulin heavy chains in B lymphocytes of patients with agammaglobulinemia. Further, they represent induction in the somatic cell hybrids of a gene product not expressed in the parental B lymphocytes.

The mechanism of action of the C3b inactivator (conglutinogen-activating factor) on its naturally occurring substrate, the major fragment of the third component of complement (C3b).

The fixation of the third component of complement (C3) results in many important biological phenomenon, among which are (a) immune adherence (1), (b) enhancement of phagocytosis (2,3), (c) the release of an anaphylatoxin which is a potent releaser of histamine (4), and (d) the feedback activation of the alternative pathway (5,6). The physiological mechanisms involving C3 fixation require the generation of a C3 convertase which may occur by two separate pathways. C3 convertase can be generated, in the form of C42, by the so-called classical pathway of activation or in the form C3b,B by the alternative or properdin pathway (7). In both cases, C3 is converted to C3b by cleavage of a small peptide, C3a. Normal human serum contains an inactivator of activated C3b. This C2b inactivator or conglutinogen-activating factor (KAF) has been shown to inhibit both immune hemolysis and the immune adherence properties of C3b and to cause cleavage of C3b in the fixed and fluid- phase stages (8-11). Although it is known that the C3b inactivator is not depleted during its reaction with C3b and that C3b treated with the C3b inactivator becomes extremely sensitive to proteolytic digestion by trypsin and "trypsin-like" enzymes (9), the exact molecular nature of the action of the C3b inactivator on C3b has not been studied. In an effort to delineate the products of this interaction, purified C3b and C3b inactivator were allowed to react for various specific lengths of time and the products of these reactions were then analyzed.

Inhibition of maturation of human precursor lymphocytes by coformycin, an inhibitor of the enzyme adenosine deaminase.

High concentrations of adenosine are known to be toxic to fibroblasts and lymphocytes under conditions of in vitro culture (1,2). Normally, accumulation of adenosine nucleotides in all mammalian cells is prevented by the presence of adenosine deaminase, an aminohydrolase which converts adenosine to inosine (3). A genetically determined deficiency of adenosine deaminase has been associated with the autosomal recessive form of severe combined immunodeficiency, a syndrome in which precursor lymphocytes fail to mature into T cells and B cells (4-7). Erythrocytes of affected infants convert exogenous adenosine to AMP and ATP at an abnormally increased rate as a consequence of the enzyme defect, and ATP at an abnormally increased rate as a consequence of the enzyme defect, and fail to form inosine from the exogenous adenosine (8). These metabolic disturbances can be mimicked in normal erythrocytes by coformycin (8), a potent competitive inhibitor of adenosine deaminase (9, 10). In this study, the effects of coformycin were examined on the in vitro function of normal lymphocytes.

A natural model of immunologic tolerance. Tolerance to murine C5 is mediated by T cells, and antigen is required to maintain unresponsiveness.

A unique experimental model is described, where natural immunologic tolerance to a well-defined soluble native antigen (murine C5) is examined in congenic strains of mice that differ only by the presence or the absence of C5. A highly sensitive hemolytic assay was developed to detect nanogram amounts of C5 as well as an assay of anti-C5 inhibition of C5 hemolytic activity. The latter was more sensitive than immunodiffusion. Two reciprocal approaches were used to study the cellular basis of tolerance in irradiated hosts of either strain. In the first, lymphoid cells from either strain were transferred to irradiated B10.D2OSN hosts that were lacking C5 and so would not hinder detection of anti-C5 antibody upon challenge with murine C5. Second, lymphoid cells from either strain were transferred to irradiated B10.D2NSN hosts, whose native C5 provided the antigenic stimulus. The immune response of whole nonadherent spleen cell suspension as well as mixtures of T and B cells (separated on the basis of surface immunoglobulin) from either strain were studied. In addition, the duration of tolerance and the antigen requirement to maintain it in irradiated C5-deficient hosts repopulated with C5-sufficient spleen cells was examined. The positive control of irradiated C5-deficient hosts repopulated with syngeneic spleen cells showed a primary and secondary response to immunization. In contrast, C5-sufficient spleen cells failed to respond both in the primary and the secondary response. Because the unresponsiveness was not caused by antigen carryover and was not antigen specific, it represents central tolerance. In C5-sufficient irradiated hosts (where immunization was not required and antigen was present in natural form and physiological concentration), transfer of C5-deficient cells mediated a drop in C5 levels to 10-20% of that noted in unreconstituted controls. T and B cell mixing experiments from the two strains into deficient or sufficient hosts demonstrated that tolerance is T cell dependent and that C5-sufficient or -deficient B cells could cooperate with nontolerant C5-sufficient T cells to produce significant anti-C5 antibody or mediate a significant drop in C5 levels. In addition, the presence of antigen was necessary to maintain tolerance. In conclusion, these results show that (a) natural tolerance to C5 is an active process that is T cell dependent and requires the presence of antigen; (b) in this natural model, clonal abortion does not seem to occur; and (c) both tolerant and nontolerant B cells retain the capacity to produce autoantibody.

Interaction of human thymus-derived and non-thymus-derived lymphocytes in vitro. Induction of proliferation and antibody synthesis in B lymphocytes by a soluble factor released from antigen-stimulated T lymphocytes.

Relatively pure populations of human T and B lymphocytes were obtained from blood and tonsils using density gradient centrifugation in bovine serum albumin. Antigen alone was incapable of triggering the B lymphocyte into blast transformation or to secrete antibody. However, supernatants from tetanus toxoid-stimulated T cells obtained from immune donors contained a factor mitogenic for B lymphocytes. 50-60% of B cells responded to this lymphocyte mitogenic factor (LMF) by proliferation, loss of C3 reactivity, and change to a secretory state. LMF-stimulated B cells exhibited a three- to fivefold increase in protein secretion and a six- to eightfold increase in gamma G globulin secretion. De novo secreted IgG had specificity directed to the tetanus toxoid present in the LMF containing T-cell supernatants. This was confirmed by an increase in the number of indirect plaque-forming cells to tetanus toxoid-coated sheep red blood cells after stimulation of B cells with LMF. It is proposed that in the course of the response to a previously encountered protein antigen, sensitized human T cells emit a signal in the form of a soluble product that, together with antigen, triggers B cells into division and antibody secretion. The experimental model utilized can be adapted to study human T-B cell cooperation under various conditions in normal individuals and in individuals with immunodeficiency diseases.

Serum-dependent phagocytosis of paraffin oil emulsified with bacterial lipopolysaccharide.

Paraffin oil containing oil red O and emulsified with lipopolysaccharide obtained from Escherichia coli was ingested rapidly by guinea pig polymorphonuclear leukocytes or human peripheral blood granulocytes and monocytes after opsonization by fresh homologous serum. The initial rate of engulfment of the particles was spectrophotometrically assayed by determination of cell-associated oil red O and reflected the opsonic activity of the serum. This activity was resistant to dialysis but labile to heat, hydrazine, and zymosan, required divalent cations, and was maximal in the presence of Ca(++) and Mg(++). It was associated with the fixation of [(125)I]C3 to the lipopolysaccharide particles. Genetically C3-deficient serum had no opsonic activity, and this activity was restored by the addition of purified C3. Normal and C4-deficient guinea pig serum and normal, C2-, and C4-deficient human sera were equally effective in opsonizing lipopolysaccharide particles and lipopolysaccharide particles sensitized with heat-inactivated lipopolysaccharide immune serum. Cord serum deficient in glycine-rich beta-glycoprotein (GBG) (properdin factor B) had diminished opsonic activity which was improved by addition of purified GBG. Thus, C3 fixation to lipopolysaccharide particles occurs by means of the properdin system, and the opsonization and ingestion of lipopolysaccharide particles constitutes a quantitative functional assay of this pathway.

The interaction between human monocytes and red cells. Specificity for IgG subclasses and IgG fragments.

Studies were carried out in order to characterize the specificity of IgG sub-classes and IgG fragments for rosette formation using red cells and human mononuclear cells. Rosette formation of red cells coated with anti-D was inhibited by free IgG(1) and IgG(3); less inhibition occurred with IgG(2) and IgG(4). Red cells specifically coated with IgG(1) and IgG(3) by chromic chloride were bound to monocytes. Rosette inhibition of anti-D-coated red cells occurred with free Fc fragment of IgG globulin, and only partly with F(ab')(2). Inhibitory capacity of Fc fragments of IgG and gamma(1) heavy chain from heavy chain disease was reversed by the cleavage of disulfide bonds. No inhibition was noted with Fab, or with pepsin components II, III, or IV. These studies indicated that the mononuclear receptor was specific for IgG(1) and IgG(3). The peptide portion of IgG globulin which attached to the mononuclear cell appeared to reside in the N-terminal portion of the Fc fragment and also appeared to require the integrity of the inter-heavy chain disulfide bond. A specific receptor for C3 was not confirmed.

The opsonic fragment of the third component of human complement (C3).

Human peripheral blood phagocytes ingest Escherichia coli 026:B6 lipopolysaccharide (LPS)-coated paraffin oil droplets containing Oil red O only if fresh serum deposits C3 on the surfaces of the particles (opsonizes them), by reactions involving the properdin system. The rate of binding of purified [125-I]C3 in serum to LPS-coated particles correlated precisely with the rate of acquisition of ingestibility assayed spectrophotometrically. Once opsonized, LPS-coated particles remained fully ingestible and retained fixed [125-I]C3 radioactivity even after exposure to extremes of temperature, pH, ionic strength, phospholipases, urea or guanidine, some nonionic and ionic detergents, and organic solvents. Trypsin, human conglutinogen-activating factor, another heat-stable activity found in human serum, and sodium dodecyl sulfate removed radioactivity and diminished ingestibility of the opsonized particles. Alkylation, reduction plus alkylation and F(ab')2 from anti-C3 blocked ingestibility but did not alter particle-bound radioactivitymelectrophoretic and tryptic peptide autoradiographic analysis of dodecyl sulfate eluates of opsonized particles, cleansed of many contaminating proteins by boiling with 2 M NaCl (yet still opsonized), revealed that the polypeptide with C3-derived radioactivity had a mol wt of approximately 140,000 and was composed of 70,000 mol wt subunits linked by disulfide bonds. Immunochemical analysis and comparison of the peptide structure of the eluate with that of C3 indicated that the opsonic fragment is not the fragment defined as C3b but a smaller derivative of C3.

T cell lines characterize events in the pathogenesis of the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a severe immunodeficiency and platelet deficiency disease arising from an X-linked defect. The disease is correctable by transplantation of hematopoietic stem cells, but the product of the defective gene is unidentified and the number of defects in patient blood cells is large. The current hurdle is the need to identify the early pathogenic event(s) that are the cause of other defects. As a step toward this goal, we have generated and examined a panel of interleukin 2-dependent allospecific T cell lines from peripheral lymphocytes of seven WAS patients and five normal individuals. WAS cell lines, like normal lines, undergo vigorous proliferation when challenged with specific allostimulant or with phorbol myristate acetate and ionomycin. Both normal and WAS T cell lines express cell surface molecules CD2, CD3, T cell receptor-alpha/beta, human histocompatibility leukocyte antigen class I, CD45 and CD11a, and varying ratios of CD4 and CD8, and are negative for natural killer cell and monocyte surface molecules. WAS T cell lines express CD43 (sialophorin/leukosialin) with molecular weight and in an amount comparable with normal T cell lines. WAS T cell lines thus do not express defects in CD43 (decreased amount, abnormal molecular weight), previously documented in WAS circulating lymphocytes. On the other hand, as detected by scanning electron microscopy, WAS cell lines exhibit severe morphological abnormalities, including decreased size and density of the microvillus surface projections. The morphological abnormalities of WAS T cell lines are similar to, or more extensive than, those previously reported for WAS peripheral lymphocytes, indicating that the generation of morphological (cytoarchitectural) defects is an early pathogenic event in this disease. The findings suggest that the gene that is defective in the WAS encodes a protein that normally functions to maintain or regulate the cytoskeletal structure of blood cells.

Structural differences between the two human complement C4 isotypes affect the humoral immune response.

An animal model has been used to address the question of the biological importance of the known structural difference between the two isotypes of human C4, i.e., C4A and C4B. Guinea pigs deficient in C4 were reconstituted transiently with either human C4A or C4B protein and immunized with the bacteriophage phi X174. Results from this study showed that C4A-reconstituted animals made a secondary response, i.e., switch from IgM to IgG; whereas the C4B-reconstituted animals did not.

The enhancement of bacterial phagocytosis by serum. The role of complement components and two cofactors.

The role of serum factors in the phagocytosis of pneumococci was studied employing a spectrophotometric assay which measures reduced nitro blue tetrazolium (NBT) dye. Dye reduction occurs within the phagocyte shortly after bacterial ingestion as measured by the phagocytic index technique and by the uptake of (125)I-pneumococci. Bacteria prepared with gammaG antibody were not phagocytosed unless a small volume of fresh normal serum was added. Using fresh sera deficient in single complement components, it was demonstrated that the first four components are necessary for optimal bacterial phagocytosis. When highly purified complement components were added to the antibody-coated pneumococci, enhancement of phagocytosis was achieved only with the sequential addition of C1, C4, C2, and C3. Evidence has been presented that human C3 bound to an immune complex exhibits peptidase activity and that this activity is essential for phagocytosis. A heat-labile, dialyzable serum cofactor which enhances C3 peptidase activity enhanced the phagocytosis of pneumococci prepared with purified complement components. A second phagocytosis-promoting cofactor, which is not a complement component, was found to be a heat-labile, 5-6S, beta pseudoglobulin. This protein may stabilize C3 peptidase activity or inhibit enzymatic inactivation of C3.

Characterization of a human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome.

gpL115 is a lymphocyte surface component that is deficient in patients with the X-chromosome-linked immune deficiency Wiskott-Aldrich syndrome (6). The glycoprotein nature of gpL115 is demonstrated through labeling in carbohydrate moieties by [3H]NaBH4 and its synthesis by lymphocytes through labeling with [35S]methionine. Native gpL115 adheres to wheat germ lectin-Sepharose and sialidase-treated gpL115 does not adhere, indicating that native gpL115 adheres via clusters of sialic acid residues. When tested on peanut lectin, which shows specificity for the disaccharide Gal beta 1-3GalNAc, gpL115 is nonadherent and sialidase-treated gpL115 is adherent, indicating the presence of the sequence sialic acid-Gal beta 1-3GalNAc, which is characteristic for O-linked (mucin-type, acidic-type) carbohydrates. A surface glycoprotein with all the above characteristics was found on the lymphoblastoid cell line CEM. CEM cells were used as immunogen to generate the monoclonal antibody L10, an IgG1, which binds native and sialidase-treated gpL115 . Sialidase-treatment of gpL115 significantly alters its physical properties, reducing its electrophoretic mobility and changing its behavior on isoelectrofocusing. Cumulatively, these findings indicate that gpL115 , like glycophorin of erythrocytes and GPIb of platelets, is a sialoglyco protein with significant quantities of O-linked carbohydrate. On treatment with limiting sialidase concentrations, gpL115 of normal lymphocytes is transformed into a series of partially desialylated species of decreasing electrophoretic mobility. This finding resembles the situation with lymphocytes of some Wiskott-Aldrich syndrome patients. Lymphocytes of eight Wiskott-Aldrich syndrome patients were found to be deficient in 125I-labeled gpL115 . Lymphocytes from three of these patients displayed an abnormal 125I-component of apparent mol wt 135,000.

Angioedema induced by a peptide derived from complement component C2.

Synthetic peptides that correspond to the COOH-terminal portion of C2b enhance vascular permeability in human and guinea pig skin. In human studies, 1 nmol of the most active peptide of 25-amino acid residues produced substantial local edema. A pentapeptide and a heptapeptide corresponding to the COOH-terminal sequence of C2b each induced contraction of estrous rat uterus in the micromole range; a peptide of 25 amino acids from this region induced a like contraction of rat uterus at a concentration 20-fold lower than the smaller peptides. The vascular permeability of guinea pig skin was enhanced by doses of these synthetic peptides in a similar fashion as that observed for the concentration of rat uterus. The induction of localized edema by intradermal injection in both the guinea pig and the human proceeds in the presence of antihistaminic drugs, suggesting that there is a histamine-independent component to the observed increase in vascular permeability. Cleavage of C2 with the enzymic subcomponent of C1, C1s, yields only C2a and C2b, and no small peptides, whereas cleavage of C2 with C1s and plasmin yields a set of small peptides. These plasmin-cleaved peptides are derived from the COOH terminus of C2b, and they induce the contraction of estrous rat uterus.

Sialophorin, a surface sialoglycoprotein defective in the Wiskott-Aldrich syndrome, is involved in human T lymphocyte proliferation.

The mAb L10 was used to determine the distribution and the function of sialophorin, the heavily glycosylated surface molecule that is deficient/defective in lymphocytes of patients with the X-linked immunodeficiency Wiskott-Aldrich syndrome. Dual-parameter FACS analysis indicated that sialophorin is expressed on CD4+ and CD8+ lymphocytes, on a subpopulation of peripheral blood B lymphocytes, on all thymocytes, and on a subpopulation of bone marrow cells. Functional studies demonstrated that L10 mAb stimulates the proliferation of peripheral blood T lymphocytes as measured by stimulation of [3H]thymidine incorporation. The time course and magnitude of increased [3H]thymidine incorporation by T lymphocytes in response to L10 mAb paralleled that induced by anti-CD3 mAb. Effective stimulation was dependent on the presence of monocytes and the Fc portion of L10 mAb. Stimulation of lymphocytes by L10, like stimulation by anti-CD3 mAb, involves increased expression of 4F2, HLA-DR, and IL-2-R. These observations suggest that sialophorin functions in T cell activation.

Beta-1C-globulin: metabolism in glomerulonephritis.

The metabolism of beta(1C)-globulin labeled with iodine-131 was studied in six normal individuals and in three individuals with glomerulonephritis who exhibited markedly reduced serum concentrations of this protein. Fractional of serum beta(1C)-globulin in glomerulonephritis appears to be chiefly secondary to decreased synthesis.

C1 inhibitor: evidence for decreased hepatic synthesis in hereditary angioneurotic edema.

Although the Cl inhibitor was detected in 5 to 10 percent of normal hepatic parenchymal cells by means of the immunofluorescent technique, none was seen in liver biopsies from two individuals with hereditary angioneurotic edema having low concentrations of Cl inhibitor in the serum. In contrast, the percentages of cells which reacted with fluorescent antiserums to C4 and transferrin were normal. These data suggest that in most subjects with hereditary angioneurotic edema, there is decreased synthesis of the C1 inhibitor but normal synthesis of C4, and that the disease results from this biosynthetic error.