Lipoteichoic acids (LTA) released by gram-positive bacteria can spontaneously bind to mammalian cell surfaces. In the present study, erythrocytes (E) sensitized with pneumococcal LTA (LTA-E) were used as a model system to determine if LTA could render host cells susceptible to damage by autologous complement. Complement (C)-mediated lysis of LTA-E from normal rats and normal humans occurred when these cells were incubated in their respective autologous sera in vitro. In addition, when LTA-E from a C2-deficient human and from C4-deficient guinea pigs were incubated in their autologous sera, there was significant lysis in vitro, demonstrating a role for the alternative pathway. The in vivo survival of 51Cr-labeled autologous LTA-E was also studied. Only 2.9% of autologous LTA-E remained in the circulation of normal rats after 90 min. In contrast, 31.2% of autologous LTA-E remained in the circulation of rats depleted of C3. Intravascular hemolysis accounted for the clearance of LTA-E in the normal rats, whereas liver sequestration was responsible for clearance in the C3-depleted rats. These results demonstrate that LTA can render the host's cells susceptible to damage by its own complement system, establishing this as a possible mechanism of tissue damage in natural bacterial infections.
D S Hummell, J A Winkelstein
A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen EBNA-1, exhibited strong reactivity with the synovial lining cells in joint biopsies from 10 of 12 patients with rheumatoid arthritis (RA) and adherent cells eluted from these tissues. No staining of RA synovial membrane frozen tissue sections or eluted synovial-lining cells was obtained with monoclonal antibodies directed against other EBV-encoded antigens (anti-p160, anti-gp200/350) or with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses, or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies only rare reactive cells were noted. Characterization of the antigen(s) in RA synovium by the Western immunoblotting technique revealed a 62,000-molecular-weight (mol-wt) protein, in contrast to the 70,000-85,000-mol-wt EBNA-1 antigen found in EBV-transformed cells. The structural basis for the cross-reactivity of the RA synovial membrane 62,000-mol-wt protein and the EBNA-1 antigen appears to reside in the glycine-alanine rich region of these molecules. A rabbit antibody directed against a synthetic peptide (IR3-VI-2) derived from the glycine-alanine-rich region of EBNA-1 reacted with the 70,000-85,000-mol-wt EBNA-1 antigen in EBV-infected cells and with the 62,000-mol-wt molecule in RA synovial membrane extracts. Since strong antibody responses to EBNA-1 are known to exist in RA patients, these results suggest that immune responses to a cross-reactive antigen may play a role in the pathogenesis of RA.
R Fox, R Sportsman, G Rhodes, J Luka, G Pearson, J Vaughan
We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in 125I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in 125I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt 125I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that 125I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. 125I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds 125I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding.
D R Clemmons, R G Elgin, V K Han, S J Casella, A J D'Ercole, J J Van Wyk
Human T lymphocytes sensitized to Candida albicans (CA) were shown to proliferate in cultures induced with mannan, a ramified polysaccharide extracted from the cell well of CA. We presently describe that, when we used strongly labeled [3H]mannan, antigen-specific T blast cells were able to bind the labeled mannan on their membrane. The observations that irrelevant blast cells did not bind [3H]mannan, and that mannan-specific blast cells did not bind tritiated pneumococcal polysaccharide SIII, indicate the specificity of mannan binding. Mannan binding was reversible and saturable. Mannan binding on T blast cells was inhibited by preincubation with monoclonal antibodies to T3 but not to other T cell-related molecules. The characteristics of this receptor suggest its identity with the T cell receptor for antigen. The direct binding of mannan could be either due to a cross-linking of the receptor by multivalent mannan or to a recognition of mannan in association with HLA-DQ molecules, as suggested by partial blocking of mannan binding using anti-HLA-DQ monoclonal antibodies.
A Durandy, A Fischer, D Charron, C Griscelli
Recent studies from our laboratory indicate that a high concentration of platelet-derived calcium-activated cysteine protease (calpain) can cleave high molecular weight kininogen (HMWK). On immunodiffusion and immunoblot, antiserum directed to the heavy chain of HMWK showed immunochemical identity with alpha-cysteine protease inhibitor--a major plasma inhibitor of tissue calpains. Studies were then initiated to determine whether purified or plasma HMWK was also an inhibitor of platelet calpain. Purified alpha-cysteine protease inhibitor, alpha-2-macroglobulin, as well as purified heavy chain of HMWK or HMWK itself inhibited purified platelet calpain. Kinetic analysis revealed that HMWK inhibited platelet calpain noncompetitively (Ki approximately equal to 5 nM). Incubation of platelet calpain with HMWK, alpha-2-macroglobulin, purified heavy chain of HMWK, or purified alpha-cysteine protease inhibitor under similar conditions resulted in an IC50 of 36, 500, 700, and 1,700 nM, respectively. The contribution of these proteins in plasma towards the inhibition of platelet calpain was investigated next. Normal plasma contained a protein that conferred a five to sixfold greater IC50 of purified platelet calpain than plasma deficient in either HMWK or total kininogen. Reconstitution of total kininogen deficient plasma with purified HMWK to normal levels (0.67 microM) completely corrected the subnormal inhibitory activity. However, reconstitution of HMWK deficient plasma to normal levels of low molecular weight kininogen (2.4 microM) did not fully correct the subnormal calpain inhibitory capacity of this plasma. These studies indicate that HMWK is a potent inhibitor as well as a substrate of platelet calpain and that the plasma and cellular kininogens may function as regulators of cytosolic, calcium-activated cysteine proteases.
A H Schmaier, H Bradford, L D Silver, A Farber, C F Scott, D Schutsky, R W Colman
We explored the effects of alterations in extracellular and intracellular calcium concentration on arginine vasopressin (AVP)-stimulated cAMP formation in cultured rat inner medullary collecting tubule cells. cAMP formation remains constant at extracellular calcium concentrations between 0.5 and 4.0 mM, which did not change intracellular calcium. Maneuvers that alter intracellular calcium concentration are associated with marked changes in cAMP generation. EGTA decreases intracellular calcium and enhances AVP-stimulated cAMP formation, while increasing cellular calcium with 2 microM A23187 decreases AVP-stimulated cAMP formation in the presence, but not in the absence, of extracellular calcium. The changes in cAMP formation observed when intracellular calcium is altered are associated with reciprocal changes in prostaglandin E2 (PGE2) synthesis. Despite greater than 95% inhibition of PGE2 synthesis with 5 microM meclofenamic acid, the changes in cAMP formation accompanying alterations in intracellular calcium concentration are still evident. These studies suggest that intracellular calcium critically influences AVP-stimulated cAMP formation. It does so by a mechanism independent of PG that is probably mediated by a direct effect of the cation on the adenylate cyclase complex.
I Teitelbaum, T Berl
The occurrence, nature, and pathogenesis of intestinal lesions were studied in a number of graft vs. host reaction (GVHR) conditions in mice, combining variations in the nature of the following: the F1 hosts (newborn or adult, normal or lethally irradiated), the injected parental T cells (mixed or selected subsets of Lyt2+ or L3T4+ cells), and the antigenic stimulus (semi-allogeneic or restricted to class I or II MHC loci). The following conclusions were drawn: Three gut alterations are always associated: donor T cell infiltration, predominating in the crypt region; acceleration of the epithelium renewal; and increased epithelial Ia expression. The initial event is T-cell infiltration, which results from stimulation within the Peyer patches followed by cyclic traffic, i.e., migration into the thoracic duct and then seeding to the whole gut mucosa. Both Lyt2+ and L3T4+ cells can infiltrate the gut wall, the extent of the infiltration by a given subset depending upon the capacity of the donor blasts to circulate in the thoracic duct (higher for L3T4+) and then to home in the gut (much higher for Lyt2+ blasts) and the nature of the alloantigenic stimulation that governs the extent of each donor subset proliferation. Both donor T-cell subsets can induce gut epithelial damage, but for a comparable amount of infiltrating cells, L3T4+ cells induce more lesions. When the antigenic stimulation is restricted to class I or class I MHC loci, gut GVHR is much more easily elicited across class II MHC differences, which stimulate preferentially L3T4+ donor cells. The main mechanism of epithelial damage is not direct cytotoxicity, but more probably lymphokine(s) release.
D Guy-Grand, P Vassalli
The effects of interferons (IFNs) on lymphocyte adhesion to cultured human vascular endothelial cells (EC) were investigated using an in vitro assay. Endothelial cells obtained from umbilical vein were first cultured at a low density with a conditioned medium (CM) from 12-O-tetra decanoylphorbol 13-acetate-concanavalin A (TPA-Con A) stimulated human peripheral blood lymphocytes (PBL), or with recombinant (r) gamma interferon (IFN-gamma) or r alpha interferon (IFN-alpha), and then were incubated with freshly isolated PBL. Natural IFN-gamma in the TPA-Con A CM and rIFN-gamma (12.5-500 U/ml) induced major histocompatibility complex-class II antigens (HLA-DR, HLA-DP, and HLA-DQ) and significant lymphocyte adhesion to the EC, whereas rIFN-alpha did not. The lymphocyte adhesion to the EC and the expression of DR antigens on the EC were well correlated in terms of both kinetics and the dose-response pattern of rIFN-gamma. When EC expressing I region associated (Ia) antigen were preincubated with monoclonal anti-DR antibody before the addition of lymphocytes, the lymphocyte adhesion was significantly inhibited in both allogeneic and syngeneic combinations, whereas anti-HLA-DP, anti-HLA-DQ, and anti-HLA-ABC antibodies did not inhibit the binding at all. Cell fractionation experiments indicated that the majority of lymphocytes adhering to Ia-expressed EC were Leu-3+ T cells, whose binding was again almost completely inhibited by anti-DR antibody. Moreover, anti-Leu-3a, but not anti-Leu-2a, antibody effectively inhibited the T cell adhesion to the EC. These results strongly suggest that the interaction of the Leu-3(T4) receptor of T cells with IFN-gamma-induced DR antigens on EC plays a central role in the selective adhesion of Leu-3+ T cell to EC.
J Masuyama, N Minato, S Kano
To determine the incidence of elevated levels of serum methylmalonic acid in patients with cobalamin deficiency, we utilized a new capillary gas chromatographic-mass spectrometric technique to measure methylmalonic acid in the serum of 73 patients with clinically confirmed cobalamin deficiency. Values ranged from 55 to 22,300 ng/ml, and 69 of the 73 patients had values above the normal range of 19-76 ng/ml as determined for 50 normal blood donors. In the cobalamin-deficient patients, serum methylmalonic acid was significantly correlated with the serum folate level and the degree of neurologic involvement. Some patients with pernicious anemia who were intermittently treated with cyanocobalamin were found to have elevated serum levels of methylmalonic acid while free of hematologic and neurologic abnormalities. A cobalamin-deficient patient is described with a normal serum cobalamin and an elevated serum methylmalonic acid. We conclude that the ability to measure methylmalonic acid in human serum will be useful in studies designed to determine the incidence of cobalamin deficiency in various patient populations.
S P Stabler, P D Marcell, E R Podell, R H Allen, J Lindenbaum
We previously demonstrated that human embryonic mesenchymal cells derived from the palate (HEMP cells) retain alkaline phosphatase (ALP) content and capacity for collagen synthesis after long-term culture, and their growth is markedly stimulated by epidermal growth factor (EGF). There was a dramatic decrease in ALP content and capacity to synthesize collagen in HEMP cells (HEMP-RV cells) persistently infected with rubella virus (RV). EGF increased ALP activity and decreased collagen synthesis in HEMP cells, whereas EGF showed no effect on these activities in HEMP-RV cells. Growth of HEMP-RV cells was slightly reduced compared with that of HEMP cells. EGF stimulated growth of HEMP cells and to a lesser extent of HEMP-RV cells. Binding of 125I-EGF to cell-surface receptors in HEMP-RV cells was, to our surprise, twice as much as that in HEMP cells. However, internalization of bound 125I-EGF in HEMP-RV cells was profoundly diminished. Thus, persistent RV infection causes not only changes in HEMP cell growth and differentiation but a decrease in or loss of HEMP cell responsiveness to EGF. The effects of persistent RV infection on palatal cell differentiation as well as growth may be responsible for the pathogenesis of congenital rubella. Furthermore, since HEMP cells appear to be closely related to osteoblasts, these results suggest a mechanism for RV-induced osseous abnormalities manifested in congenital rubella patients.
T Yoneda, M Urade, M Sakuda, T Miyazaki