The Journal of Experimental Medicine

Peterson, K M; Alderete, J F

doi: 10.1084/jem.160.5.1261pmid: 6333482

Human plasma low density lipoprotein uptake by the urogenital pathogen, Trichomonas vaginalis, was examined. Rapid binding and internalization of 125I-labeled low density lipoproteins by live T. vaginalis was observed at 37 degrees C. Data showing parasite degradation of the internalized apoproteins and lipid accumulation following low density lipoprotein uptake was obtained. Acquisition of low density lipoproteins was by a trichomonad surface protein that possessed a molecular weight of greater than 250,000. The receptor is specific for apolipoprotein CIII, a component of high, low, and very low density lipoprotein subfractions. Low density lipoproteins in a semi-defined medium of trypticase, nucleic acid precursors, vitamins, and maltose promoted T. vaginalis growth and multiplication at rates and levels equal to the yeast extract-trypticase-serum complex medium routinely used for culture of trichomonads. HeLa cell membranes as a source of lipids were unable to sustain T. vaginalis organisms. These data demonstrate host lipoprotein internalization by T. vaginalis via a specific uptake mechanism.

van Furth, R; Diesselhoff-den Dulk, M M

doi: 10.1084/jem.160.5.1273pmid: 6491600

The present study concerns the isolation, characterization, origin, and kinetics of spleen macrophages. The spleen was first perfused in situ to remove monocytes from the vascular bed and then dissected and treated with collagenase. The macrophages in the cell suspension thus obtained were characterized morphologically and cytochemically and then quantitated. The spleen cell suspension was incubated for 24 h in Leighton tubes to obtain an enriched glass-adherent population of macrophages for characterization and 3Hthymidine-labeling studies. Almost all of the adhering macrophages were esterase positive, had Fc and C3b receptors, and ingested EIgG and opsonized bacteria. In vitro labeling with 3Hthymidine showed that approximately 5% of the mononuclear phagocytes in the spleen synthesize DNA and must be considered to be dividing cells. The course of the number of labeled monocytes and macrophages after a single injection of 3Hthymidine indicates migration of monocytes into the spleen, where they become macrophages. Calculation of the influx of monocytes into the spleen and of the local production of macrophages by DNA-synthesizing mononuclear phagocytes showed that under steady-state conditions, 55% of the population of spleen macrophages is supplied by monocyte influx and 45% by local production. This means that there is a dual origin of spleen macrophages. The mean turnover time calculated with the value for the efflux of spleen macrophages is 6.0 d.

Weiss, A; Stobo, J D

doi: 10.1084/jem.160.5.1284pmid: 6208306

The association between T3 and the T cell antigen receptor was examined using the T3 bearing T cell leukemic line Jurkat. A monoclonal antibody, C305, was produced, which reacted with idiotypic-like determinants expressed on Jurkat. The molecule with which this antibody reacted was a disulfide-linked heterodimer of 90 kD, composed of polypeptides of 42 and 54 kD. Thus, C305 reacted with a molecule with characteristics of the putative T cell antigen receptor described by others. A series of mutants of Jurkat, induced with ethyl methane sulfonate or radiation, was selected for T3 or antigen receptor negativity. In every instance, there was a concomitant loss of both T3 and the antigen receptor as assessed by quantitative absorption, indirect immunofluorescence, and antibody plus complement-mediated cytotoxicity. The absence of antigen receptor molecules was confirmed on diagonal gels, excluding the possibility that conformational changes of the antigen receptor on such T3-negative mutants were responsible for the failure of such mutants to react with C305. Moreover, in a mutant that expressed a marked decrease in the level of T3 expression, there was a comparable decrease in the expression of antigen receptor determinants. These results suggest that there is an obligate requirement for the coexpression of T3 and the T cell antigen receptor. Furthermore, attempts to activate such mutants with the lectin phytohemagglutinin suggested that the expression of T3 and/or the antigen receptor was required for activation of these cells.

Waters, S J; Luzzatti, P R; Bona, C A

doi: 10.1084/jem.160.5.1300pmid: 6208307

Four keyhole limpet hemocyanin (KLH)-specific clones prepared from the lymph node of CB6F1 mice immunized with KLH had a proliferative response restricted to parental major histocompatibility complex (MHC)-encoded antigens. These clones provided help for CB6F1 trinitrophenyl-ovalbumin (TNP-OVA)-primed B cells to mount IgM and IgG plaque-forming cell (PFC) responses in the presence of KLH-TNP conjugate. In addition, two of these clones (A12.11 and F6) proliferated in response to allogeneic cells from mice strains bearing H-2k or H-2q haplotypes, respectively. However, they did not provide help for C3H/He or B10.Q primed B cells. The clonal nature of A12.11 and F6 was demonstrated by subcloning and in BUdR-suicide experiments. The proliferative response to KLH was ablated by anti-Iad antibodies, whereas the proliferation induced by C3H/HeJ stimulating cells was ablated by anti-Iak antibodies. Furthermore, both responses were inhibited by a monoclonal anti-clonotype (idiotype) antibody. Taken together, these results strongly support the hypothesis that the same receptor recognizes alloantigens and KLH associated with self-antigens.

Norcross, M A; Bentley, D M; Margulies, D H; Germain, R N

doi: 10.1084/jem.160.5.1316pmid: 6436430

To study the relationship between the structure and function of Ia antigens, as well as the physiologic requirements for antigen presentation to major histocompatibility complex-restricted T cells, class II A alpha and A beta genes from the k and d haplotypes were transfected into Ltk- fibroblasts using the calcium phosphate coprecipitation technique. Individually transfected genes were actively transcribed in the L cells without covalent linkage to, or cotransformation with, viral enhancer sequences. However, cell surface expression of detectable I-A required the presence of transfected A alpha dA beta d or A alpha kA beta k pairs in a single cell. The level of I-A expression under these conditions was 1/5-1/10 that of Ia+ B lymphoma cells, or B lymphoma cells expressing transfected class II genes. These I-A-expressing transfectants were tested for accessory cell function and shown to present polypeptide and complex protein antigens to T cell clones and hybridomas in the context of the transfected gene products. One T cell clone, restricted to I-Ak plus GAT (L-glutamic acid60-L-alanine30-L-tyrosine10), had a profound cytotoxic effect on I-Ak- but not I-Ad-expressing transfectants in the presence of specific antigen. Assays of unprimed T cells showed that both Ia+ and Ia- L cells could serve as accessory cells for concanavalin A-induced proliferative responses. These data indicate that L cells can transcribe, translate, and express transfected class II genes and that such I-A-bearing L cells possess the necessary metabolic mechanisms for presenting these antigens to T lymphocytes in the context of their I-A molecules.

Parmely, M J; Iglewski, B H; Horvat, R T

doi: 10.1084/jem.160.5.1338pmid: 6208308

To aid in understanding the role of cellular immunity in limiting Pseudomonas aeruginosa infections, we have identified some of the principal antigens of the organism that are recognized by human T cells. Clones of T cells were selected in such a manner that they would provide information not only about the identity of Pseudomonas antigens, but also the T cell repertoires of immune donors. Most clones were found to be specific for Pseudomonas alkaline protease (AP). Such clones could be physically isolated by selecting with crude Pseudomonas antigens or purified AP. In either case, their fine specificities were the same when tested against a panel of Pseudomonas antigens. The conclusion that AP is the principal immunogen for many donors was confirmed by measuring the absolute frequencies of proliferating T cells committed to AP and all other Pseudomonas antigens. Frequencies of AP-specific clones (1.5-2.7 X 10(-5 were comparable to those from the same donors that were specific for all secreted Pseudomonas antigens (1.3-6.0 X 10(-5. These results provide a model system for studying human T cell-mediated immunity to bacteria by identifying discrete antigens and measuring the repertoire diversities of cells responding to them.

Johnson, J P; Wank, R

doi: 10.1084/jem.160.5.1350pmid: 6208309

The monoclonal antibodies Genox 3.53, S1, and R1 define polymorphic epitopes localized to DQ molecules. All three antibodies showed a strong association with the HLA-DR antigens 1, 2, and w6 when tested on a panel of 68 unrelated individuals, suggesting that they all recognized the DQw1 allospecificity. However, segregation analysis and binding studies with a panel of HLA-D/DR homozygous cells indicated that these monoclonal antibodies defined two different alloantigens. Cells homozygous for DR 1, 2, or w6 expressed the epitopes defined by all three antibodies (i.e., S1, R1, and Ge) while cells homozygous for DR4 and DRw8 expressed only the S1 and R1 epitopes. Sequential immunoprecipitation analyses in S1+/R1+/Ge+ individuals, in which the three epitopes were shown by segregation analysis to be encoded by the same chromosome, revealed two distinct DQ-like molecules. While R1 and S1 appeared to reside on the same molecule, the epitope defined by Genox 3.53 was on a different molecule. Identical results were obtained with DR1-, DR2-, or DRw6-bearing cells. Thus it appears that DQw1-bearing individuals express two cis-encoded DQ-like molecules that carry distinct alloantigenic specificities.

Janusz, M J; Chetty, C; Eisenberg, R A; Cromartie, W J; Schwab, J H

doi: 10.1084/jem.160.5.1360pmid: 6387033

A single intravenous injection into rats of 0.4 mg of the muralytic enzyme mutanolysin, given as long as 3 d after an arthropathic dose of peptidoglycan-polysaccharide polymers derived from group A streptococci (PG-APS), resulted in a complete resolution of acute arthritis and the prevention of chronic joint disease. When administration of mutanolysin was delayed until 14 d after the injection of PG-APS, a great reduction in the severity of chronic inflammation was still observed. Quantitation of the amount of PG-APS present in the limbs, spleen, and liver by a solid phase enzyme-linked immunoassay indicated that the tissues of mutanolysin-treated rats contained as much PG-APS as tissues of PBS-treated control rats. In addition, rats treated with mutanolysin immediately after receiving an intraperitoneal injection of PG-APS developed a transient limb edema similar to that seen in rats after the injection of PG-APS digested to a small fragment size in vitro with mutanolysin. We hypothesize that mutanolysin acts in vivo by degrading PG-APS to small fragments that persist but are no longer arthropathic.

Ghebrehiwet, B; Silvestri, L; McDevitt, C

doi: 10.1084/jem.160.5.1375pmid: 6436431

We have shown previously that an activity which is capable of precipitating purified C1q and inhibiting some of the C1q-dependent biologic reactions could be solubilized from the membranes of both normal human peripheral B lymphocytes and a B cell-derived lymphoblastoid cell line (Raji), both of which are known to possess receptors for human C1q. In this report we present evidence that this membrane-associated C1q inhibitor is a chondroitinase-insensitive macromolecule and is the receptor for human C1q. The receptor was solubilized from membranes of Raji cells with Nonidet P-40 and purified to homogeneity using C1q-Sepharose 4B affinity chromatography. Equilibrium density gradient centrifugation analysis revealed that the complex could be resolved into a protein-rich, low density fraction and a carbohydrate-rich, high density fraction. The large hydrodynamic size, coupled with the high buoyant density, suggests that a proteoglycan is a constituent of the complex and indicates that the receptor might be a macromolecular complex of a proteoglycan portion noncovalently linked to a 60-70 kD glycoprotein. The glycoprotein moiety, in turn, consists of two or more identical (70,000 mol wt) polypeptide chains held together by disulfide bonds and constitutes the C1q receptor (C1qR). Sucrose density ultracentrifugation analysis showed that the isolated receptor sediments with an apparent rate of 4.2 S. Immunochemical analyses demonstrated that a typical preparation of the C1qR complex consists of approximately 23% uronic acid and approximately 21% galactosamine with a galactosamine-to-glucosamine ratio of 3.2. Binding of C1q to the receptor was found to be optimal at low ionic strength and neutral or near-neutral pH (7-7.4). The isolated receptor was found to inhibit C1q hemolytic function, abrogate C1q-dependent rosette formation, and block the C1q-dependent, cell-mediated cytotoxicity, all of which are activities mediated by the receptor.

Welte, K; Andreeff, M; Platzer, E; Holloway, K; Rubin, B Y; Moore, M A; Mertelsmann, R

doi: 10.1084/jem.160.5.1390pmid: 6092510

We investigated the effect of OKT3 antibody and interleukin 2 (IL-2) on Tac antigen expression and the proliferation of human peripheral blood mononuclear leukocytes. OKT3 monoclonal antibody at low, nonmitogenic concentrations (25 pg/ml) or IL-2 alone at optimal concentrations (20 U/ml) did not induce IL-2 receptor expression, as measured by Tac antibody or by T cell proliferation. However, costimulation with these concentrations of OKT3 antibody and IL-2 led to Tac antigen expression and T cell proliferation. These data suggest that the T cells are activated in two steps: OKT3 antibody at 25 pg/ml does not induce Tac antigen expression, but preactivates T cells to become responsive to IL-2. The addition of exogenous IL-2 then leads to expression of the IL-2 receptor, as recognized by Tac antibody, and to subsequent proliferation.