The Journal of Experimental Medicine

Kupfer, A; Singer, S J; Dennert, G

doi: 10.1084/jem.163.3.489pmid: 3081676

In mixtures of two CTL of the type a anti-b and b anti-c, only the latter is lysed; i.e., killing is unidirectional. Here, we show that two profound types of changes occur in the effector CTL but not in the target CTL upon formation of couples between them. One is that the microtubule organizing center (and presumably the Golgi apparatus that is invariably colocalized with it) is reoriented inside the effector CTL to face the bound target CTL. This unidirectional reorientation, it is proposed, serves to direct putative cytotoxic secretory components derived from the Golgi apparatus of the effector cell to the site of cell-cell binding. The second unidirectional change is in the membrane-associated cytoskeleton of the effector CTL in the area of target cell binding. The cytoskeletal protein talin, but not any of four other such proteins assayed, is highly concentrated at the contacting membrane of the effector CTL, while it is uniformly distributed over the entire membrane of the bound target CTL. This localized, massive cytoskeletal reorganization may reflect a mechanism to protect the membrane of the effector CTL from the effects of putative cytotoxic components secreted by the effector cell into the intercellular space between it and the target cell.

Meek, R L; Hoffman, J S; Benditt, E P

doi: 10.1084/jem.163.3.499pmid: 3950541

The deposits of fibrils found in amyloidosis of the A type are derived from only one of the three serum amyloid A (SAA) gene products, namely SAA2. In order to explore the mechanism of SAA isotype-specific amyloid protein AA deposition, the molecular kinetics of the serum amyloid proteins were examined in CBA mice during casein induction of amyloidosis. The presence of SAA mRNA in spleen was searched for; hepatic SAA1 and SAA2 mRNA levels, rates of specific protein synthesis and secretion by hepatocytes, and serum levels were measured during a 20-d period of amyloid induction. We observed the following: small amounts of amyloid substance appeared in the spleen by day 5 and increased steadily over the ensuing 15 d to occupy nearly 30% of splenic volume by day 20. No SAA mRNA was detected in spleen at any time during induction of amyloid formation. Total serum SAA levels peaked 1 d after we began casein treatment, and thereafter declined. This decline was accounted for entirely by a dramatic fall in SAA2, while SAA1 levels remained nearly constant throughout. The ratios of hepatic SAA2:SAA1 mRNA, as determined by in vitro translation, remained constant during the 20-d period, as did amounts of SAA1 and SAA2 synthesized and secreted by freshly isolated hepatocytes. These data indicate that the deposition of amyloid A protein derived from SAA2 is not due to local SAA production in spleen, nor excessive SAA2 production compared with SAA1, but involves the selective and accelerated removal of SAA2 from the circulating pool of both SAA1 and SAA2.

Roberts, N J; Prill, A H; Mann, T N

doi: 10.1084/jem.163.3.511pmid: 3485170

Respiratory viral infections are commonly associated with altered immune responses, such as proliferative responses to mitogens and antigens. To examine potential mechanisms, we examined production of IL-1 and IL-1 inhibitors by purified human peripheral blood-derived macrophages exposed to influenza virus or respiratory syncytial virus (RSV). IL-1 and IL-1 inhibitor activities in supernatant fluids from macrophages exposed to the viruses 24 h previously were measured using the standard mouse thymocyte comitogen assay. Crude fluids from macrophages exposed to influenza virus contained substantial IL-1 activity, whereas crude fluids from macrophages exposed to RSV contained marked IL-1 inhibitor activity. Assays with gel filtration-separated fractions revealed that both influenza virus and RSV induced production of both IL-1 and IL-1 inhibitors. Neither IL-2 nor IL-2 inhibitor activities were detected. Thus, effects of human macrophage-derived factors on thymocyte proliferation, or potentially on human lymphocyte proliferation, may reflect the total or net activity of multiple composite factors, the balance of which varies according to the challenge. The data raise the possibility that marked production of IL-1 inhibitor activity in response to RSV plays a role in the clinical recurrence of RSV infection despite the absence of clear evidence for antigenic shift or drift of the virus.

Rabinovitch, M; Zilberfarb, V; Ramazeilles, C

doi: 10.1084/jem.163.3.520pmid: 3950542

Leishmania amastigotes parasitize almost exclusively the mononuclear phagocytes of mammals. The organisms survive and multiply within acidified vacuoles (parasitophorous vacuoles; p.v.) akin to phagolysosomes. Certain amino acid esters are known to accumulate in and disrupt lysosomes. We postulated that, since Leishmania possess lysosome-like organelles, they may be susceptible to the potentially high ester concentrations attained in the p.v. We report here that L-amino acid esters can rapidly destroy intracellular Leishmania at concentrations that do not appear to damage the host cells. L-leu-OMe, which cured greater than or equal to 90% of infected macrophages at 0.8 mM concentrations, was used in most of the experiments. L-leu-OMe was only active after infection, implying inefficient transfer from secondary lysosomes to the p.v. Parasite destruction had several features in common with lysosomal and leukocyte damage induced by the esters, i.e., inactivity of D-amino acid esters, a marked pH dependence and increased killing after ester pulses at lower temperatures. Killing depended on the amino acid and on the ester substitution. The most active of the methyl esters assayed was that of leucine, followed by those of tryptophan, glutamic acid, methionine, phenylalanine, and tyrosine. Methyl esters of seven other amino acids were inactive when tested at up to 10 mM concentrations. Among leucine esters studied, benzyl ester was sixfold more active than the methyl homolog. The dipeptide L-leu-leu-OMe produced 90% cure at 0.08 mM concentrations. Leishmanicidal activity could be related to penetration of the parasites by the esters or to toxic ester hydrolysis products released in the p.v. The first hypothesis is supported by the pH-dependent destruction of isolated amastigotes by the esters. Furthermore, relatively high concentrations of L-leucine, methanol, or benzyl alcohol were not demonstrably toxic to the amastigotes. We postulate that ester concentrations sufficient to damage the intracellular amastigotes may be obtained within the p.v. after exposure of infected macrophages to the esters. Esters preferentially hydrolyzed by parasite enzymes may be expected to be leishmanicidal, but less damaging to the host.

Keene, W E; Petitt, M G; Allen, S; McKerrow, J H

doi: 10.1084/jem.163.3.536pmid: 2869098

FPLC anion-exchange and chromatofocusing chromatography were used to purify the major neutral proteinase from secretions of axenically cultured Entamoeba histolytica trophozoites. HM-1 strain trophozoites, which were more proteolytically active than the less virulent HK-9 strain, were used for purification of the enzyme. It is a thiol proteinase with a subunit Mr of approximately 56,000, a neutral pH optimum, and a pI of 6. The importance of this enzyme in extraintestinal amoebiasis is suggested by its ability to degrade a model of connective tissue extracellular matrix as well as purified fibronectin, laminin, and type I collagen. The enzyme caused a loss of adhesion of mammalian cells in culture, probably because of its ability to degrade anchoring proteins. Experiments with a peptide substrate and inhibitors indicated that the proteinase preferentially binds peptides with arginine at P-1. It is also a plasminogen activator, and could thus potentiate host proteinase systems.

Fujii, M; Sugamura, K; Sano, K; Nakai, M; Sugita, K; Hinuma, Y

doi: 10.1084/jem.163.3.550pmid: 3005464

Receptor-mediated internalization and degradation of IL-2 were investigated in cell lines carrying human T cell leukemia/lymphoma (lymphotrophic) virus type I (HTLV-I) and PHA-treated normal PBL. The HTLV-I-carrying cell lines ILT-Yan and TL-Mor, and the PBL expressed both high- and low-affinity IL-2-R. However, another HTLV-I-carrying T cell line, MT-1, expressed mainly low-affinity receptors. Greater than 50% of the IL-2 bound to high-affinity receptors was internalized within 10 min when these cells were incubated at 37 degrees C. The internalized IL-2 was rapidly degraded and the products were excreted into the culture fluid. The t1/2 of IL-2 degradation in these cells was estimated as 60-80 min at 37 degrees C. The internalization and degradation of IL-2 were both temperature dependent. Light-microscopic autoradiography with 3H-labeled IL-2 confirmed the internalization of IL-2, and suggested that some IL-2 might be carried to the nucleus.

Sawada, Y; Urbanelli, D; Raskova, J; Shenk, T E; Raska, K

doi: 10.1084/jem.163.3.563pmid: 2936861

Viable recombinant adenoviruses that carry a portion of the type 12 E1A and E1B transcription units in a type 5 background were used to identify genes controlling expression of the adenovirus tumor-specific transplantation antigen (TSTA). The TSTA immunity is not crossreacting between the group A and group C adenovirus serotypes. Viruses carrying the E1A region (sub370-12E1A), or both E1A and E1B (sub370-12E1AB) regions of Ad12, induce a strong transplantation immunity against tumors induced by syngeneic cells transformed with adenovirus 12, but fail to induce any protection against syngeneic cells transformed with adenovirus 2. Immunization with the virus carrying only the E1B region (sub370-12E1B) of adenovirus 12 induces no immunity to adenovirus 12 transformed cell line, but confers a strong protection against cells transformed with adenovirus 2. These results provide strong evidence that the adenovirus tumor-specific transplantation antigen is a function of the E1A early region.

Schiff, C; Milili, M; Hue, I; Rudikoff, S; Fougereau, M

doi: 10.1084/jem.163.3.573pmid: 3005465

Ig germline genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA either in pBR328 plasmid or in EMBL 3 phage. Three clones that gave a very strong positive hybridization signal with a VH anti-GAT-specific probe were completely characterized and sequenced. All three were greater than 95% homologous, with the exception of the 5' noncoding region, which was only 85% homologous but contained characteristic regulatory signals. One of these genes, H10, had a sequence that was completely identical to that of a cDNA derived from a GAT-specific BALB/c hybridoma. Southern blot analysis using Eco RI-digested DNA from rearranged GAT-specific hybridomas revealed that the same gene was used for other GAT-specific VH regions, including one differing from the H10 sequence by 12 nucleotides, which must have been generated by a somatic mechanism. The same H10 germline gene was also used, in most cases without any nucleotide substitution, in hybridomas of the Ab1' set of the GAT idiotypic cascade, suggesting that immunization with Ab2 (antiidiotypic) antibodies preferentially stimulates the direct expression of VH germline genes. Finally, the previous hypothesis that NPa and GAT VH genes were derived from the same germline gene was definitively confirmed, both from sequence data and Southern blot analysis.

Kefalides, N A; Pegg, M T; Ohno, N; Poon-King, T; Zabriskie, J; Fillit, H

doi: 10.1084/jem.163.3.588pmid: 3950543

Sera from patients with poststreptococcal glomerulonephritis (PSGN) known to have antibodies to proteoglycans were studied for the presence of antibodies against other basement membrane (BM) components. BM collagen (type IV) was isolated in the native state by extracting bovine anterior lens capsule (ALC) with 0.5 M acetic acid. The 7-S (collagenous) domain and the NC-1 (noncollagenous) domain of type IV collagen were obtained after bacterial collagenase digestion of ALC followed by gel filtration. Laminin was isolated from the mouse EHS tumor and fibronectin from human plasma. Immunologic studies, using an ELISA and electroimmunoblot, revealed the presence of antibodies that reacted with intact, native type IV collagen and the 7-S collagenous domain of this molecule. Reaction with the NC-1 (noncollagenous) domain was minimal, and not higher than that obtained with control sera. Laminin reaction strongly with the patients' sera, but fibronectin did not. Unlike sera from patients with Goodpasture syndrome, which contain antibodies primarily against the NC-1 (noncollagenous) domain of type IV collagen, sera from patients with acute PSGN contain antibodies against all the major macromolecular components of BM. This difference in immunologic reactivity may account for the observed differences in the pathologic picture at the glomerular level.

Mizuochi, T; Ono, S; Malek, T R; Singer, A

doi: 10.1084/jem.163.3.603pmid: 2936862

This study has characterized the primary T cell subpopulations that secrete IL-2 in response to recognition of either class I or class II MHC encoded determinants. The addition to culture of anti-IL-2-R mAb inhibited the consumption of IL-2 by activated lymphocytes during the response period, permitting a much more accurate assessment of the amount of IL-2 produced in the response cultures. Using this response system, we found that primary T cell populations contain two IL-2-secreting T cell subsets that express reciprocal phenotypes and different MHC recognition specificities: an L3T4+, Lyt-2- T cell subset responsive to both class I and class II MHC alloantigens, and an L3T4-Lyt-2+ T cell subset responsive only to class I MHC alloantigens. The L3T4+ T cell subset expressed a broad functional response repertoire in that L3T4+ T cells were triggered to secrete IL-2 upon recognition of unmodified self-Ia determinants, allogeneic Ia determinants, and class I alloantigens presented by self-Ia determinants. The activation of L3T4+ IL-2-secreting T cells, even those responsive to class I MHC alloantigens, could be blocked completely by anti-Ia mAbs, confirming that the L3T4+ T cell subset was in fact class II restricted. In contrast, the Lvt-2+ T cell subset expressed a narrow functional response repertoire in that they were triggered to secrete IL-2 only in response to allogeneic class I MHC determinants, and were not triggered to secrete IL-2 even in response to TNP-modified self-MHC determinants. The specificity of Lyt-2+ IL-2-secreting T cells for class I MHC allodeterminants was confirmed by the observations that: (a) their activation could be blocked completely by anti-class I mAbs, (b) they could be triggered by Ia- cell lines which expressed class I MHC alloantigens and possessed accessory function, and (c) they responded to class I MHC alloantigens but failed to respond to class II MHC alloantigens, even in the presence of exogenously added second signals that circumvented the requirement for alloantigen-bearing accessory cells. Finally, the frequency of primary Lyt-2+ T cells that secreted IL-2 in response to class I (Kbm1) MHC alloantigens was shown to be only minimally lower than that of L3T4+ T cells that secreted IL-2 in response to class II (I-Abm12) MHC alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)