The Journal of Experimental Medicine

Hara, I; Izui, S; Dixon, F J

doi: 10.1084/jem.155.2.345pmid: 7057139

A single intraperitoneal injection of bacterial lipopolysaccharide (LPS) or its lipid A component induced high levels of glycoprotein, gp70, in sera of several strains of mice within 24 h. This serum gp70 response induced by LPS was independent of the activation of B cells and the presence of T cells. However, serological and immunohistochemical studies demonstrated the production of gp70 by hepatic parenchymal cells and its subsequent release into the circulating blood. The expression of gp70 in the serum was enhanced not only by LPS but also other inducers of acute phase reactants (APR) such as turpentine oil or polyriboinosinic-polyribocytidylic acid. Further, the serum gp70 response was kinetically identical to those of APR. These results strongly suggest that (a) the liver may be the major source for serum gp70, (b) serum gp70 behaves like an APR, (c) its expression may be controlled by a mechanism similar to that for other APR, and (d) this glycoprotein apparently behaves as a normal host constituent and not a product of a viral genome.

Ceredig, R; Glasebrook, AL; MacDonald, HR

doi: 10.1084/jem.155.2.358pmid: 6120202

The correlation between surface phenotype and function in subpopulations of murine thymocytes has been investigated using flow microfluorometry (FMF). C57BL/6 thymocytes stained with monoclonal antibodies directed against Lyt-2, H-2K(b), and Thy-l.2 and passed on an FACS II flow cytometer could be resolved into at least four distinct subpopulations on the basis of fluorescence and forward light scatter (FLS) measurements. (a) Medium-sized Lyt-2(+) cells that stained strongly with H-2K(b) and weakly with Thy-l.2 (5 percent of total cells); (b) medium-sized Lyt-2(-) cells with other properties as in (a) (10 percent); (c) small Lyt-2(+) cells that stained weakly with H-2K(b) and strongly with Thy-l.2 (60 percent); and (d) large Lyt-2(+) cells that stained weakly with H-2K(b) and very strongly with Thy- 1.2 (23 percent). Cortisone-resistant thymocytes (CRT) were found to correspond phenotypically to populations (a) and (b). The distribution of cytolytic T lymphocyte precursors (CTL-P) directed against H-2(d) alloantigens in subpopulations of C57BL/6 thymocytes that had been sorted according to the phenotypic criteria described above was then investigated. CTL-P in sorted and control populations were quantitated by limiting dilution analysis of mixed leukocyte microcultures established in an excess of interleukin 2 (IL-2). These studies established that all thymus CTL-P could be quantitatively recovered in a subpopulation of cells that was cortisone-resistant, medium-sized, Lyt-2(+), H-2K(b+), and weakly stained with Thy-l.2. In parallel studies, the production of IL-2 by subpopulations of C57BL/6 thymocytes was quantitatively assessed using a recently developed sensitive microassay system. Graded numbers of sorted or control thymocytes were stimulated with irradiated T cell-depleted allogeneic cells and assayed for their ability to support the growth of an IL-2-dependent cytolytic T lymphocyte clone. Using this method, IL-2 production was found to reside entirely in a subpopulation of cortisone-resistant, medium-sized Lyt-2(-) thymocytes. Further phenotypic analysis of this subpopulation of cells indicated that it was homogeneously H-2K(b+) and weakly staining with Thy- 1.2. Taken together with the CTL-P results, these data directly demonstrate that a subpopulation of thymocytes with a mature phenotype (i.e., cortisone- resistant, medium-sized, H-2K(b+), and weakly staining with Thy-l.2) accounts for all the functional activity in the thymus. Reasons for the apparent discrepancy between these results and other recent studies will be discussed.

Sherman, L A

doi: 10.1084/jem.155.2.380pmid: 6173455

Superimposed on the heterogeneous anti-H-2Kb cytolytic T lymphocyte (CTL) receptor repertoire of allogeneic murine strains are reactivities that recur with high frequency amongst individuals of any given strain. These receptor specificities represent phenotypic markers of the CTL repertoire and, as such, have been used to compare receptor repertoires of genetically disparate strains. The results demonstrate that congenic strains differing only in the MHC (B10.D2 and B10.BR) differ significantly in their H-2Kb-specific CTL repertoires. This finding clearly demonstrates a role for the MHC in determination of the CTL precursor repertoire. The mechanism by which MHC influences CTL specificity was explored through analysis of the anti-H-2Kb repertoire of (B10.BR X B10.D2)F1 hybrids. Because at least one recurrent parental specificity has found to be recurrent in F1 progeny as well, the findings indicate that MHC-specific tolerance cannot be solely responsible for repertiore differences between MHC-disparate strains. In addition, the F1 repertoire is characterized by the emergence of several nonparental recurrent specificities.

Jörg, A; Henderson, W R; Murphy, R C; Klebanoff, S J

doi: 10.1084/jem.155.2.390pmid: 6120203

Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spectra, spectral shift on treatment with lipoxygenase, incorporation of 14Carachidonic acid, gas chromatography-mass spectrometry, and comparison of retention times on HPLC to authentic standards. The eosinophil products characterized were 5-(S), 12-(R)-dihydroxy-6-cis-8, 10-trans-14-cis-eicosatetraenoic acid (leukotriene B4) and its 5-(S), 12-(R)-6-trans and 5-(S), 12-(S)-6-trans isomers, 5-(S)-hydroxy-6-(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (leukotriene C4) and its 11-trans isomer, and 5-(S)-hydroxy-6-(R)-S-cysteinylglycine-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4).

Wylie, D E; Sherman, L A; Klinman, N R

doi: 10.1084/jem.155.2.403pmid: 6173456

The capacity of the antibody repertoire to recognize complex antigens on viral-infected cells was investigated at the level of monoclonal B cell responses. A majority of primary B cells responsive to PR8(H1N1)-infected H-2 syngeneic cells produced antibody that bound viral determinants only in the context of infected cells and not the isolated virion. An examination of the fine specificity of such antibodies revealed that most could be distinguished by a panel of cells infected with closely related heterologous H1 influenza strains. Indeed, most antibodies bound hemagglutinin determinants of PR8 exclusively, and few were broadly cross-reactive. An examination of the same antibodies for their recognition of cell surface antigens revealed that the majority recognized MHC-encoded antigenic determinants. Thus, most BALB.K (H-2k) primary B cells responsive to PR8-L919 (H-2k) cells produced monoclonal antibodies that bound PR8-antigens only in the context of H-2Dk-infected cells. Most C57BL/10 (H-2b) B cells responsive to PR8-EL4 (H-2b) cells produced monoclonal antibodies that bound PR8 antigens only in the context of H-2Kb-infected cells. These latter antibodies were further shown to recognize that H-2Kb molecule by virtue of their capacity to be discriminated by a panel of PR8-infected H-2Kb mutant cells. These findings demonstrate that much of the antibody repertoire is capable of highly specific complex recognition of viral antigenic determinants in the context of the appropriate MHC alloantigen.

Rabinovitch, M; Dedet, J-P; Ryter, A; Robineaux, R; Topper, G; Brunet, E

doi: 10.1084/jem.155.2.415pmid: 7057140

Exposure of macrophages infected with Leishmania mexicana amazonensis to phenazine methosulfate (PMS) resulted in rapid damage and disappearance of the intracellular amastigotes without obvious ill effects to the host cells. The reduction of the percent infection was related to the concentration of PMS and to the duration of the pulse. Most Leishmania disappeared within 2 h of a 2-h pulse with 10 μM of the drug. In contrast, pretreatment of the macrophages with PMS followed by removal of the drug before infection did not result in disappearance of the parasites. The pH of the PMS medium markedly influenced the disappearance of Leishmania: maximum effect was observed at pH 8.0, while the effect was negligible at pH 6.3. The pH effect may be related to pseudobase formation by the PMS cation. Dose-response curves for PMS were similar for resident, elicited, or activated macrophages. Observations by time-lapse cinemicrography documented the explosion-like fragmentation of the amastigotes within 1-2 h of exposure of infected macrophages to the drug. Parasite-derived granules and vacuoles were seen to scatter within the parasitophorous vacuoles. This early damage to the parasites was confirmed by transmission electron microscopic observations. Infected macrophages incubated with PMS displayed detectable vacuolar fluorescence, indicating that PMS or a metabolite of PMS had access to the vacuoles. A series of other electron carriers, including phenyl methanes, phenazines, oxazines, a xanthene, and a naphthoquinone, given continuously for 18 h, also induced the disappearance of the Leishmania. The most potent was crystal violet, active at 70 nM. The presence of apolar substituents enhanced activity and this is probably related to increased permeation of the dyes. Finally, PMS, as well as other electron carriers examined, also reduced the growth of Leishmania promastigotes in culture. The results are compatible with a direct effect of the drugs on the intracellular amastigotes, involving only a permissive participation of the macrophages. We propose that the diverse agents destroy the amastigotes by redox-cycling generation of active oxygen metabolites at or near the parasites. Alternatively, the effect of the drugs could be mediated by toxic free radical reduction species of the drugs or by interference with electron flow or with the intermediary metabolism of Leishmania.

Ross, EAM; Anderson, N; Micklem, HS

doi: 10.1084/jem.155.2.432pmid: 7035599

The mouse hematopoietic system was subjected to repeated depletion and regeneration either by serial transfer of bone marrow cells through lethally irradiated recipients or by repeated treatment with the cycle-active drug hydroxyurea (HU). The capacity of surviving stem cells to proliferate and self-renew was assayed at intervals by two methods: (a) the spleen colony method; and (b) competitive repopulation of irradiated recipients using chromosome markers, with normal bone marrow cells as an internal control. The progressive decline in stem cell function that occurred during serial transfer of bone marrow and that had already begun after a single transfer was not seen during HU treatment; up to 25 pairs of HU injections given over more than 1 yr had no discernible effect on the number of stem cells present 3 wk after the final injection or on their capacity to self-renew. Within 2 d after exposure to HU, the average self-renewal capacity of surviving stem cells was enhanced. This implies that the drug selectively eliminates poorly self-renewing stem cells and hence that these enter cycle more readily than stem cells with a high self-replicative potential. However, the fact of being in cycle at the time of injection did not of itself affect self-renewal. The results show that serial transfer of bone marrow is not a valid method for studying clonal aging phenomena because it does not fulfill the assumptions on which such studies are based. No evidence was obtained for any intrinsic limitation in the capacity of bone marrow populations for repeated regeneration after HU-induced depletion. However, this does not necessarily imply that individual hematopoietic clones are capable of indefinite expansion because hematopoiesis may (as suggested by the relative resistance of highly self-replicative stem cells to mitogenic signals) proceed on the basis of clonal succession.

Glimcher, L H; Kim, K J; Green, I; Paul, W E

doi: 10.1084/jem.155.2.445pmid: 6460073

Several Ia-positive BALB/c B cell tumor lines were screened for their ability to present alloantigen and protein antigens to alloreactive and antigen-reactive T cells. Of six Ia-positive tumor lines studied, three were found to be effective as antigen presenting cells (APC). Indeed, on a per cell basis, one of the stimulatory lines, A20.3, was substantially more effective than whole spleen cells. The other three lines, although Ia-positive, were nonstimulatory. A20.3 was chosen for further study. This tumor appeared to behave like the conventional APC because (a) the tumor cells presented alloantigen, (b) they presented protein antigen in an MHC-restricted fashion to both primed donor T cells and to long-term continuous T cell lines, (c) alloantigen presentation was blocked by the inclusion of an anti-Ia antibody in the culture system, and (d) A20.3 cells could be effectively pulsed with antigen, although the continuous presence of antigen in the culture system resulted in a superior response. The addition of an exogenous source of interleukin 1 proved necessary to obtain an alloreactive but not an antigen-specific T cell response, although its inclusion did enhance the magnitude of antigen-stimulated proliferation. These tumor cells should prove useful in studying the biochemical events that occur during antigen processing and the requirements for T cell triggering by processed antigen in association with Ia molecules.

Oite, T; Batsford, SR; Mihatsch, MJ; Takamiya, H; Vogt, A

doi: 10.1084/jem.155.2.460pmid: 6460074

Cationized human IgG can bind to the rat glomerular basement membrane (GBM), act as planted antigen, and induce in situ immune complex formation accompanied by severe glomerulonephritis. Perfusion of highly cationized human IgG (isoelectric point {more than} 9.5) via the left renal artery resulted in preferential localization within the perfused kidney (up to 56 percent of dose injected); after intravenous administration, only 4 percent was bound to the kidneys. The planted antigen was localized along the glomerular capillary walls and was accessible for antibody administered intravenously 1 h after perfusion, when virtually no antigen remained in the circulation. Persistence of cationized human IgG in the perfused kidney was markedly prolonged when complexed with antibody; one-half the cationized human IgG was still present after 12 d. There was a difference in the disappearance rates of antigen and antibody, as cationized human IgG was removed faster from the kidney than the antibody, the binding of which remained almost unchanged during the first week. Renal perfusion of a minimum of 20 μg of cationized human IgG, followed by intravenous injection of antibody, regularly induced severe glomerulonephritis with a proteinuria of at least 100 mg/24 h. The degree and the persistence of proteinuria induced depended on the dose of cationized human IgG perfused. Experiments using radiolabeled antigen and antibody showed that after renal perfusion of 20 μg cationized human IgG, 11.1 μg was kidney bound at the time of antibody injection. At the onset of proteinuria, 4.0 μg of antigen and 31.9 μg of anti-human IgG antibody were present in the perfused kidney. Immunofluorescence revealed immune deposits consisting of cationized human IgG and rabbit IgG (anti-human IgG) along the GBM. The staining pattern was linear (confluent) during the first 2 d and became granular during the course of the disease. Electronmicroscopically, a prominent finding was the accumulation of dense deposits, mainly in the subepithelial space and beneath the slit pores.

Centifanto-Fitzgerald, Y M; Yamaguchi, T; Kaufman, H E; Tognon, M; Roizman, B

doi: 10.1084/jem.155.2.475pmid: 6276491

The pattern of ocular disease produced in the rabbit eye by HSV-1 (F) and HSV-1(MP) strains and recombinants F(MP)A, F(MP)B, F(MP)C, F(MP)D, F(MP)E, and F(MP)F was studied. The characteristics of ocular herpetic disease such as morphology of dendritic ulcers, severity of epithelial disease and incidence and duration of stromal disease produced in the rabbit eye are genetically determined by the virus strain. Our studies show that transfer of a defined part of the genome of the stromal disease-producing virus, HSV-1(MP), to the genome of an epithelial disease-producing virus, HSV-1(F), yielded recombinants with one or more of the disease characteristics of the donor strain. Specifically, recombinant F(MP)D produced lesions characteristic of the donor HSV-1(MP) strain; recombinants F(MP)C and F(MP)E produced stromal disease approaching the severity of the disease produced by the donor HSV-1(MP) strain, and only recombinants F(MP)A and F(MP)B retained the typically elongate lesions of the recipient HSV-1(F), whereas the recombinant strain F(MP)F produced no disease. The viral functions pertaining to the ocular disease pattern map between 0.70 and 0.83 map units in HSV-1 DNA within the BglII F DNA fragment. The pattern of stromal disease is independent of the production of glycoprotein C and fusion of HEp-2-infected cells. The functions relating to these aspects of ocular disease segregate but are closely linked.