Virus-induced alterations of lymphoid tissue. IV. The effect of Newcastle disease virus on the fate of transfused thoracic duct lymphocytes

⁵¹Cr-labeled thoracic duct lymphocytes were briefly incubated at 4 °C with Newcastle disease virus (NDV) and then infused into syngeneic rats. Virus diverted the homing of many donor cells from lymph nodes and spleen to the liver. Evidence was obtained suggesting that some NDV-treated lymphocytes initially trapped in the liver subsequently migrated into the lymph nodes. The results imply that NDV transiently interrupts the normal route of lymphocyte migration. Alterations in lymphocyte distribution were mediated by attachment of virus to the cell surface and were the same as those induced by incubating lymphocytes with V. cholera neuraminidase before infusion. It appears that reactions involving 2–3′ and/or2–8′ linked sialyl residues on the surface of recirculating lymphocytes can markedly affect their distribution in the body.     

Influenza A virus interaction with murine lymphocytes. I. The influence of influenza virus A/Japan 305 (H2N2) on the pattern of migration of recirculating lymphocytes.

The effect of influenza virus A/Japan 305 (H2N2) on the path of migration of recirculating lymphocytes has been studied. 51Cr-labeled rat thoracic duct lymphocytes (TDL) were incubated with virus at 37 degrees C for 1 hr and then infused i.v. into syngeneic recipients which were killed 1 hr later. Virus-treated TDL accumulated in the liver and their recovery in lymph nodes and spleen was severely reduced. Changes in lymphocytes induced by virus developed rapidly and were evident after incubation for only 15 min. UV-irradiated virus altered the pattern of lymphocyte localization but attachment of heat-inactivated virus to lymphocytes in vitro had no effect on their distribution in vivo. Evidence was obtained that some virus-treated TDL, initially sequestered in the liver, subsequently recovered their ability to circulate normally. Recovery was not complete and a population of cells failed to regain their ability to home into lymph nodes. Evidence is also presented demonstrating that influenza virus affected the homing properties of both T and B cells. It is suggested that aberrations in lymphocyte homing were mediated by the viral neuraminidase which induces changes in the cell membrane leading to their accumulation in the liver.     

Tolerance induction to a thymus-dependent antigen in vitro: treatment of nonadherent cells with tolerogen biologically filtered in vitro

Highly tolerogenic bovine gamma globulin (BGG), a thymus-dependent antigen, was prepared by biologic filtraration in vitro. It readily induced tolerance in vivo in BALB/c mice and also rendered their nonadherent lymph node cells tolerant after in vitro incubation. Biologic filtration in vitro was carried out by incubating 2.5 × 10⁷ lymph node cells with 10 mg of nontolerogenic BGG in 10 ml of Eagle's medium containing 2% normal mouse serum at 37 °C for 6 hr. The BGG-containing medium was clarified by centrifugation and was used without further dilution.For tolerance induction in vitro, lymph node cells were separated into adherent and nonadherent populations on Falcon plastic. These cells were incubated for 0–18 hr at 37 °C with biologically filtered BGG (bBGG). After incubation, the cells were washed three times and (2–2.5) × 10⁷ nonadherent or 4 × 10⁶ adherent cells were injected iv with their untreated counterpart into lethally irradiated mice which had received 10⁶ bone marrow cells. The recipients were then challenged with 300 μg of aggregated BGG, and tolerance was assayed by the elimination of labeled BGG, rosette formation, and passive hemagglutination. Spleen cells were similarly treated for comparison. Our findings show that tolerance was not induced in vitro in adherent lymph node cells. However, in the nonadherent populations, those from the lymph node but not the spleen were rendered tolerant. The acquisition of tolerance in vitro was gradual. It was dependent upon the length of exposure to bBGG and required at least 6 hr.     

Migratory patterns of B lymphocytes. IV. Effect of anti-Ig on follicular localization of radioactively labeled murine spleen and bone marrow cells.

A study was made of the localization of nylon-wool-adherent (AD) and nonadherent (NA) murine spleen cells in lymphoid tissue of irradiated syngeneic recipients. Cells were labeled in vitro with [³H]uridine or ⁵¹Cr and injected intravenously. Localization in recipient tissues was expressed as percent of injected radioactivity. NA and AD [³H]uridine labeled cells gave spleen to lymph node (S:LN) ratios of 1.0 and 2.7, respectively. After treatment of AD cells with rabbit anti-mouse Fab + C at 37 °C, localization in S decreased markedly.NA cells primarily localized in LN paracortex and splenic periarteriolar sheaths. Untreated and NRS-treated AD cells localized in lymphoid follicles, whereas anti-Fab-treated AD cells did not. When ⁵¹Cr-labeled AD cells were treated with anti-Fab at 4 °C without C, there was a transient decrease in splenic localization at 24 hr followed by a recovery to the normal pattern at 48 hr after transfer. [³H]uridine-labeled bone marrow (BM) cells showed less localization in lymphoid tissue than did S cells. Some BM cells were seen in LN follicles, particularly at 48 hr after transfer, but this localization was not affected by prior treatment with anti-Fab + C. The possible role of surface Ig in the determination of follicular localization of B lymphocytes is discussed.     

Studies on the recognition of xenogeneic cells by nonimmune macrophages. I. Destruction of chicken fibroblasts in vitro by murine macrophages.

Human cord blood lymphocytes were compared with adult lymphocytes with regard to proportions of cells with surface markers for surface immunoglobulin (Ig), receptors for C′3 and the Fc-portion of IgG, as well as two types of erythrocyte rosettes (rapid and late E-rosettes). A significant decrease (P < 0.02 ? 0.05) in both early and late E-rosettes was noted when cord cells were compared to adult lymphocytes. After 20 hr of incubation at 37 °C, proportions of cells bearing Fc receptors in cord blood samples showed striking increments (P < 0.001) when compared with adult lymphocytes. T cell enrichment studies and sequential depletion of cells bearing Fc receptors as well as E-rosette forming cells indicated that the precursors of cells generating Fc receptors in vitro did not arise from cells with Fc receptors or T cell markers.     

The stability of L-phenylalanine mustard in bile

L-phenylalanine mustard (L-PAM) was incubated at 37° C in bile of bovine, canine and human origin. Recovery rate constants of L-PAM from bile were 0.1/hr for canine bile (0–3 hours); 0.18/hr for bovine bile; 0.45/hr for human bile. No significant hydrolysis of L-PAM in canine bile was noted for the period of 3 to 6 hours at 37° C. The incubation of L-PAM in sodium taurocholate solution (1000 molar excess) gave a recovery rate constant 0.15/hr at 37° C. However, the incubation of L-PAM in bilirubin solution (2.5 mg/ml H₂O) gave a recovery rate constant of 0.52/hr at 37° C. The high concentration of the parent compound L-PAM seen $\text{in vivo}$ in canine bile after i.v. administration may be related to its low $\text{in vitro}$ degradation rate in canine bile.     

Effects of concanavalin A on the migration of radioactively labeled lymphoid cells

The effects of the jackbean globulin Concanaalin A (Con A) on the distribution of radioactive ⁵¹Cr-labeled lymph node cells was studied in CBA mice. Lymph node cells treated in vitro with Con A in subagglutinating noncytotoxic doses were unable to “home” to the lymph nodes of syngeneic recipients after intraenous injection. The effect was almost immediate and seemed unrelated to mitogenesis. The inhibitory effect of Con A on lymphocyte migration could be partially reersed by alpha-methyl mannoside; the degree of migratory impairment was related to the amount of Con A bound to the lymphocyte surface at the time of transfer. The membrane site at which Con A binds to the lymphocytes is similar to that which is bound by heterologous antilymphocyte serum but is probably distinct from the theta antigenic site. These data lend support to the hypothesis that surface lymphocyte carbohydrate determinants are involved in the specific lymphocyte “homing” receptor.     

Lymphocyte-mediated cytolysis of allogeneic tumor cells in vitro. III. Enzyme sensitivity of target cell antigens.

Target tumor cells pretreated with high concentrations of papain or Pronase were resistant to lysis by cytotoxic T lymphocytes (CTL), whereas treatment with trypsin or neuraminidase had no protective effect. Parallel determinations of the H-2 content of target cells following enzyme treatment showed that approximately 80% of surface H-2 was removed by papain or Pronase, 40% by trypsin, and virtually none by neuraminidase treatment. Both susceptibility to lysis by CTL and content of surface H-2 after papain treatment were fully restored by 6 hr at 37 °C in nutrient medium. These findings suggest that lymphocyte-mediated cytolysis (LMC) determinants (target cell antigens bound by CTL) are sensitive to degradation by papain and Pronase but are resistant to the enzymatic action of trypsin and neuraminidase. That a similar pattern of enzyme sensitivity is shown by serologically defined H-2 antigens indicates that both functional classes, LMC and H-2, may have a structural association.     

Immunoglobulin on activated T cells detected by indirect immunofluorescence

A high proportion of H2 antigen-activated thymus-derived thoracic duct lymphocytes stained positively with rabbit anti-mouse immunoglobulin reagents as detected by indirect immunofluorescence. In view of the specificity of the reagents used and the fact that T cells from other sources e.g., thymus, were not stained by this technique, it was concluded that the material detected on H-2 antigen-activated thymus-derived thoracic duct lymphocytes was indeed immunoglobulin. When H-2 antigen-activated thymus-derived thoracic duct lymphocytes were cultured in vitro at 37 °C for 18 hr, with or without prior treatment with chymotrypsin, surface immunoglobulin could no longer be detected on the cells. This suggested that immunoglobulin had not been synthesized by the cells but absorbed from elsewhere.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

Separation of induction and expression of tight junction formation mediated by proteinases

The formation of tight junctions can be induced in the human adenocarcinoma cell line HT 29 by treatment with trypsin at 37°C. In contrast, after treatment of the cells with trypsin at low temperature (3°C), no tight junctions were observed. However, abundant formation of tight junctions occurred when cells were treated with trypsin at 3°C, washed with soybean trypsin inhibitor, and subsequently incubated at 37°C. Thus, this protocoi allows for the first time the temporal separation of the induction and assembly of tight junctions.     

Formation of rosettes by nonspecific lymphoid cells which have adhered to antigen-antibody complexes

Rat lymph node-sheep red cell rosettes have been formed through the adherence of nonspecific small lymphocytes and larger cell types to antibody-sheep red cell complexes. Enough antibody to form a significant number of rosettes has been synthesized in cultures, containing draining lymph node cells of immunized rats, within 1 hr of incubation at 37 °C, or during overnight storage at 4 °C. This suggests that conditions which differentiate between specific and nonspecific rosette-forming cells are essential when immunocyto-adherence phenomena are utilized for the study of the specificity of the immune response.     

Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes

The tissue localization or "homing" of circulating lymphocytes is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. In peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches and appendix), and sites of chronic inflammation, for example, lymphocytes leave the blood by adhering to and migrating between those endothelial cells lining postcapillary high endothelial venules (HEV). Functional analyses of lymphocyte interactions with HEV have shown the lymphocytes can discriminate between HEV in different tissues, indicating that HEV express tissue-specific determinants or address signals for lymphocyte recognition. We recently described such a tissue-specific "vascular addressin" that is selectively expressed by endothelial cells supporting lymphocyte extravasation into mucosal tissues and that appears to be required for mucosa-specific lymphocyte homing (Streeter, P. R., E. L. Berg, B. N. Rouse, R. F. Bargatze, and E. C. Butcher. 1988. Nature (Lond.). 331:41-46). Here we document the existence and tissue-specific distribution of a distinct HEV differentiation antigen. Defined by monoclonal antibody MECA-79, this antigen is expressed at high levels on the lumenal surface and in the cytoplasm of HEV in peripheral lymph nodes. By contrast, although MECA-79 stains many HEV in the mucosal Peyer's patches, expression in most cases is restricted to the perivascular or ablumenal aspect of these venules. In the small intestine lamina propria, a mucosa-associated site that supports the extravasation of lymphocytes, venules do not stain with MECA-79. Finally, we demonstrate that MECA-79 blocks binding of both normal lymphocytes and a peripheral lymph node-specific lymphoma to peripheral lymph node HEV in vitro and that it also inhibits normal lymphocyte homing to peripheral lymph nodes in vivo without significantly influencing lymphocyte interactions with Peyer's patch HEV in vitro or in vivo. Thus, MECA-79 defines a novel vascular addressin involved in directing lymphocyte homing to peripheral lymph nodes.     

Spontaneous rosette formation of rat thymus cells with guinea pig erythrocytes.

C57 black mice were immunised intraperitoneally with a DBA2 lymphoma (SL2). Fourteen days later spleen cells were prepared. These cells lysed the specific target (SL2) in vitro. Spleen cells were cultured for 24 hr at 37 ° C. Cell-free culture supernatants contained IgG and lysed SL2 cells either in the presence of a source of complement or in the presence of a monolayer of macrophages (a good source of antibody-dependent effectors). The cells producing cell-dependent antibody adhered to nylon wool and were unaffected by anti-theta serum. It was found that the production of antibody in vitro did not make a significant contribution to the observed cytolysis of SL2 by sensitised spleen cells. This effect was mediated by thymus-derived lymphocytes.     

Characterization of canine T-lymphocyte subpopulations: the detection of T mu and T gamma lymphocytes with homologous and heterologous immunoglobulins and the requirements for Fc receptor expression

Erythrocyte antibody (EA) rosette techniques employing sheep red blood cells sensitized with canine (homologous) and rabbit (heterologous) IgM and IgG antibodies were used to determine the number of cells with Fc receptors for IgM (Tμ) and IgG (Tγ) among T lymphocytes isolated from peripheral blood and lymph nodes of dogs. The percentages of Tμ and Tγ lymphocytes detected were found to be independent of the species origin of sensitizing antibody. Among peripheral blood T lymphocytes there were 53.0 ± 2.7% Tμ cells and 18.4 ± 3.6% Tγ cells. T lymphocytes obtained from lymph nodes were 62.1 ± 5.4% Tγ and 15.7 ± 2.6% Tγ. The number of Tμ cells detected increased from 20.0% when freshly isolated to 49.1 ± 4.1% after in vitro culture for 2–16 hr. The expression of the Fc-μ receptor in culture was inhibited by cycloheximide, demonstrating a requirement for active protein synthesis. In contrast, the number of Tγ lymphocytes detected did not vary between freshly isolated cells and those which had been cultured for 16 hr. Expression of the Fc-γ receptor during this time period was not inhibited by cycloheximide.     

E rosette formation at 37°:A property of mitogen-stimulated human peripheral blood lymphocytes

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C.Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture.This observation also demonstrates technical problems associated with the use of SRBC to quantitate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.     

Application of temperature-sensitive mutants for single-cell protein production

Cell-division-cycle, temperature-sensitive mutants of Saccharomyces cerevisiae were investigated as a means of altering the morphological characteristics and subsequent physical properties of single-cell protein (SCP). Strain 4471, harboring mutation cdc 4, formed a visible complex mass at the nonpermissive temperature, after being grown at 30°C and then transferred to 37°C for 8 hr. Microscopic observation showed that the mother cell was unable to complete the budding process at the nonpermissive temperature, which caused the cells to enlarge. Viscosity measurements were used to establish and characterize optimum morphological changes in the yeast. The Maximum increase in viscosity occurred when cells were incubated at 30°C and then shifted to 37°C for 8 hr. Strain 4471 exhibited yield stress, whereas A364A did not. Maximum change in yield stress occurred when cells were shifted from 30 to 37°C for 8 hr. No significant loss of protein or RNA occurred in strain 4471, as compared to strain A364A, when incubated at the nonpermissive temperature.     

Radiation-induced alterations in murine lymphocyte homing patterns: I. Radiolabeling studies

In vitro X-irradiation of ⁵¹Cr-labeled spleen, lymph node, bone marrow, or thymus cells was found to alter their subsequent in vivo distribution significantly in syngeneic BDF₁ mice. Irradiated cells demonstrated an increased distribution to the liver and a significantly lower retention in the lungs. Cells going to the lymph nodes or Peyer's patches showed a significant exposure-dependent decrease in homing following irradiation. Irradiated lymph node cells homed in greater numbers to the spleen and bone marrow, while irradiated cells from other sources showed no preferential distribution to the same tissues. Sampling host tissues at various times after irradiation and injection did not demonstrate any return to normal patterns of distribution. The alterations in lymphocyte homing observed after in vitro irradiation appear to be due to the elimination of a selective population of lymphocytes or membrane alterations of viable cells, and the detection of these homing changes is in turn dependent upon the relative numbers of various lymphoid subpopulations which are obtained from different cell sources. Radiation-induced alterations in the normal homing patterns of lymphoid cells may thus be of considerable importance in the evaluation of subsequent functional assays in recipient animals.     

Surface immunoglobulin on rabbit lymphoid cells. II. Ultrastructural distribution of b4 allotypic determinants and morphology of spleen lymphocytes

Surface immunoglobulin allotypic determinants on rabbit spleen lymphoid cells are ultrastructurally localized by labeling with antiallotype antisera and soluble complexes of ferritin and rabbit antiferritin of a given allotype. At 0 °C surface Ig is visualized in patches on the membrane of 54% of the spleen lymphocytes examined. Four morphologically distinct categories of spleen lymphocytes display different amounts of labeled surface Ig. Type I cells are essentially identical to peripheral blood lymphocytes and demonstrate rapid endocytosis of surface Ig at 37 °C. Type II cells greater amounts of surface Ig, demonstrate little endocytosis, and are consistent with lymphoblast cells. Type III cells have the greatest amount of surface Ig, reveal some endocytosis, and are morphologically consistent with proplasmacytes and plasmablast cells. Type IV cells are immature plasma cells and have very little detectable surface Ig. The percentage of each cell type making up the labeled population is Type I, 28%; Type II, 21%; Type III, 50%, and Type IV, 1%. Immunoferritin labeled Ig determinants may be modulated from the surface of these cells at 37 °C by endocytosis and/or by shedding after reaction with anti-Ig antisera.