Binding and endocytosis of erythrocytes sensitized with rabbit IgG via Fc gamma receptors of human monocytes.

The properties of the monocyte Fc gamma receptors (FcR) were investigated with monoclonal antibodies (mAb) against FcRI (10.1) and FcRII (IV3). mAb against FcRI inhibited partially the binding of sheep red blood cells (SRBC) sensitized with anti-SRBC rabbit IgG (EA) at 37 degrees to monocytes pretreated with N-ethyl maleimide, which inhibits the EA ingestion. The erythrocytes (E) were sensitized with varying concentrations of anti-E rabbit IgG. The EA binding to different FcR depends on the concentration of specific antibody used to sensitize the erythrocytes. At high levels of sensitization a high proportion of rosettes form via FcRII which can be inhibited with mAb IV3. As sensitization decreases it is more difficult for FcRII to form rosettes, so an increased percentage of them is mediated via FcRI. Sensitization of SRBC with 1-1.5 x 10(3) anti-SRBC rabbit IgG molecules per erythrocyte is the threshold to allow FcRII to mediate rosettes. At the lowest levels of sensitization the total number of rosettes is even lower and all rosettes are mediated via FcRI, hence mAb 10.1 is fully inhibitory. In addition, our data strongly support the view that the ingestion of EA takes place mainly via FcRII. We show in this study that while binding of slightly sensitized erythrocytes was blocked efficiently by mAb 10.1, the ingestion of the equivalent EA was hardly inhibited by it.     

Carbohydrate surface constituents of T cells mediating delayed-type hypersensitivity that control entry into sites of antigen deposition

Peritoneal exudate T lymphocytes (PETLs) that mediate delayed-type hypersensitivity (DTH) to sheep red blood cells in mice were modified by in vitro treatment methods which modify surface carbohydrate constituents. Neuraminidase treatment resulted in the release of both N-acetylneuraminic acid and N-glycolyl-neuraminic acid, and periodate treatment in the formation of the corresponding C7 analogues. Treatment of PETLs with neuraminidase led to a transient reduction of DTH reactions in syngeneic cell recipients. After treatment with neuraminidase plus galactose oxidase, adoptive mediation of DTH was more markedly reduced. Incubation of PETLs with periodate caused a permanent loss of DTH transferring capacity. The oxidation-induced effects following treatment of PETLs with neuraminidase plus galactose oxidase or with periodate could be reversed by subsequent reduction with borohydride of the previously formed aldehyde moieties. Decreased DTH reactions observed after the various treatment procedures were paralleled by reduced immigration of PETLs into sites of antigen deposition, indicating that the reduction/oxidation state of cell surface carbohydrates is crucial for induction of inflammatory processes by T cells. Trapping of PETLs in the liver could not be the sole mechanism since neuraminidase- and periodate-treated PETLs, but not neuraminidase plus galactose oxidase-treated PETLs, accumulated in the liver. It therefore appears that alterations in the reduction/oxidation state of the cell surface can lead to unresponsiveness of T cells to inflammatory signals.     

Secondary delayed-type hypersensitivity to sheep red blood cells and minor histocompatibility antigens in mice: transfer of memory by recirculating thoracic duct lymphocytes

Secondary delayed-type hypersensitivity (DTH) in mice to sheep red blood cells (SRBC) and minor histocompatibility (H) antigens is dependent on long-lived memory T cells. In this paper we investigated whether these memory T cells recirculate. It was shown that late phase "immune' thoracic duct lymphocytes (TDL) from mice which were immunized with SRBC or non-H-2-incompatible spleen cells several weeks previously could adoptively transfer secondary DTH to these antigens. Passing the immune TDL through intermediate recipients demonstrated that these SRBC- or minor H-antigen-reactive memory T cells recirculate from blood to lymph. In contrast to mice immunized with minor H antigens, no secondary type DTH reactivity could be demonstrated in mice immunized with H-2-incompatible spleen cells. Also, after adoptive transfer of TDL from mice immunized with H-2-alloantigens, it was impossible to demonstrate an accelerated DTH reactivity.     

Antigen-specific suppressor factors produced by T cell hybridomas for delayed-type hypersensitivity.

This study describes the generation of T hybridoma lines which secret factors specifically suppressing delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC). AKR strain-derived T lymphoma BW 5147 cells were fused with spleen cells from mice primed with SRBC and containing antigen-specific T suppressor cells for DTH. Supernants from the derived hybridomas were tested for suppression of either expression of induction of DTH to SRBC. Six lines produced specific suppressor activity for the expression of DTH; 4 lines produced suppressor activity for the induction of DTH of which only one line was antigen-specific. These lines were passaged in normal AKR mice, and the serum obtained had activity up to 10(-4) dilution. The factor was effective across the H-2 barrier.     

Regulation of delayed-type hypersensitivity IV. Antigen-specific suppressor cells for delayed-type hypersensitivity induced by lipopolysaccharide and sheep erythrocytes in mice.

Mice injected subcutaneously with 1 x 10(8) sheep red blood cells (SRBC) developed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after injection. Such DTH was suppressed when 100 microgram lipopolysaccharide (LPS) was injected intravenously 1-2 days before or at the time of SRBC injection. This suppression of DTH was transferable by spleen, lymph node, thymus and bone marrow cells to sensitized or normal syngeneic recipients, but could not be transferred by serum. Suppressor cells were not induced by LPS alone or SRBC alone, and they were antigen-specific since DTH to chicken red blood cells was not affected. The suppressor cells appeared in the spleen in optimum number 3-4 days after induction. They were theta-negative and Ig-positive as judged by antiserum plus complement treatment and by Ig rosette separation. Attempts to obtain soluble suppressor factor from the suppressor cells by sonication or in vitro incubation were unsuccessful. Mitomycin C treatment of the suppressor cells completely abolished the suppressor activity. Thus, LPS, in conjunction with antigen, appears to induce a population of specific suppressor B cells which are capable of regulating T cell function.     

Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. I. Production and characterization as to function and phenotype.

T lymphocytes which mediate DTH reactions to sheep red blood cells (SRBC) in mice enter casein-induced peritoneal exudates from which they can be recovered and assayed in a passive transfer system. Peritoneal exudates need not contain specific antigen for inducement of T-cell immigration. The amount (or biological activity) of DTH-transferring peritoneal exudate lymphocytes is enhanced by the previous use of immune modulating agents, such as cyclophosphamide (Cy) (200 mg/kg 2 days prior to sensitization), or BCG (10(7) live organisms i.v. 14 days prior to sensitization). SRBC-specific peritoneal exudate lymphocytes phenotypically are Thy 1+ and Ly 1+, 2-. In vivo, peritoneal exudate T cells from Cymodulated donors persist in circulation for a short period only and are subject to the suppressive mechanisms acting in anergic mice. Cells from BCG-plus-Cy-modulated donors, on the other hand, persist in circulation for a longer period and appear to be less susceptible to immune suppression.     

Cellular immune response to sheep erythrocytes: interrelationship between proliferation of popliteal lymph node cells and footpad swelling

Mice were sensitized with graded doses of sheep erythrocytes by the intravenous or subcutaneous route and challenged for delayed-type hypersensitivity (DTH) at different times thereafter. The DTH response as assessed by footpad swelling (FPS) was compared to the spontaneous proliferative response of the popliteal lymph node cells (PLNC). Proliferation of PLNC was optimal after sensitization regimens resulting in optimal FPS. The same was true for mice sensitized under cyclophosphamide modulation. Proliferation of PLNC induced by SRBC was antigen-specific, although some crossreactivity with horse red blood cells was observed. Proliferation of PLNC could be abrogated by treatment with anti-Thy-1.2 antiserum plus complement demonstrating the T cell nature of proliferating cells. In accordance with published data, FPS of mice presensitized with a high dose of SRBC as well as FPS of recipients of spleen cells from high-dose-sensitized donors was suppressed. In marked contrast, PLNC proliferation was not diminished in these mice. Although proliferation of PLNC did not parallel FPS under all circumstances, it seems to be a correlate of the cellular immune response to SRBC.     

Isolation and characterization of Fc-receptors shed from human peripheral mononuclear cells.

Supernatants of human peripheral mononuclear cells containing membrane components shed in consequence of 4--37 degrees temperature shift were used as source for isolation of Fc-receptors (FcR). Aggregated IgG1 myeloma protein and TMV-anti-TMV complexes proved suitable sorbents to adsorb quantitatively and specifically the FcR-s. The isolated FcR interacts only with IgG and not with IgM. No haemagglutination was observed when the isolated FcR was incubated with sensitized human Rh+ red blood cells. Complement dependent lysis of sheep red blood cells was not inhibited by the isolated FcR-s. The interaction between IgG and SpA from Staphylococcus aureus (Cowan I) bacteria was not inhibited when red blood cells sensitized with IgG were preincubated with the isolated FcR-s. The differences between the FcR-like material isolated from supernatants of peripheral human mononuclear cells and those secreted by stimulated T cells or produced by lymphoblastoid cell lines are discussed.     

Dextran sulphate: an adjuvant for cell-mediated immune responses.

The effect of high mol. wt dextran sulphate (DS) on cell-mediated immune responses was studied. Three criteria were used to assess cell-mediated delayed-type hypersensitivity: footpad swelling, i.d. skin tests and macrophage migration inhibitory factor (MIF) production. Guinea-pigs sensitized with egg albumin (EA) and treated with DS showed strong positive delayed skin tests. Control animals given only EA showed negative skin tests. Lymphocytes from mice sensitized s.c. with EA and treated with DS showed an increase in MIF production. Delayed footpad swelling responses in mice sensitized s.c. with sheep red blood cells (SRBC) and treated with DS were increased when the mice were challenged 8 days after sensitization. Doses of DS which were effective in increasing delayed footpad swelling ranged from 50-200 mg DS/kg body weight. DS was only capable of increasing delayed footpad swelling responses when both SRBC and DS were injected s.c. at the same site. Intraperitoneal injection of both SRBC and DS obeebction of both s.c. but at different sites did not result in increased delayed footpad swelling. DS was capable of augmenting footpad swelling responses when given s.c. as much as 6 days before SRBC. The optimal time for administration of DS was 2 days before SRBC. Injection of DS 2 days or more after SRBC resulted in no increase in delayed footpad swelling responses. The results of this study indicate that dextran sulphate is a potent adjuvant for cell-mediated delayed-type hypersensitivity immune responses in both mice and guinea-pigs.     

Effects of contact sensitization and delayed hypersensitivity reactions on immune responses to non-related antigens. Modulation of Immune responses.

Guinea pigs were immunized intracutaneously into the ears with sheep red blood cells (SRBC). Application of a sensitizing dose of the contact allergen dinitrochlorobenzene (DNCB) onto the same ears was shown to suppress or enhance the humoral response to SRBC depending on the time of application. When guinea pigs were sensitized to a contact allergen, application of a sensitizing dose of a non-related allergen on the same ears either had no effect or caused a clear enhancement of the development of delayed type hypersensitivity (DTH). Strongest enhancement was found when both sensitizations were performed on the same day. Further experiments on the effects of a concomitant DTH reaction elicited at the site of application of a contact allergen showed a strong potentiation of DTH when B-cell suppression was minimized by pretreatment with cyclophosphamide (CY). It was considered that CY-DTH-immunopotentiation might be a useful tool for achieving a higher level of sensitivity after epicutaneous sensitization.     

Altered circulation of lymphocytes mediating delayed-type hypersensitivity when primed in Wf/Wf mice.

When mice were homozygous for the Wf allele at W locus, the delayed-type hypersensitivity (DTH) reaction observed in mice primed i.v. or s.c. with optimal doses of sheep red blood cells was greatly decreased. The number of sensitized splenic cells able to transfer a delayed-type hypersensitivity reaction was identical in +/+ and Wf/Wf primed mice. At site of local transfer of +/+ sensitized cells and antigen, the recruitment of monocytes was identical in +/+ and Wf/Wf mice. In contrast, the number of blood cells able to transfer a DTH reaction was decreased by a factor 40 in Wf/Wf primed mice compared to +/+ ones. This decreased number of sensitized cells in blood of Wf/Wf mice revealed a specific defective circulation of Wf/Wf DTH mediating cells whatever the genotypes (+/+ or Wf/Wf) of recipients used to measure this property of circulation.     

Regulation of delayed-type hypersensitivity. II. Specific suppressor factor for delayed-type hypersensitivity to sheep erythrocytes in mice.

An antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is described. Lymph node cells and spleen cells from mice injected intravenously with 1 x 10(9) SRBC 4 days previously were incubated in vitro for 48 h in culture medium. Supernatant obtained from the culture inhibited the induction of DTH to SRBC in normal mice. It also suppressed the expression of DTH in presensitized mice. The suppression is specific as the suppressor factor had no effect on the DTH to noncross-reacting antigen, chicken red blood cells. Treatment of the spleen cells with anti-theta serum and complement prevented the production of the suppressor factor, whereas treatment with anti-Ig serum and complement had no effect. Suppressor factor produced by H-2k mice suppressed the DTH in H-2b mice. The factor thus seems to act across the H-2 barrier. The suppressor factor was not removed by adsorption with goat anti-mouse immunoglobulin immunoadsorbent, but could be adsorbed by SRBC. It was stable at 56 degrees C for 1 h, but was partially inactivated by freezing and thawing. The factor has a molecular weight of less than 35 000 daltons.     

The role of the Fc receptor (FcR) of thymus-derived lymphocytes. II. Presence of FcR on suppressor cells and direct involvement in suppression.

Alloantigen-activated T cells (ATC) were prepared by injecting C3H/He thymocytes into host-irradiated BALB/c mice. ATC were taken from the spleen of the recipients at day 5 and tested for their activity on “In vitro” primary antibody production to sheep red blood cells (SRBC) induced in splenic C3H/He lymphocytes. ATC added 24 h after antigen were found to be suppressive. Separation of ATC by velocity sedimentation, according to cell size showed that suppressor cells were found in each population, medium and large lymphocytes, however, being more active. Separation of ATC, according to the presence of Fc receptor (FcR) at their membrane was achieved by velocity sedimentation after rosetting with IgG-sensitized SRBC (EA). Suppressor cells were then found in FcR⁺ and not in FcR^? fractions, the degree of suppression being parallel to the ratio of FcR⁺ cells. At both extremes, pure populations of FcR⁺ cells were highly suppressive, while populations of ATC, completely devoid of FcR⁺ cells, were inactive. Since FcR is a very labile molecule at the T cell surface and shed within 3 h at 37°C, but not at 4°C, ATC were pre-incubated at each temperature and added 24 h after antigen to the spleen cell cultures. In these cases, ATC having released their FcR, were much less suppressive than control ATC, and in previous work we have shown that the suppressive activity was found in the cell supernatant associated with an FcR-like molecule (immunoglobulin-binding factor). The data therefore allow the conclusion that FcR may be a marker of nonantigen-specific suppressor T cells and seems to be involved itself in the suppression phenomenon.     

Contrasuppressor cells induced by Junin virus.

Intracerebral inoculation of Junin virus (JV) in all susceptible mouse models available induces a lethal meningoencephalitis compatible with a delayed-type hypersensitivity (DTH) immune response. In contrast, adult BALB/c mice prove resistant to infection and no DTH response is seen. JV inoculation in adult BALB/c mice induces DTH suppression to unrelated antigens such as sheep red blood cells. (SRBC). This suppression is mediated by JV-induced T cells (Tsv), which are operative from 1 to 24 days post-infection (p.i.), and seems to be related to adult mouse survival. The presence of JV-induced contrasuppressor cells (CS) bearing the Thy-1+, Ly 1+2- phenotype, able to abrogate Tsv cells-mediated suppression, is described here. Thus, CS cells may be involved in the mechanism by which mice avoid over-exposure to Tsv-mediated DTH suppression. Such CS cells were found in the spleen of inoculated animals and may also be induced by transferring JV-free Tsv cells to 'naive' recipients, in which JV inoculation then induces morbidity.     

Maturation of bone marrow lymphocytes. I. Quantitative rosetting methods of detecting Fc and complement receptors and surface immunoglobulin.

Cytocentrifuge rosetting procedures were developed for use with bone marrow cells to quantitate Fc and complement receptors (FcR, CR) and surface IgM on marrow small lymphocytes. Cell suspensions from 9–11-week-old C3H mice were mixed (1 : 30) with washed sheep red blood cells (SRBC) coated with either mouse anti-SRBC serum (for FcR), rabbit anti-SRBC stroma serum plus mouse serum (for CR) or goat anti-mouse IgM serum (for IgM). Centrifuged cell pellets were incubated for 30 min at either 37°C (FcR, CR) or 0°C (IgM) and examined in stained cytocentrifuge preparations. Small lymphocytes were defined as non-DNA-synthesizing lymphocytes smaller than 10 μm nuclear diameter. Many of these cells in the bone marrow formed specific rosettes for FcR (25%), CR (18%) and surface IgM (39%). The incidence of FcR- and CR-bearing small lymphocytes relative to IgM-bearing small lymphocytes was lower in the marrow than in the blood, spleen and lymph nodes. Rosette size ranged from 4 to > 20 SRBC per lymphocyte but tended to be smaller in the marrow and blood than in the spleen and lymph nodes. The methods and results are discussed as a basis for studies of B lymphocyte maturation and lymphocyte heterogeneity in the bone marrow.     

Regulation of delayed-type hypersensitivity. I. T suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

Mice injected with 1 X 10(8) sheep red blood cells (SRBC) into the footpad showed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after the injection. In contrast, mice injected intravenously with 1 X 10(9) SRBC were unresponsive to DTH induction through 1 X 10(8) SRBC injected into the footpad. This suppression of DTH was maintained for at least 6 weeks and was transferable spleen, lymph node and thymus cells to normal syngeneic recipients. Bone marrow cells, on the other hand, did not contain the suppressor cells. The suppression of DTH was antigen-specific in that DTH to chicken red blood cells and contact sensitivity to 2,4-dinitrofluorobenzene was not affected. The suppressor cells were theta-positive and Ig-negative. They appeared in the spleen in optimum number 3-4 days after induction. The suppressor cells affected both the induction and manifestation of DTH. The presence of suppressor and effector cells for DTH inducible by different routes of antigenic presentation reflects the dynamic balance in the regulation of DTH.     

Identification of lymphocyte subpopulations in E-RFC-enriched and E-RFC-depleted cell fractions of fresh and cryopreserved lymphocytes.

Fresh lymphocytes and frozen-stored lymphocytes were separated into E-RFC-enriched and E-RFC-depleted cell fractions by density gradient centrifugation of sheep red blood cell (SRBC) rosette-forming cells (E-RFC), since the ability to form rosettes is primarily a T cell characteristic. Subpopulations of lymphocytes were identified, demonstrating the presence of cell surface markers: T cell specific antigens (T+), receptors for SRBC on T cells (E-RFC), Fc-receptors (FcR) for IgG type antibodies, and surface Ig (sIg). Our results indicate that, although the E-RFC-depleted fraction contains virtually no cells capable of binding SRBC, there is still a considerable proportion of T cells present in that fraction, as detected with the anti-T cell antiserum. Moreover, data are presented to indicate that the E-RFC-enriched fraction does not consist exclusively of T lymphocytes. Since this separation procedure is used frequently for the identification of the nature of effector cells in cell-mediated (CMC) and antibody-dependent cellular cytotoxicity (ADCC) assays, the identification of T cells in purified lymphocyte fractions by means of SRBC rosette formation may lead to a false conclusion as to the nature of the effector cells.     

Long-lasting enhancement of the delayed-type hypersensitivity response to heterologous erythrocytes in mice after a single injection of cyclophosphamide.

Previous reports have indicated that cyclophosphamide (CY) treatment can enhance delayed-type hypersensitivity (DTH) reactions by abrogating suppressor T cell functions. Such findings have suggested that cells in the suppressor lineage may be particularly sensitive to this alkylating agent. The experiments reported here demonstrate that a single injection of CY before sensitization can induce a long-lasting state of enhanced DTH responsiveness to sheep red blood cells (SRBC) in mice. This enhancement required concurrent antigenic stimulation and appeared to be antigen-specific. Additionally, CY treatment of sensitized mice before the first antigenic challenge for DTH resulted in suppressed responses to that challenge, followed by enhanced DTH to subsequent challenge with the same antigen. The suppressed response was achieved with a lower dose of CY than the subsequent enhancement and also required concurrent antigenic stimulation. These results indicate that the effects of CY on both effector and suppressor mechanisms are critically dependent upon antigenic stimulation, and suggest that apparent suppressor sensitivity to CY may be a function of differential ability to recover from CY treatment in a context of antigenic stimulation.     

T and B lymphocytes in myasthenia gravis.

Peripheral blood lymphocytes from seventeen non-thymectomized and nine thymectomized patients with myasthenia gravis (MG) and thirteen healthy controls were examined for the presence of surface markers characteristic of T and B lymphocytes by rosette formation with sheep red blood cells (SRBC). T cells were identified by their capacity to spontaneously form rosettes with SRBCs. The percentage of B lymphocytes was determined by the erythrocyte antibody complement (EAC) rosette-forming test. The EAC complex was prepared with either whole rabbit anti-SRBC serum or with the IgM fraction of rabbit anti-SRBC serum. The two kind of erythrocyte complement rosette-forming cells (EAC-RFC) are designated erythrocyte-haemolysin-complement RFC (EA(H)C-RFC), and erythrocyte-IgM-complement RFC (EA(M)C-RFC). The percentage of total lymphocytes and T cells was not altered in MG patients. The percentage of 'active' T cells, which have been considered to be more actively involved in cellular immunity, was also similar in MG patients and controls. A significant increase in EA(H)C-RFC occurred in both thymectomized and non-thymectomized MG patients, while in B cells detected by EA(M)C-RFC no alterations were found. The increase in EA(H)C-RFC in lymphocytes from MG patients may be due to an increase in the 19S antibody-forming B lymphocytes or to an increase in T cells which have Fc receptors on their surface.     

Immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with vaccinia virus (neurotropin); enhancing effect on delayed type hypersensitivity response through the induction of Lyt-1+2- T cells

To clarify the immunopharmacological action of an extract isolated from inflamed skin of rabbits inoculated with vaccinia virus (Neurotropin), its effect on delayed type hypersensitivity (DTH) response to sheep red blood cells (SRBC) in mice was examined. Neurotropin enhanced the DTH response in C57BL/6 mice which were low responders to SRBC, but not in either BALB/c or C3H/He mice (high responders) when administered i.p. for 4 consecutive days prior to sensitization. However, Neurotropin did not affect the formation of plaque-forming cells to SRBC in C57BL/6 mice under the condition where it enhanced the DTH response. We further examined the mechanism by which Neurotropin enhanced the DTH response in C57BL/6 mice by means of cell transfer experiments. Spleen cells from mice administered Neurotropin i.p. for 4 days, but not saline, could enhance the DTH response when transferred i.v. into normal syngeneic mice just before sensitization. However, the treatment of the spleen cells with anti-Thy-1.2 + complement (C) or with anti-Lyt-1.2 + C, but not with anti-Lyt-2.2 + C, abrogated its enhancing effect. The depletion of macrophages from the cells had no effect. On the other hand, the spleen cells from mice administered Neurotropin had no enhancing effect in the effector phase of DTH response, and they showed a helper T cell activity in a DTH helper T cell assay system in which cyclophosphamide-treated mice were used as recipients. These results suggest that Neurotropin enhances the DTH response in low responder mice through the induction of Lyt-1+2- DTH helper T cells.