Interferon production and lymphocyte stimulation in human leucocyte cultures stimulated by Corynebacterium parvum

Killed C. parvum organisms stimulated a lymphoproliferative response in human peripheral leucocyte cultures of both adult and cord blood origin. They also induced high titres of interferon in cultures of adult leucocytes, but there was no correlation between the degree of lymphocyte stimulation and of interferon production. A considerable variability between donors was seen in both assays. The amount of interferon produced in C. parvum-stimulated cultures was considerably higher than that stimulated by the T-cell mitogens PHA and Con A and that induced by LPS. The anti-viral protein induced by C. parvum fulfilled the criteria of interferon and appeared to represent type II interferon.     

B8, DR3 antigens and production of human leucocyte migration inhibitory factor (LIF) by mononuclear cells stimulated with concanavalin A (Con A)

LIF release by Con A stimulated mononuclear cells was evaluated in 67 randomly selected healthy Sicilians typed for HLA antigens. The results show that B8 and/or DR3 positive subjects release less LIF than negative ones, suggesting that this immunological response might be controlled by HLA-linked immune response (Ir) gene(s).     

Cellular collaboration in the production of human leucocyte migration inhibition factor.

The participation of cell subpopulations in the expression of leucocyte migration inhibition factor (LMIF) in response to Concanavalin A and Protein A was evaluated for cells isolated from the peripheral blood of five healthy subjects. LMIF activity could not be attributed to the function of T cells, B enriched cells, or monocytes acting alone, or to a combination of B enriched cells and monocytes. The LMIF response was the result of a collaborative event that occurred between T cells and B enriched cells, or between T cells and monocytes.     

The kinetics of T lymphocyte subpopulations in guinea-pigs sensitized with allogeneic transplantation antigens.

The kinetics of specifically sensitized T lymphocytes in the circulation and lymphoid tissues of guinea-pigs immunized with allogeneic transplantation antigens or with synthetic peptide sequence known to induce delayed-type hypersensitivity were documented by the antigen-stimulated active rosette-forming T cell (AgARFC) assay. The results show that immunologically functional cells sensitized to a particular antigen do not remain in the circulation when the antigenic source has been withdrawn. These cells become sequestered in lymphoid tissue and may be recalled into the circulation shortly after the de novo administration of sensitizing antigen. The detection of antigen-sensitive T cells in the circulation was indicative of the presence of and failure to detect these cells and their eventual appearance in lymphoid tissues was related to depletion of the antigenic source.     

Lactoferrin stimulates the production of leucocyte migration inhibitory factor by human peripheral mononuclear leucocytes.

The effect of lactoferrin on the migration of human polymorphonuclear cells was investigated. High concentrations of lactoferrin (greater than 250 micrograms/ml) markedly inhibited the migration of granulocytes under agarose. This migration inhibition could not be neutralized by an antibody against leucocyte migration inhibitory factor (LIF), suggesting a direct effect of lactoferrin on the granulocytes. Low concentrations of lactoferrin were, however, indirectly capable of inhibiting neutrophil migration. The overnight culture of mononuclear leucocytes with low concentrations of lactoferrin (10 micrograms/ml) resulted in the release of granulocyte migration inhibiting factors in the cell free culture supernatant. Strong evidence indicating that the migration inhibiting factors were due to LIF, was obtained in experiments whereby the inhibitory activity could be completely neutralized by anti-LIF antibodies. The lactoferrin-mediated stimulation of LIF release by mononuclear leucocytes could be neutralized by an anti-lactoferrin serum, but not by an anti-albumin serum, whereas PPD-induced LIF release was not affected by either antiserum. These findings suggest that lactoferrin besides its well known anti-microbial properties, may also play a regulatory role in the migratory response of polymorphonuclear cells during inflammatory conditions.     

Specific blastogenesis and lymphokine production in DNCB-sensitive human leucocyte cultures stimulated with soluble and particulate DNP-containing antigens.

A soluble hapten (dinitrobenzene sulphonic acid (DNBSO3)) and particulate antigens consisting of DNCB, DNFB or DNBSO3 complexed with erythrocytes, all induce a specific blastogenic response and lymphokine production in leucocyte cultures from human subjects topically sensitized to dinitrochlorobenzene (DNCB). While low concentrations (50-100 mug/ml) of DNBSO3 could be left in human leucocyte cultures the entire 4 or 5 days of culture and result in reasonable levels of blastogenesis, it was found that consistently higher degrees of blast transformation resulted when DNCB-sensitive leucocytes were exposed to high concentrations of DNBSO3 (500 mug/ml) for a short period (2 hr). Cell-free supernatants from DNCB-sensitive leucocyte cultures harvested after 48 hr induced blastogenesis and DNA synthesis in secondary target leucocyte cultures from subjects not sensitized to DNCB. Such a blastogenic factor or lymphokine appeared to stimulate even in the absence of any residual antigen, since DNCB complexed to erythrocytes was removed by simple filtration through a 0-45 mum Millipore filter. In contrast to DNCB complexes, the antigenic activity of DNBSO3 complexed with erythrocytes was not removed by such filtration. Thus, several DNP-containing haptens (DNCB, DNFB, DNBSO3) induce specific lymphocyte transformation and lymphokine production when exposed in several different manners to leucocytes from humans sensitized to DNCB. The ability to use either a particulate or soluble stimulant in vitro offers a versatile system for studying cell-mediated immunity in humans with a broad range of potential applicability in both investigate and clinical medicine.     

Monocyte subsets in the production of inhibitory factor by Candida albicans-activated human T cells.

Macrophages are essential for the proliferative response of human T lymphocytes to a purified polysaccharide extract from Candida albicans (MPPS). The role of macrophages as antigen-presenting cells in the production of an antigen non-specific inhibitory factor (nsINH) by MPPS-activated T cells was also investigated. Fc receptor positive (FcR+) or negative (FcR-) plastic-adherent mononuclear cells were used as MPPS-presenting cells, and the results show that the FcR- subset is mainly responsible for the release of nsINH from activated T cells.     

Differentiation antigens in rat lymphocyte subpopulations recognized by rabbit antisera against electrophoretically separated cells.

Rat lymphocytes of high (H cells) and low (L cells) electrophoretic mobility (EPM) from lymph node (LN), spleen (Spl) and thymus (Thy) were separated by free flow electrophoresis and antisera against these cells raised in rabbits. Eight distinct lymphocyte-specific xenoantigens could be characterized by appropriate cross-absorption of three antisera: anti-Thy-L cell IgG, anti-LN/-Spl-H cell IgG and anti-LN/Spl-L cell IgG. Cross-absorption of anti-Thy-L cell IgG with unseparated LN or Spl cells revealed a thymus-specific antigen complex which was absent from peripheral rat lymphocytes but present on mouse thymocytes and peripheral T cells. This complex could be further separated into brain-associated and nonassociated antigens. The brain-associated antigens consisted of a rat-specific component, a component shared by the rat and all mice which carry the Thy-1.1 and Thy-1.2 allogenic specificity, and a third component shared by rat and Thy-1.1 mice. This Thy-1 antigen complex showed a 26-fold larger antibody-binding capacity to thymocytes of low EPM than to those of high EPM. The thymic antigens which were not absorbed by brain tissue showed similar antibody-binding capacity to all thymocytes whether of low or high EPM. Anti-LN/Spl-H cell IgG cross-absorbed with LN/Spl-L cells detected antigens which were localized on all rat peripheral H cells and thymocytes but which were absent from peripheral L cells. Further absorption with Thy-L cells revealed a distinct subclass of antigens present on peripheral H cells and Thy-H cells but absent from Thy-L cells. On the other hand, cross-absorption of anti-LN/Spl-L cell IgG with peripheral H cells provided specificities which were directed against all peripheral L cells but which did not react with peripheral H cells and thymocytes. The relationship of these different xenoantigens to known lymphocyte subpopulations is discussed.     

Response of sensitized and unsensitized human lymphocyte subpopulations to Plasmodium falciparum antigens.

Antigen preparations derived from Plasmodium falciparum-infected erythrocytes (but not from uninfected erythrocytes) can stimulate the in vitro proliferation of peripheral blood lymphocytes from malaria-sensitized as well as nonsensitized donors. The possibility that the nonspecific responses might be due to a parasite-derived B-cell mitogen has been previously suggested since polyclonal hypergammaglobulinemia is a frequent accompaniment of malaria infection. To test this hypothesis, we investigated the in vitro proliferative responses of purified T- and B-cell populations to malaria antigens. T but not B cells responded to the antigens. The addition of small numbers of T cells restored the ability of purified B cells to respond to lectin mitogens but not to malaria antigens. Falciparum malaria infection was associated with an increase in T-cell but not in B-cell proliferation in vivo, as assessed by the spontaneous tritiated thymidine incorporation of lymphocytes during a brief incubation in vitro. Our observations suggest that extracts of malaria parasites do not contain a B-cell mitogen but are antigenic as well as mitogenic for T cells.     

Antibody production and DNA synthesis of human lymphocyte subpopulations induced by PPD tuberculin.

Purified protein derivative (PPD) of tuberculin was found to induce antibody secretion and DNA synthesis in human lymphocytes from blood, spleen, tonsil and lymph node. Antibody secretion was measured as plaque-forming cells (PFC) against fluorescein isothiocyanate (FITC) coupled sheep red blood cells (SRBC) in a haemolysis-in-gel assay. Peak antibody secretion by 100 micrograms/ml of PPD was usually seen on day 6 for blood lymphocytes, and varied from day 3 to day 6 for spleen cells. Peak DNA synthesis for blood lymphocytes stimulated by various concentrations of PPD ranged from day 4 to day 7. The highest number of PFC in tonsil and spleen cells was induced by PPD compared to Staphylococcus aureus bacteria Cowan 1, lipopolysaccharide (LPS) and pokeweed mitogen (PWM). Antibody secretion by PPD was not affected when phagocytic cells were removed by iron treatment. PPD stimulated a higher DNA synthesis in unseparated lymphocytes or mixtures of T and B cells than in enriched T or B cell suspensions. PPD did not induce PFC in B cells enriched by the removal of sheep erythrocyte rosette-forming cells (E-RFC). However, more PFC were stimulated by PPD in enriched E-RFC compared to unseparated lymphocytes, which may indicate that most of the FITC-SRBC reactive B cells also form rosettes with SRBC.     

The production of migration inhibitory factor and reproductive capacity in allogeneic pregnancies.

Migration inhibitory factor (MIF) is produced during allogeneic pregnancies but not during syngeneic pregnancies. Removal of the paraaortic or paraaortic and renal lymph nodes significantly decreased MIF production whereas splenectomy did not. Removal of these regional lymph nodes decreased the mean litter size and increased the variance in the weights of the offspring, with the greatest changes occurring when both the paraaortic and renal lymph nodes were removed; splenectomy did not alter either parameter. None of the surgical procedures affected the gestation period significantly, but removal of the paraaortic and renal lymph nodes greatly reduced the rate of conception and increased the incidence of stillbirths. These findings support the proposition that a vigorous immune response occurs during allogeneic pregnancies and that this response provides reproductive advantages to the offspring.     

Kinetics and characterization of interferon production by murine spleen cells stimulated with Legionella pneumophila antigens.

Formalin-killed Legionella pneumophila bacterial cells, as well as a purified cell wall preparation (designated F-1 antigen) containing lipopolysaccharide (LPS), stimulated production of interferons (IFNs) in mouse spleen cell cultures. L. pneumophila whole-cell vaccine induced an IFN that was pH 2 labile and neutralized by anti-IFN-gamma indicating that IFN-gamma was the dominant form present. F-1 antigen induced a mixture of IFNs, depending upon the age of the culture and cell types present. In freshly prepared whole-spleen cultures and in 2-h adherent cultures, F-1 induced predominantly IFN-alpha/beta. In whole-spleen cultures that were allowed to age for 24 to 48 h before stimulation, F-1 was seen to induce mostly IFN-gamma, with low levels of IFN-alpha/beta present. Since only IFN-alpha/beta was produced in T-cell-depleted populations (at 2 h or at 48 h), it is suggested that T cells are responsible for IFN-gamma production in aged cultures. Additionally, heat-treated F-1, Escherichia coli LPS, and heat-treated E. coli LPS all induced similar levels of IFN-gamma in whole-splenocyte or nonadherent cell cultures which were incubated 48 h before stimulation. This suggests that LPS present in F-1 is responsible for IFN-gamma production and that an activated cell population is required. These results show that L. pneumophila antigens can induce the production of various types of IFN in mouse spleen cell cultures through several mechanisms.     

Evolutionary development of lymphocyte heterogeneity: leucocyte subpopulations in the Pacific hagfish

Flow cytometric analysis of forward angle versus 90 degree scatter patterns of hagfish peripheral blood revealed two distinct leucocyte populations with size characteristics analogous to mammalian monocytes/granulocytes (hagfish large leucocytes) and small lymphocytes (hagfish small leucocytes). A cell population enhanced for the small leucocytes was obtained by density gradient centrifugation. Over 70% of the small leucocyte population consistently stained with a rabbit antiserum directed against polypeptide determinants on hagfish immunoglobulin, while staining of the large cell population was greatly reduced (less than 10%). A panel of monoclonal antibodies raised against a crude hagfish leucocyte preparation distinguished the two cell populations and revealed the existence of subpopulations of both small and large leucocytes.     

The effects of cyclic AMP on leucocyte inhibitory factor (LIF) production and on the inhibition of leucocyte migration.

The effect of drugs known to increase intracellular levels of cyllic AMP were studied in the leucocyte migration ihibition system. It was found that cyclic AMP, dibutyryl cyclic AMP, theophyline, and prostaglandins E1 and E2 inhibited the production of leucocyte inhibiting factor by HA pulsed lymphocytes Inhibition only occured when the drugs were present during or after the PHA pulse. In addition it was found that these drugs enhanced the migration of polymorphonuclear leucocytes (PMN), in this system. Electrophoretic mobility of PMN cells was not altered by these drug indicating that the effect is not due to changes in membrane charge. However, granulocyte adhesion was reduced in the presence of these drug suggesting that adhesion is of primary importance in the migration of polymorphonuclear leucocytes out of capillary tubes. The findings show that cyclic AMP is important in modulating both cell-mediated and inflammatory responses.     

Tumor necrosis factor production by human T-cells stimulated with bacterial superantigens

Tumor necrosis factor (TNF) production from T-cells stimulated with superantigenic exotoxins, staphylococcal enterotoxin B and streptococcal pyrogenic exotoxin A was investigated in the presence of cells bearing distinct isotypes of HLA class II molecules. The main T-cell subset for TNF production was investigated in parallel. Similarly high levels of TNF production were induced upon stimulation with the toxins in the presence of DR⁺ or DQ⁺ cells, but only marginal levels of TNF production were induced in the presence of DP⁺ cells. Although both CD4⁺ T-cells and CD8⁺ T-cells produced TNF-α and TNF-β in response to toxin stimulation in the presence of HLA class II⁺ cells, the former T-cell subset was the major source of producers of TNF-α and TNF-β.     

Distribution of Ia antigens and T lymphocyte subpopulations in rat lungs.

In order to examine the mechanism of specific immunity in the lung, the distribution of Ia antigens and T lymphocyte populations was determined using immunoperoxidase-staining of cryostat sections of lungs from specific pathogen-free rats. BALT was found to be divided into three regions of lymphoid tissue. The central region was primarily composed of B cells, and was surrounded by a peripheral region of T cells (MRC OX-19+) which included both T helper (W3/25+) and T suppressor/cytotoxic (MRC OX-8+) cells. The subepithelial region contained a dense network of W3/25+, non-T cells. A majority of BALT cells, including the lymphoepithelial cells, were Ia+. The alveolar walls were found to contain numerous Ia+ dendritic-shaped cells. Alveolar macrophages found in sections, as well as those collected using bronchoalveolar lavage, were Ia- and W3/25-. Mechanisms for the induction of immunity within both BALT and the alveolar region are proposed.     

Lymphocyte blastogenesis and interferon production in adult human leukocyte cultures stimulated with cytomegalovirus antigens.

Lymphocyte blastogenesis and interferon production were measured in adult human leukocyte cultures stimulated with purified or crude cytomegalovirus antigens. Leukocytes from seropositive adults underwent blastogenesis when stimulated with purified or crude Towne strain antigen, whereas neither antigen stimulated blastogenesis in cultures from seronegative donors. The concentrations of antigens yielding maximal blastogenesis varied among the individuals tested. When cultures from seropositive individuals were stimulated with antigens prepared from three different CMV strains--AD-169, Towne, and Davis--comparable levels of blastogenesis were detected. Type 1 interferon was detected in supernatants of cultures stimulated with crude antigens regardless of the immune status of the donor. In contrast, when purified antigen was used as the stimulant, only cultures obtained from seropositive individuals produced detectable levels of interferon, which appeared to be predominantly type 2 or immune interferon.