Immunocompetent Cells in Man. IV. The Inhibition of Pokeweed Mitogen-Induced Blastogenesis of Normal Human Peripheral Leukocytes by Trypsin: A B-Cell Mitogen of Mouse, but not of Human, Lymphocytes

Normal human peripheral blood lymphocytes were tested for their blastogenic response in vitro to trypsin. Although it has been shown by other investigators that mouse B lymphocytes are stimulated by trypsin to undergo blastogenesis, we have shown that human lymphocytes are not stimulated by trypsin. Paradoxically, trypsin inhibits the response of human lymphocytes to pokeweed mitogen, a B-cell stimulant, without significantly affecting the response of these cells to phytohemagglutinin, a T-cell stimulant. These results serve to underline the functional dissimilarity of mouse spleen cells and human circulating lymphocytes, at least with respect to their blastogenic responses to mitogenic agents.     

Effects of Simple Imidazoles on Human Peripheral Blood Lymphocytes Stimulated by Mitogen or Allogeneic Cells

Five imidazole compounds were added to cultures of human lymphocytes which had been stimulated to undergo blast transformation by exposure to phytohaemagglutinin, poke-weed mitogen or allogeneic cells. Two compounds, clotrimazole and dacarbazine (DTIC) produced a dose related suppression of these responses. Nirnorazole was largely inactive whereas metronidazole and tinidazole actually enhanced the response -at least in those cultures stimulated by the plant mitogens. It is suggested that experiments of this kind are helpful in identifying those imidazole compounds that could be used as immunosuppressants in vivo.     

Differential Morphology of Mouse Spleen Cells Stimulated In Vitro by Endotoxin, Phytohemagglutinin, Pokeweed Mitogen and Staphylococcal Enterotoxin B

The in vitro mitogenic effects of endotoxin (LPS), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and staphylococcal enterotoxin B (SEB) on mouse spleen cells were studied by light and electron microscopy. By light microscopy, LPS was found to “transform” a culture largely composed of small lymphocytes into “large lymphocytes” which did not synthesize DNA and into blast cells which did synthesize DNA. The blast cells were morphologically similar to those transformed by PHA, PWM and SEB. By electron microscopy, a large percentage of the endotoxin-transformed cells developed extensive dilated rough ER. Their appearance was similar to that reported by others for cells stimulated by PWM, but in our hands the development of rough ER was more conspicuous in LPS-stimulated cells. In contrast, PHA and SEB produced few blast cells which contained significant rough ER. The morphologic observations are consistent with the hypothesis that the mitogenic effect of endotoxin is on bone-marrow-derived lymphocytes. This effect may relate to the known adjuvant effect of endotoxin in vivo.     

Dendritic Cells from Human Rheumatoid Synovial Inflammatory Tissue and Peripheral Blood as Accessory Cells in Mitogen Stimulation of T Lymphocytes.

Dendritic cells (DC) were purified from the peripheral blood (PB) of normal individuals and from the synovial fluid (SF) and synovial tissue (ST) of patients with rheumatoid arthritis. These cells are strongly HLA-DR positive and lack B-cell, T-cell, and monocyte markers as well as Birbeck granules. The DC were compared with monocytes and non-T cells from PB for their ability to act as accessory cells for T-cell responses to concanavalin A (Con A) and phytohaemagglutinin (PHA). DC from PB, SF and ST were much more efficient accessory cells for the mitogenic responses than autologous monocytes from PB. The mean PHA responses in cpm obtained with DC from the various compartments were 4-20 times greater than the responses obtained with monocytes from PB. The Con A responses obtained when the various DC populations were used as accessory cells were 3-13 times greater than those obtained with monocytes from PB. The mitogenic responses seen with monocytes were very low. The non-T cells, which comprise a mixture of cells obtained after removal of T cells, also gave low T-cell responses to PHA and Con A compared with DC as accessory cells.     

Destruction of Dextran-Coated Target Cells by Normal Human Lymphocytes and Monocytes.

A human anti-dextran serum, EAK, with IgG antibodies restricted to subclass IgG2, was tested for its capacity to induce lysis of dextran-coated chicken erythrocytes by normal human lymphocytes or monocytes. Another human anti-dextran serum, RGM, with most antibodies belonging to sublass IgG1, and a hyperimmune rabbit anti-dextran serum were used for reference. In lymphocyte-mediated erythrolysis, serum EAK gave rise to 51-Cr release varying from 20% to 80% in different experiments. The hyperimmune rabbit serum was 100 to 1000 times more active, whereas serum RGM was consistently negative. These results correlated well with the concentration of anti-dextran antibodies in these sera. In monocyte-mediated erythrolysis serum EAK had a somewhat higher titer than in lymphocyte-mediated lysis, and serum RGM had a weak but significant activity at low dilutions. Serum EAK also induced erythrophagocytosis by monocytes. Ultracentrifugation did not significantly decrease the inductive capacity of this serum. The results show that antibodies of human sublass IgG2 are efficient inducers of effector functions in both lymphocytic and monocytic cells. Myeloma proteins of the four IgG subclasses were tested for inhibitory capacity in lymphocyte- or monocyte-mediated erythrolysis. Either serum EAK or the rabbit reference serum was used for induction of erythrolysis. Individual myeloma proteins within and between the subclasses varied considerably in inhibitory power. However, whereas IgG1, IgG2, and IgG3 proteins inhibited lymphocyte-mediated erythrolysis induced by either type of antiserum, the two IgG4 proteins tested were essentially negative. These results suggest a lack of specificity of the Fc receptor for subclasses IgG1, IgG2, and IgG3 in both heterologous and homologous inhibition. In monocyte-mediated erythrolysis, IgG1 and IgG3 were strong inhibitors, whereas inhibition by IgG2 and IgG4 was weak and inconsistent. This pattern was seen regardless of whether and inducing antiserum was of rabbit or human origin. Similar results were obtained in monocyte-induced erythrophagocytosis induced by serum EAK. These and previous results suggest that effector cells of the lymphocytic (K cell) variety have Fc receptors different from those of monocytic cells. However, the basis for the differences observed in the inhibition tests remains to be elucidated.     

Polyclonal Activation of Human B Lymphocytes in Vitro by Pokeweed Mitogen: a Simple Technique for the Simultaneous Assessment of Cell Proliferation, Generation of Plasma Cells, Plaque-forming Cells and Immunoglobulin Production

A simple technique has been worked out for the simultaneous assessment of cell proliferation, generation of plasma cells, plaque-forming cells and immunoglobulin production from single cultures of 1 X 10(6) human peripheral blood lymphocytes stimulated with pokeweed mitogen (PWM). Kinetic studies showed that number of cells, thymidine incorporation rate, number of plaque-forming cells against fluorescein-isothiocyanate-haptenated sheep erythrocytes and concentration of IgM and IgG in the supernatant peaked on the average between day 5 and 7 of culture. This technique is particularly suitable for the analysis of hypogammaglobulinaemias in infancy.     

Immunocompetent cells in man. IV. The inhibition of pokeweed mitogen-induced blastogenesis of normal human peripheral leukocytes by trypsin: a B-cell mitogen of mouse, but not of human, lymphocytes.

Normal human peripheral blood lymphocytes were tested for their blastogenic response in vitro to trypsin. Although it has been shown by other investigators that mouse B lymphocytes are stimulated trypsin to undergo blastogenesis, we have shown that human lymphocytes are not stimulated by trypsin. Paradoxically, trypsin inhibits the response of human lymphocytes to pokeweed mitogen, a B-cell stimulat, without significantly affecting the response of these cells to phytohemagglutinin, a T-cell stimulant. These results serve to underline the functional dissimilarity of mouse spleen cells and human circulating lymphocytes, at least with respect to their blastogenic responses to mitogenic agents.     

A Trypanosoma cruzi Alkaline Antigen Induces Polyclonal B-Cell Activation of Normal Murine Spleen Cells by T-Cell-Independent, BCR-Directed Stimulation

We have previously reported that a cytosolic alkaline fraction (FI) obtained from epimastigotes of Trypanosoma cruzi promotes the activation, proliferation and differentiation of normal murine B cells into antibody-secreting plasmocytes. Neither the mechanism nor the cells involved in the FI-induced polyclonal B-cell activation were established. In this work we report that accessory cells are required for FI-induced polyclonal B-cell activation as no proliferative responses were obtained following treatment of normal spleen mononuclear cells (NSMC) with L-leucine methyl ester. Furthermore, FI did not induce the expression of CD25 on T cells and it promoted the proliferation of a T-cell-depleted population, indicating that it acts in a T-independent manner. We observed that NSMC were stimulated in vitro by FI-released cytokines, such as interleukin (IL)-4, IL-6 and IL-10, which are involved in B-cell proliferation and differentiation. Interestingly, while significant amounts of interferon-gamma (IFN-gamma) were found in culture supernatants we did not observe detectable levels of IL-2. Additionally, we found that B-cell receptor (BCR) and major histocompatibility complex (MHC) class II antigens were involved in the proliferative response induced by FI because antibodies directed against cell-surface immunoglobulin M (IgM), CD45 and MHC class II molecules inhibited the FI-induced B-cell proliferation. CD40 ligand (CD40L) did not participate in such a phenomenon.     

Inhibition of Chemical Mediator Release from Human Leukocytes and Lung in Vitro by A Novel Antiallergic Agent, Kb-2413

The effect of KB-2413 on IgE-mediated histamine and LTC₄, release from leukocytes obtained from asthmatic patients who were sensitive to mites, and from human lung tissues passively sensitized with IgE myeloma serum was studied. KB-2413 inhibited the IgE-mediated chemical mediator release concentration dependently at a range of 10^-4 to 3±10^-3 M. KB-2413 did not enhance histamine release at a higher concentration unlike ketotifen. These findings suggest that the inhibitory effect of KB-2413 on the release of chemical mediators contributes to the anti-allergic activity of this compound.     

Serum activity of prolyl endopeptidase, but not of dipeptidyl peptidase IV, is decreased by immunotherapy with IFN-alpha in high-risk melanoma patients.

Immunotherapy with interferon-alpha (IFN-alpha) induces neuropsychiatric side effects, most notably depression. In hepatitis patients treated with IFN-alpha, severity of depression correlates with a decrease in serum activity of dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5), a membrane-bound protease involved in the cleavage of cytokines and neuroactive peptides. Abnormal serum activity of the cytosolic peptidase prolyl endopeptidase (PEP, EC 3.4.21.26, postprolyl cleaving enzyme, prolyl oligopeptidase) has been documented in patients with a variety of psychiatric disorders, most consistently in mood disorders. The serum activity of PEP and DPP-IV was measured before and after 4 weeks of high-dose induction treatment with IFN-alpha in 18 patients with high-risk melanoma. In this exploratory study, we show a clear decrease in the serum activity of PEP after 4 weeks of treatment with IFN-alpha. This decrease was not related to changes in hematologic parameters. In contrast, serum activity of DPP-IV did not change. Further studies focusing on a possible role of PEP in the pathophysiology of IFN-alpha-induced depression are warranted.     

The Availability for Recognition of Normal H-2 Antigens by Cytotoxic T Lymphocytes on a Rauscher-Virus-transformed Cell, RBL-5A

The availability for recognition of cell surface H-2Db antigenic determinants on RBL-5A cells in comparison to normal C57BL/L cells was investigated by an in vitro immunological assay. No differences in the immunological recognition of the H-2Db antigens on RBL-5A cells compared to normal C57BL/L cells were detected. This assay system, however, readily detected changes in the H-2Kb determinant profile on target cells from the C57BL/L H-2Kb region mutant strains H(zl) and H(zl70) when compared to normal C57BL/6 mice. The result obtained with RBL-5A target cells therefore suggests that Rauscher murine leukaemia virus transformation does not induce, either genotypically or phenotypically, an immunologically recognizable alteration of the H-2Db antigen profile on this cell.     

The Marginal Blood Pool of the Rat Contains not only Granulocytes, but also Lymphocytes, NK-Cells and Monocytes: a Second Intravascular Compartment, its Cellular Composition, Adhesion Molecule Expression and Interaction with the Peripheral Blood Pool

To leave the blood, leucocytes marginate to the vessel wall. Granulocytes thereby form the so-called marginal pool. It is unclear to what extent such a second intravascular compartment also exists for lymphocyte subsets, NK-cells and monocytes. Samples of the peripheral blood and the marginal pool of the LEW rat were analysed by flow cytometry. In the marginal pool the percentage of granulocytes and monocytes was significantly higher compared to that of the peripheral blood, and the proportion of 'naive' T and B lymphocytes was decreased. The expression of LFA-1 was higher on all leucocyte subsets of the marginal pool except the granulocytes, whereas no differences were seen for the expression of other adhesion molecules (α₄-integrins, ICAM-1, CD2, L-selectin, and CD44). In addition, splenectomy influenced the cellular composition of peripheral blood and marginal pool differently and, after injection of blood leucocytes, these cells were found in both compartments showing its characteristic cellular composition. Thus, not only granulocytes, but also B and T lymphocyte subsets, NK-cells and monocytes form a second distinct intravascular compartment. This marginal pool probably influences the cellular composition of leucocyte subsets available for entry into the tissues.     

The protein tyrosine kinase inhibitor herbimycin A, but not genistein, specifically inhibits signal transduction by the T cell antigen receptor.

Several lines of evidence implicate a regulatory tyrosine phosphorylation in the activation of phospholipase C (PLC) by the T cell antigen receptor (TCR). These include studies using inhibitors of protein tyrosine kinases (PTKs). In Jurkat T cells expressing the heterologous human muscarinic receptor (HM1), PLC activity can be induced by either the TCR or HM1. HM1 activates PLC via a guanine nucleotide binding protein. We have studied the selectivity of the effects of the PTK inhibitors, herbimycin A and genistein, in this system. The results indicate that these inhibitors have different mechanisms of action, and suggest that herbimycin A, but not genistein, is a specific inhibitor of PTKs in T cells. Herbimycin A markedly inhibited both the resting and induced levels of phosphotyrosine-containing proteins, including the gamma 1 isozyme of PLC and the zeta chain of the TCR, and prevented activation of PLC by anti-TCR mAb. Herbimycin A did not inhibit activation of PLC by HM1. Genistein had a much less pronounced effect than herbimycin A on the appearance of tyrosine phosphoproteins. Moreover, genistein inhibited activation of PLC by both the TCR and HM1, and inhibition was only partial. Genistein was cytotoxic and markedly inhibited protein synthesis in both Jurkat cells and human peripheral lymphocytes. Herbimycin A was not cytotoxic. These findings confirm the role of a regulatory tyrosine phosphorylation in activation of PLC by the TCR. Herbimycin A was a selective inhibitor of a subclass of PTKs in Jurkat cells. In contrast, inhibition of signal transduction and later events in T cells by genistein may be due to effects other than direct inhibition of PTK activity.     

Mitogen-Induced Interleukin 2 and Gamma Interferon Production by CD4+ and CD8+ Cells of Patients with Inflammatory Arthritides. A Comparison between Cells from Synovial Fluid and Peripheral Blood

The purpose of this study was to investigate interleukin 2 (IL-2) and gamma interferon (IFN-gamma) production by purified CD4+ and CD8+ cells isolated from peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and other inflammatory arthritides (non-RA). CD4+ and CD8+ cells were selected positively by immunomagnetic separation. Supernatants of unstimulated CD4+ and CD8+ cells from both compartments did not contain any detectable IL-2 or IFN-gamma, while supernatants of CD4+ and CD8+ cells stimulated with phytohaemagglutinin and irradiated Raji cells mostly contained both cytokines. In vitro stimulated SF CD4+ cells gave supernatants with significantly less IL-2 than supernatants from PB CD4+ cells, while in vitro stimulated SF CD4+-cell supernatants contained significantly more IFN-gamma. SF CD4+-cell supernatants contained significantly more IL-2 than the parallel CD8+ supernatants, while there was no significant difference with regard to IFN-gamma content. The pattern of differences between SF- and PB-derived T cells was the same for the two groups of patients, but the SF CD4+ cells from RA patients produced significantly less IL-2 than the corresponding cells from the non-RA group. The difference between SF and PB T cells with regard to lymphokine production is probably related to various degrees of in vivo pre-activation. The results do not indicate a major T-cell deficiency in relation to lymphokine production in RA.     

The interferon-inducible 204 gene, a member of the Ifi 200 family, is not involved in the antiviral state induction by IFN-alpha, but is required by the mouse cytomegalovirus for its replication.

To examine whether Ifi 200 genes are involved in antiviral state induction by IFNs we expressed mutant forms capable of inactivating the endogenous p204 and analyzed replication of both RNA and DNA viruses following IFN-alpha treatment. Inactivation of p204 does not impair replication of vesicular stomatitis virus, encephalomyocarditis virus, ectromelia virus, and herpes simplex virus 1 and does not alter an IFN-alpha induced antiviral state. By contrast, in cells lacking functional p204, mouse cytomegalovirus (MCMV) replication is strongly inhibited and is not further modulated by IFN-alpha. These results suggest that p204, a member of the Ifi 200 gene family, is not involved in the IFN-alpha-induced antiviral activity against some RNA or DNA viruses, but is required by MCMV for its replication.     

Brefeldin A, but not monensin, completely blocks CD69 expression on mouse lymphocytes: efficacy of inhibitors of protein secretion in protocols for intracellular cytokine staining by flow cytometry.

Flow cytometry is increasingly used for cytokine detection where it serves to complement ELISA (enzyme-linked immunosorbent assay) and ELISPOT assays. Since it is possible to stain both extracellular epitopes and intracellular cytokines on the same cells, this is a powerful technique for analysing cytokine expression in defined cell populations. However unstimulated cells do not express cytokines. Thus, appropriate stimulation is a prerequisite for studying cytokine expression. Here phorbol 12-myristate 13-acetate (PMA)/ionomycin in vitro stimulation has been applied. In order to accumulate the cytokines within the cells, protein secretion needs to be inhibited, by the addition of reagents that inhibit protein secretion during the stimulation. The two most widely used reagents are monensin and brefeldin A (BFA). These reagents differ somewhat in their mode of action, which might explain their different effects. Monensin is an inhibitor of trans-Golgi function, while BFA inhibits protein transport between the endoplasmic reticulum (ER) and the Golgi. CD69, a very early activation marker on lymphocytes and neutrophils, was monitored in order to measure the efficacy of the protein secretion inhibition. Here we report that: (a) BFA, but not Monensin, is able to completely block extracellular CD69 expression on mice splenocytes after in vitro stimulation with PMA/ionomycin; (b) Monensin is more toxic than BFA and increases the relative amount of CD4+ cells due to a more profound increase in dead cells in the CD4- population; (c) CD69 is a useful marker when setting up intracellular staining of cytokines for flow cytometry.