Search

The assessment of intracellular cytokines at the single-cell level by flow cytometry has recently become a potent tool in many areas of cell biology and in defining the role of cytokines in various human diseases. Three-color flow cytometry for detection of intracellular cytokines combined with simultaneous determination of lymphocytes (CD3(+) and CD4(+)) or monocytes (CD33(+) and CD14(+)) was used for comparison of phytohemagglutinin (PHA)-and phorbol myristate acetate (PMA)-ionomycin-induced production of intracellular cytokines in peripheral blood mononuclear cells (PBMCs) of healthy donors. We found that the number of PBMCs stained for tumor necrosis factor alpha and gamma interferon after 6 h of activation was higher when PMA-ionomycin was used for stimulation, while the frequencies of cells positive for interleukin 4 (IL-4) were similar for both stimulators. However, PMA-ionomycin stimulation caused prominent alterations of cell morphology and membrane expression of CD4 and CD14. In contrast, PHA did not cause downregulation of surface markers and resulted in less pronounced alterations in both forward and side scatter signals during flow cytometry analysis. Moreover, during 48 h of culture PHA stimulated tumor necrosis factor beta and IL-10 production, which was not observed when PMA-ionomycin was used. We conclude that the use of PHA for cell activation may limit in vitro artifacts and allow more precise analysis of intracellular cytokine production in various disease states.

Mycobacterium tuberculosis and human immunodeficiency virus type 1 (HIV-1) are virulent intracellular pathogens that invade and multiply within macrophages. The effect of M. tuberculosis on HIV-1 infection and replication was analyzed in vitro using human monocyte-derived macrophages (MDM) isolated from peripheral blood mononuclear cells by countercurrent centrifugal elutriation. Preinfection of MDM with M. tuberculosis followed by HIV-1 infection resulted in an increase in p24 release, reverse transcriptase activity, and infective virus production. In contrast, no increase in HIV-1 production was observed when MDM were infected with Mycobacterium avium complex or heat-killed M. tuberculosis. Coinfected MDM were potent stimulators of T cell proliferation, while HIV-1-infected MDM failed to present exogenous tuberculin to T cells. Furthermore, coinfected MDM showed an increased capacity to transmit HIV-1 to activated T cells. These results suggest that M. tuberculosis infection can both up-regulate HIV-1 infection and replication within MDM and increase the efficiency of virus transmission from infected MDM to T cells.

The question was asked whether tumour necrosis factor alpha (TNF) is involved in regulation of interferon gamma (IFN gamma) production by T cells. Monocytes were exposed to exogenous TNF or to TNF synthesis inhibitors (pentoxifylline, PTX and adriamycin, ADR) and then used as antigen (PPD) presenting cells for autologous T cells. The ability of T lymphocytes to release IFN gamma was assessed after 3 days of culture. Preincubation of monocytes with rTNF enhanced their ability to induce IFN gamma production while TNF synthesis inhibitors decreased it. Anti-TNF and anti-TNF-R2 monoclonal antibodies (mAbs) inhibited monocyte ability to present PPD for IFN gamma production which suggested that endogenously produced TNF by monocytes had to be released and acted on TNF-R2 on the monocyte surface. The enhancing effect of exogenous TNF was also abrogated by anti-TNF-R2 mAb. Pretreatment of monocytes with rTNF enhanced, while pretreatment with PTX decreased, PPD-induced IL-6 production. An increased production of IL-4 was found in cultures of PTX-treated, PPD-pulsed monocytes with T cells. This may indicate that in the relative absence of monocyte costimulatory signal(s), probably IL-6, Th2 cells are stimulated. These results indicate that TNF is involved in control of monocyte-mediated regulation of cytokine production by T cells.

T lymphocytes can kill antigen-presenting cells (APC) in the presence of antigen or lectin. The subject of this study was to investigate whether the state of activation or phenotype of monocytes, influence their susceptibility to killing by T cells activated with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody. The data are presented which show that monocytes activation with cytokines (IFN-gamma, IL-4, or IL-2), PPD, phorbol ester or phagocytic stimulus, have no influence on monocyte susceptibility to killing by T lymphocytes. Furthermore, flow cytometry data suggest that monocytes eliminated from culture have no characteristic phenotype. In conclusion, our data indicate that elimination of monocytes by activated T lymphocytes does not depend on the state of activation of monocytes.

Monocyte subpopulations which differ in the expression of Fc receptor for human IgG (FcRI) differentially regulate the T-cell-dependent, pokeweed mitogen (PWM)-induced, polyclonal B-cell response. We, thus, studied the cytokine production in human peripheral blood monocyte and T-lymphocyte cultures activated with this lectin. Monocytes or their FcR+ and FcR- subpopulations stimulated with PWM were cultured with or without T lymphocytes or their CD4+ and CD8+ subsets. Both monocyte subpopulations cultured alone produced similar amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), but FcR- monocytes showed significantly enhanced ability to secrete interleukin-1 (IL-1). T cells, especially CD4+, added to monocyte cultures enhanced IL-1 production. This enhancement was presumably due to interferon-gamma (IFN-gamma) release by T lymphocytes, since this lymphokine enhanced IL-1 secretion when added to PWM-stimulated cultures of monocytes. Addition of monocytes, in particular the FcR+ subpopulation, greatly enhanced production of IFN-gamma by T lymphocytes. Although both T-cell subsets produced IFN-gamma, the CD4+ cells were more efficient. These results indicate that in PWM-stimulated cultures subpopulations of monocytes differ in secretion of cytokines, which might explain their differential effect on T-cell-dependent immune responses in vitro.

Monoclonal anti-cryptococcal TsF (which inhibits phagocytosis by macrophages) and anti-picryl TsF use the same two circuits to block the transfer of contact sensitivity (CS). Both arm macrophages which then release a macrophage suppressor factor (MSF) when exposed to antigen. This MSF depresses the transfer of CS. The evidence suggests that a single molecular species of TsF (MW ca. 70 kDa), which bears an antigen-binding site and I-J determinant, is responsible for MSF production and inhibition of phagocytosis. Anti-cryptococcal TsF also arms the T acceptor cell which then releases nsTsF-1 after triggering with a specific antigen (SCPA). This nsTsF-1, which depresses the transfer of contact sensitivity, was authentic, as shown by its I-J positivity (in contrast to MSF) and its role in the production of nsTsF-2. As anti-picryl TsF also inhibits phagocytosis, it was concluded that anti-cryptococcal TsF, originally detected by the inhibition of phagocytosis, and anti-picryl TsF, originally detected by inhibition of CS, are functionally equivalent.

The data presented show that the production of interferon gamma (IFN-gamma) by pokeweed mitogen (PWM)-activated T lymphocytes requires monocytes and that the amount of lymphokine produced depends on the number of monocytes present in the culture. Accessory function of monocytes was independent from their ability to secrete IL-1 but required cell-cell contact, since blocking of adhesion molecules reduced the IFN-gamma production. Furthermore, production of IFN by lectin-preactivated T lymphocytes could not be triggered by IL-2 but also required monocyte-T cell interaction.

Monocyte-T-cell interactions were studied in pokeweed mitogen (PWM)-activated cell cultures. We addressed the question of monocyte changes in PWM-stimulated cultures of T cells and monocytes and found, by flow cytometric analysis, that PWM activation led to a loss of cells with monocyte or macrophage phenotype (CD14, HLA-DR, HLA-DQ) within 48 hr of culture in the presence of T cells (CD4+ T cells), but not in cultures of pure monocytes. Chemiluminescence measurements revealed that phagocytic stimulation of monocytic superoxide release was impaired in PWM-stimulated cultures of monocytes plus T cells, but not in PWM-stimulated cultures of pure monocytes. Furthermore, PWM induced the secretion of interferon-gamma (IFN-gamma) in primary cultures of T cells supplemented with 20% of monocytes, whereas in subsequent secondary cultures of these cells PWM induced IFN-gamma only when monocytes were added. We conclude from these flow cytometric and functional analyses that monocytes are efficiently eliminated from PWM-activated T-cell/monocyte cultures by CD4+ T lymphocytes.

The skin of CBA mice was painted with the contact sensitizing agent 4-ethoxymethylene-2-phenyloxazolone (oxazolone). One day later the regional lymph node cells were injected into the footpads of normal recipients. The recipients were tested 6 days later for contact sensitivity by challenging the ears with oxazolone and measuring the increase of ear thickness at 24 h. T cells and macrophages in the regional lymph nodes each independently gave rise to contact sensitivity in the recipient following injection into the footpad. This activity of T cells and macrophages was found in lymph nodes taken 1, 3 and 4 days after painting the donors. The role of T cells in the injected population was shown by purifying T cells by nylon-wool filtration and rosetting with sheep red cells coated with antibody and complement (EAC rosetting) and by destroyed T cells with anti-0 serum and complement. The activity of purified T cells resisted 2000 rad in vitro. The activity of cells from T-deprived (B) mice showed that a second cell type was important in the footpad transfer. This cell behaved like a macrophage, and not like a B cell, on EAC rosetting in the presence or absence of divalent cations and on treatment with silica and carrageenan--agents which damage macrophages. Our working hypothesis is that the footpad transfer may be caused independently by macrophages or T cells with oxazolone (probably linked to major histocompatibility complex antigens) on their surface and that these cells act by collaborating with T cells in the recipient which give rise to the effector cells for contact sensitivity.

The relative number of T/B lymphocytes and the response of peripheral blood mononuclear cells to PHA in the migration inhibition test was studied in a group of patients with idiopathic nephrotic syndrome. It has been found that the level of T lymphocytes and the response to PHA was decreased in patients in particular those with frequent relapses. The possible implications of these finding for pathogenesis of idiopathic nephrotic syndrome is briefly discussed.

The production of a nonspecific inhibitor of DNA synthesis (nsINH) appears to be one of the final events in the T-suppressor cell circuit in mice exposed to contact sensitizers. We report here that: The nsINH suppresses the proliferative response to a polyclonal T-cell mitogen, concanavalin A (Con A), regardless of the dose of Con A used. It also suppresses DNA synthesis in lymphoid cells stimulated with alloantigens. This suppression can be completely eliminated by adding exogenous interleukin 2 (IL-2). DNA synthesis in lymphoid cells exposed to nsINH before the proliferative stimulus is uninfluenced so that activation of the lymphoid cells at the same time as exposure to nsINH seems to be a requirement for its action. Since the activity of nsINH can be absorbed by activated Lyt-1+ or Lyt-2+ lymphocytes, the early activated T cell appears to be a target of the action of nsINH. The production of nsINH is abolished or severely reduced by adult thymectomy. Natural killer (NK) cells are resistant to nsINH action and no interferon (IFN)-like activity can be demonstrated in nsINH preparation using a conventional assay for IFN.

The phenomenon of associative recognition, i.e., the recognition of antigen together with major histocompatibility complex products (MHC) was studied in a model system. T-acceptor cells armed with antigen-specific T-suppressor factor (TsF) released a nonspecific inhibitor of the transfer of contact sensitivity when exposed to antigen together with MHC. The MHC product occurred in a KCl extract of cells and behaved genetically and serologically as I-J. Cells armed with anti-picryl or anti-"oxazolone" TsF could be triggered by the corresponding "bis-picryl-L-lysine" and "bis-oxazolone-L-lysine" together with MHC. This suggested that cross-linking of antigen recognition sites on separate molecules of TsF might be required. To investigate this possibility the bifunctional "mixed" hapten "N alpha-picryl-N epsilon-oxazolone-L-lysine," which is univalent with respect to the picryl and oxazolone haptenic groups, was synthesized. This triggered cells armed with a mixture of anti-picryl and anti-oxazolone TsF but not cells armed with either TsF alone. It was concluded that both occupancy of the I-J recognition site and the cross-linking of separate molecules of TsF was required for triggering. Moreover the hapten and the KCl extract could be given sequentially and in either order. This finding suggested that the triggering of the release of nonspecific inhibitor was due to the separate recognition of I-J and antigen and not to new antigenic determinants produced by their interaction.

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.

The spontaneous migration in vitro, production of T cell migration inhibitory factor (TIF) and the response to TIF of OKT4+ and OKT8+ human T cell subsets were studied. The OKT4+ lymphocytes migrated far better than the OKT8+ cells although the movement of both subsets was comparably inhibited by TIF. The OKT8+ subset was found to be a major source of TIF, while OKT4+ cells were responsible for macrophage migration inhibitory factor (MIF) production. The implications of lymphokine production by OKT8+ cells for the regulation of inflammatory responses are discussed.

Lyt-1+2+ hapten-specific T suppressor cells (Ts) from mice injected and then painted with picryl or oxazolone derivatives produce hapten-specific T suppressor factors (TsF) in vitro. Stimulation by painting with contact sensitizer (which need not be specific) gives rise to Lyt-1-2+, I-J+, cyclophosphamide-sensitive T acceptor cells (Tacc). When the Tacc population is armed with TsF and then is exposed to specific antigen in the context of I-J-controlled determinants (antigen-presenting, haptenized spleen cells and Ts sharing the same I-J subregion), a nonspecific inhibitor of DNA synthesis (nsINH) appears in the supernatant. This inhibitor suppresses the primary DNA synthetic response to concanavalin A, lipopolysaccharide, and alloantigens in both syngeneic and allogeneic lymphocytes. The nsINH is only effective when added to lymphocyte cultures less than 8 hr after the stimulation with concanavalin A. The nsINH, however, affects neither primary nor secondary cytotoxicity in vitro. These data suggest the mouse immune system is capable of selective regulation of the response to specific antigen by the production of nonspecific soluble suppressor factor(s).

The lymph node and spleen cells of mice painted on the skin with the contact sensitizing agent, picryl chloride, transfer contact sensitivity. Their ability to transfer reaches a peak 4 days after immunization and is absent by day 6 providing the recipient mice are challenged shortly after transfer (Chase type transfer). In contrast, when challenge of the recipients is delayed for 6 days (adoptive transfer), lymph node and spleen cells show the greatest ability to transfer 8-12 days after immunization. When cells taken 4 days after immunization (which transfer contact sensitivity) are mixed with cells taken at 6-11 days (which fail to transfer), the mixture shows little ability to transfer. This provides evidence for the occurrence of suppressor cells. Lymph node and spleen, and thymus cells show suppressor activity. The suppression is specific and cells from donors immunized with the contact-sensitizing agent oxazolone will not suppress passive transfer of contuse of the loss of ability of lymph node and spleen cells in transfer later than day 6 after immunization. Experiments on the loss of radioactivity from lymph nodes labelled with 125I-iododeoxyuridine (IUDR) suggest that loss of cells from the lymph nodes may be a contributory factor.

The inhibition of migration of human peripheral blood cells in the presence of PPD was studied. It was found that migration inhibition of peripheral blood mononuclear cells (MN) from Mantoux-positive donors was far greater than the migration inhibition of peripheral blood leucocytes (PBL). Moreover, MN cells and T lymphocytes showed larger and more uniform areas of migration. In contrast, the migration of B lymphocytes and monocytes was poor. Further analysis using purified subpopulations of MN cells showed that PPD inhibited the migration of T lymphocytes but not of B lymphocytes and monocytes. Corresponding to these findings, lymphokine-containing supernatants also inhibited the migration of purified T cells from Matoux-negative donors. It was concluded that the T lymphocyte was the predominant cell in the MN cell population, which migrated, and was subject to inhibition by PPD or lymphokines. These results imply that the movement of human T lymphocytes may be influenced by soluble factors from antigen-activated sensitized cells.

Supernatants from 24 hr cultures of PHA-pulsed human T lymphocytes inhibit the migration of human peripheral blood T lymphocytes and guinea pig macrophages in vitro. The factor responsible for the inhibition of T lymphocytes provisionally called TIF (T cell migration inhibitory factor) was separated from MIF by preparative PAGE, had apparent molecular weight (m.w.) of 1,000-10,000 daltons and isoelectric point of 3.1. TIF activity was resistant to treatment with trypsin, chymotrypsin and neuraminidase but sensitive to PMSF (phenyl-methyl-sulfonyl-fluoride). This suggests that TIF is presumably different from human MIF and may represent a novel lymphokine which preferentially affects T cell migration in vitro.

We have examined the ability of macrophages (Mphi) to transmit T-cell derived suppressor signals to other T cells. The suppressor signal studied is an antigen-specific factor which suppresses the ability of adoptively transferred, sensitized lymphocytes to express contact hypersensitivity in normal recipients. We have found that this factor binds to peritoneal exudate Mphi via cell surface structures which can be blocked with heat-aggregated gamma globulin. Dead (HK) Mphi bind the factor but fail to present it in a functional way to assay (immune) T cells, whereas live (L) Mphi perform both functions. Further, L Mphi can retrieve the factor in an active form from the surfaces of HK Mphi. Based on these and other findings (1-5), we discuss the possibility that Mphi may play as important a role in presenting T-cell communication signals to the cells of the immune system as they do in presenting antigen.

There is considerable confusion over whether the antigen-specific T suppressor factors (TsF) described by different authors are indeed equivalent. This paper investigates whether monoclonal TsF3, obtained from hybridomas derived from mice injected subcutaneously with NP derived spleen cells, is functionally equivalent to the conventional T suppressor factor, produced by mice injected intravenously with chemically reactive, water soluble haptene (picrylsulphonic acid and oxazolone thioglycolic acid). Comparison of monoclonal anti-NP TsF3 with conventional anti-picryl and anti-oxazolone T suppressor factor showed that both armed the non-specific T acceptor cell (Tacc) which was sensitive to cyclophosphamide and adult thymectomy. Moreover, non-specific inhibitor (nsINH) of the transfer of contact sensitivity was released when antigen, together with major histocompatibility complex products (MHC), reacted with conventional or monoclonal TsF on the surface of the non-specific T acceptor cell. The interaction of monoclonal TsF3 with antigen, which led to the release of NsINH, required the presence of MHC and was I-J restricted. However, there was no Igh-1 restriction. The equivalence of conventional anti-picryl and anti-oxazolone TsF has been demonstrated by arming the Tacc with a mixture of these two suppressor factors, and then triggering the release of nsINH with the mixed haptene 'picryl-oxazolone-lysine' which crosslinks separate molecules of TsF. A similar equivalence of conventional anti-oxazolone TsF and monoclonal anti-NP TsF3 was demonstrated using the mixed hapten 'NP-oxazolone-lysine' to trigger the release of nsINH. It was concluded that monoclonal TsF3 and conventional TsF were equivalent, and that both had an indirect mode of action through the non-specific T acceptor cell which led to the production of non-specific inhibitor.

Suppressor cells, which depress the passive transfer of contact sensitivity appear in the lymph nodes and spleen of mice injected with picryl sulfonic acid (PSA). These cells produce a soluble suppressor T cell product (s-TCP), and immune lymph node cells incubated in s-TCP fail to transfer contact sensitivity. This paper shows that the appearance of suppressor T cells following the injection of PSA was prevented by adult thymectomy (ATx). ATx also limited the production of s-TCP. However, ATx had no effect on the DNA synthesis which occurs in the lymph nodes of mice injected with PSA. The adverse effect of ATx on suppressor cells was completely reversed by a neonatal thymus graft placed under the renal capsule and partially reversed by grafts given 600 r in vitro and to a limited extent by grafts given 1000 r. The injection of thymus extract also reversed the effect of ATx whereas splenic extract was inactive. It is suggested that the suppressor T cell which depresses contact sensitivity is dependent on the presence of the thymus because it requires a thymus hormone, and not primarily because it belongs to a short-lived population which is rapidly renewed by cells coming from the thymus.

The effect of the lymphocytes from picryl sulfonic acid treatment animals on primary and secondary humoral immune response to TNP hapten was investigated. These cells known to inhibit contract sensitivity reactions were also able to depress primary IgM response. When peritoneal exudate cells from normal mice incubated in suppressor supernatant were transferred into primed mice the suppression of IgM but no IgM secondary response was observed. In contrast peritoneal exudate cells incubated in control supernatant augmented IgM but didn't affect IgG response. It is postulated that normal peritoneal exudate cells (presumably macrophages) can stimulate IgM response. This enhancing activity could be partially blocked by soluble suppressor factor.

Cellular immune reactivity was investigated in 49 newly diagnosed children with Wilms' tumor and compared to age-matched control. The level of total T (T4 degrees) and B lymphocytes was normal while the relative number of T lymphocytes with high affinity receptors for sheep erythrocytes (T29 degrees) was significantly decreased in the patients studied. The lymphocyte response to PHA in vitro was diminished but PHA-induced lymphokine production was not altered. The depression of T29 degrees level and lymphocyte reactivity to PHA was associated with high grade tumor rather than with the clinical stage. Lymphocytes of 42-47% patients reacted with autochthonous and allogeneic KCl tumor extracts in the migration inhibition test and the degree of reactivity was related to the histological differentiation of the tumor.

In the T suppressor circuit which affects contact sensitivity, the T acceptor cell (Tacc) armed with T suppressor factor (TsF) and then triggered by antigen and major histocompatibility complex products (I-J) releases non-specific inhibitor (nsINH). These non-specific inhibitor(s) affect both the efferent and afferent stage of the contact sensitivity reaction and were originally detected by the inhibition of the passive transfer of contact sensitivity. The nsINH also blocks the induction of contact sensitivity when given intravenously at the time of immunization but has no effect when given at the time of challenge. Similarly, it blocks proliferation in the regional lymph nodes induced by contact sensitizer in a dose-dependent fashion; it acts when given at the time of immunization but not 1 day later. This effect is antigen non-specific and H-2 unrestricted. The nsINH bears I-J determinants as shown by affinity chromatography on monoclonal antibody. The nsINH comes from the Tacc and is not a breakdown product of the TsF. This is shown by the fact that, when the Tacc and TsF have I-J of different genotypes, the genotype of the nsINH corresponds to that of the Tacc. Parallel measurements of inhibition of lymphoproliferation and of passive transfer show that the nsINH has a molecular weight of 50-60 Kd and a pI around 6.8 and suggest that similar or identical molecules block both the afferent and efferent stage of the contact sensitivity reaction.

Antigen-specific T-suppressor factor (TsF), which acts at the expression stage of the contract sensitivity reaction, was produced by culturing the lymphoid cells of mice injected with picryl-sulphonic (trinitrobenzenesulphonic) acid and then painted with picryl chloride. Supernatant activity was found around 50-60 and 90 kDa on Sephadex gel filtration. The activity at 50-60 kDa was due to two separate (or readily separable) molecules, one antigen binding and the other bearing I-J determinants as shown by affinity chromatography on insolubilized antigen and anti-I-J. These two separate molecules were inactive alone but complemented each other and may be designated as the variable chain of TsF (TsFv) and the I-J+ chain. The use of gel filtration and sequential absorption of individual pools on anti-I-J antibody followed by antigen, together with a complementation assay, also showed a TSFv chain at 30-40 kDa and an I-J+ chain at 20-30 kDa. The higher-molecular-weight activity around 90 kDa was due to a single molecule which was both antigen binding and I-J+. This molecule dissociated on treatment with the reducing agent dithioerythritol followed by alkylation into two separate chains, one antigen binding and the other I-J+, both of which were required for activity. There was a requirement for genetic matching between the antigen-binding chain (TsFv) and the I-J+ chain for biological activity. These data support a two-chain model of TsF in which TsFv and the I-J+ chain occur as a single disulphide-bonded molecule around 90 kDa, or as two separate (or readily separable) chains of lower molecular weight which were inactive alone but complemented each other.