Presence Of Monocytes Research Articles

Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells.

Human peripheral blood T cells were purified by a four-step procedure which included depletion of plastic-adherent cells, rosetting with sheep red blood cells, nylon wool passage, and treatment with mouse monoclonal antibodies to human Ia antigens plus complement. The purified T cells completely failed to proliferate to phytohemagglutinin (PHA). Bacterially derived recombinant human interleukin 2 (IL 2) reconstituted the proliferative response of resting T cells to PHA. The optimal concentration of IL 2 required was 100 to 200 U/ml. IL 2 alone caused no T cell proliferation. Both PHA and IL 2 needed to be present together for the proliferation of T cells to occur. Incubation of T cells with either PHA or IL 2 alone for up to 18 hr, followed by washing, then by the addition of the reciprocal reagent, resulted in no T cell proliferation. Expression of IL 2 receptors and of Ia antigens, as assessed by indirect immunofluorescent staining, revealed that both PHA and IL 2 needed to be present for Tac and Ia antigen expression by T cells. T cells incubated with PHA and IL 2 for 18 to 42 hr acquired responsiveness to IL 2. These T cells remained absolutely dependent on IL 2 for proliferation to occur. In contrast to T cells stimulated with PHA in the presence of monocytes, T cells stimulated with PHA and IL 2 released no detectable IL 2. The failure of IL 2 secretion was not caused by down-regulation of IL 2 production by IL 2 itself, because the addition of IL 2 to cultures of T cells stimulated with PHA in the presence of monocytes did not interfere with IL 2 production. These results indicate that IL 2 is a sufficient signal to induce the expression of its receptor in PHA-stimulated T cells and subsequent proliferation but is not sufficient to cause endogenous IL 2 release.

Subsets of patients with aplastic anemia identified by flow microfluorometry.

We used flow microfluorometry to analyze peripheral-blood mononuclear cells from 50 patients with aplastic anemia, to determine whether patients who would recover after immunosuppressive therapy could be distinguished before treatment from those who would not recover. Cells were labeled with murine monoclonal antibodies that are relatively specific for B cells, T cells, T-cell subsets, and monocytes. The data suggested that the number of lymphocytes and the ratios of various subclasses of T cells were not useful in identifying patients who were likely to recover. The complete absence of monocytes was found to identify patients who would not recover, but the presence of monocytes was also sometimes associated with lack of recovery. An unexpected finding was the significant (P less than 0.0001) association between clinical recovery and the presence of a population of small cells (4 to 8 micron) that were phenotypically associated with the erythroid lineage. If this association is confirmed, flow microfluorometry may be useful in selecting the optimal treatment for individual patients with aplastic anemia.

Failure of OKT3 monoclonal antibody to induce lymphocyte mitogenesis: a familial defect in monocyte helper function.

Monoclonal antibodies to the T3 molecule on human T cells have mitogenic activity. Although anti-T3 antibodies of the IgG1 subclass (e.g., UCHT1) induce mitogenesis in lymphocyte cultures from only 60 to 70% of normal donors, antibodies of Ig2a subclass (e.g., OKT3) invariably have been found to be mitogenic in all subjects tested up to the present. This paper describes a family (a mother, six daughters, and one son) in which five members failed to respond mitogenically to OKT3 although the proportion of OKT3-reactive cells in their peripheral blood was normal. Mitogenic responses to PHA, Con A, and PWM were normal. Five members comprising four OKT3 nonresponders were also unresponsive to UCHT1. Unresponsiveness to OKT3 and unresponsiveness to UCHT1 were not absolutely linked to each other, nor were they linked to an HLA haplotype inherited from the mother. Upon stimulation by OKT3, lymphocyte preparations from OKT3-nonresponders failed to produce interleukin 2 (IL 2) and to display IL 2 receptors. OKT3 unresponsiveness was due to defective monocyte help: thus, responsiveness to OKT3 of T cells from an OKT3-nonresponder was restored by the addition of monocytes from an HLA-identical sister who had a normal response to OKT3. Inversely, T cells from the OKT3 responder had no reactivity to OKT3 when cultured in the presence of monocytes from an HLA-identical, OKT3-nonresponsive sister. Unresponsiveness to OKT3 could not be overcome by the addition of phorbol myristate acetate to the cultures. These data on a familial, non-HLA-linked deficiency of monocytes to exert their auxiliary function provide better insight into the mechanism of anti-T3-induced T cell activation.

Enhancing effects of monocytes on modulation of a lymphocyte membrane antigen.

Redistribution, or modulation, of some cell surface antigens occurs in the presence of specific antibody. The phenomenon of antigenic modulation may therefore affect the use of antibodies as therapeutic agents. This study was undertaken to investigate modulation of the 65,000 dalton T65 antigen, present on normal and malignant T cells and some malignant B cells, which is recognized by the monoclonal antibody T101. To induce cell surface antigenic modulation, normal or leukemic lymphoid cells were cultured in the presence of monoclonal antibody T101 for 3-hr periods. Removal of monocytes from mononuclear cell preparations resulted in significantly lower degrees of T65 antigenic modulation. The degree of antigenic modulation could be increased by adding monocytes back to monocyte-depleted lymphocyte suspensions. Furthermore, maximal modulation occurred in the presence of monocytes at T101 concentrations that were 3 logs lower than in the absence of monocytes. The enhancing effect of monocytes was dependent on the Fc portion of the T101 antibody molecule, and presumably was mediated by cross-linking of antigen-antibody complexes on the surface membrane of the modulating cell by Fc receptors present on monocytes. Further experiments performed to examine the characteristics of this enhancement of antigenic modulation by monocytes indicated that autologous as well as allogeneic monocytes were effective, indicating that the enhancing phenomenon was not dependent upon recognition of major histocompatibility antigens. Viable monocytes were required, but pretreatment of monocytes with sodium azide to inhibit energy production, or indomethacin to inhibit prostaglandin synthesis had no effect on this phenomenon. Polymorphonuclear leukocytes did not mediate similar enhancement, although monocytic and myeloid cell lines U937, THP-1, and HL-60 did. Spent culture medium from modulated cultures and preparations containing IL 1 activity did not enhance modulation of the T65 surface antigen on lymphocytes, suggesting that direct contact between lymphocytes and monocytes is required to mediate the effect. The finding that leukemic cells from patients with CLL undergo modulation of the T65 antigen to a much lower degree in vitro than observed in vivo, and that this difference can be overcome by the addition of monocytes, suggests that monocytes or the reticuloendothelial system may augment antigenic modulation in vivo.

Rapid separation of human monocytes and lymphocytes by Sephadex G-10

Many of the interesting functions of human lymphocytes in vitro are known or suspected to be dependent on the presence of monocytes. A practical limiting factor in cell interaction experiments is the ability to separate and recover functional monocytes and lymphocytes rapidly from the same blood specimens. Here we describe a simple, rapid and inexpensive solution to this problem. Incubation at 37 °C on Sephadex G-10 columns consistently depleted monocytes to low levels (<0.1%) and gave maximum lymphocyte yields (> 60%) in a short time. Half the esterase-positive monocytes entering the column may be easily recovered from the beads by a 4°C chilling/shaking technique; by using 0.5% lidocaine, an additional 15% can be recovered. Simultaneously, the G-10-nonadherent lymphocytes are being further separated into T and B cells by E-rosetting. These recovered G-10-adherent cells were above 90% esterase positive, and showed a cell size distribution on the Coulter channelyzer distincly larger than nonadherent lymphocytes. To illustrate the use of this protocol we present a monocyte titration experiment which demonstrated a plateau of optimal pokeweed mitogen-stimulated Ig production at 5–10% monocytes.

Paradoxical production of mouse thymocyte activating factor by ouabain-treated human mononuclear cells

At concentrations as low as 10 −7 M, the cardiotonic glycosteroid ouabain, a specific inhibitor of the membrane Na +,K +-ATPase, is known to inhibit in vitro human lymphocyte proliferation produced in mixed lymphocyte cultures or induced by various stimulating agents (PHA, Con A, PWM, soluble antigens), while mouse lymphocyte proliferation is unaffected at this concentration. Ouabain inhibits most of proliferative response parameters at all stages of the transformation. This observation prompted us to suggest that ouabain could also act through inhibition of interleukin production which is known to occur during the first hours after T-cell stimulation in the presence of monocytes. In order to check the possible influence of ouabain on interleukin production, conditioned media from stimulated human mononuclear cells, prepared in the presence or in the absence of inhibitor, were tested for their ability to promote a mouse thymocyte response to PHA. Instead of the expected inhibition, we found that ouabain, even at high concentrations (2 × 10 −6 M) enhanced the stimulatory effect and/or the production of murine thymocyte activating factor(s). Moreover conditioned media from serum-free cultures of unstimulated human mononuclear cells exposed for 24 hr to low ouabain concentrations (10 −8 to 10 −7 M) showed a high activating effect on the response of murine thymocytes to PHA. This soluble factor produced upon ouabain treatment is produced by adherent cells and appears to be functionally similar to interleukin 1.

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism.

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.

Mitogen-induced stimulation and suppression of erythroid burst promoting activity production by human mononuclear cells.

Exposure of human peripheral blood mononuclear cells or highly enriched monocytes to various plant lectins substantially alters their production of erythroid burst promoting activity (BPA). Neither unstimulated, nor mitogen stimulated, enriched T lymphocytes produced demonstrable BPA. Each of the lectins tested resulted in a different pattern of alteration of BPA production by mononuclear cells. Increasing concentrations of phytohaemagglutinin (PHA) caused a progressive increase in BPA production up to a plateau level at concentrations above 0.25-0.5 microliter/ml. Concanavalin A (Con A) at concentrations of 0.05-0.1 micrograms/ml stimulated BPA production, but Con A concentrations greater than 1 microgram/ml never augmented BPA production by mononuclear cells. Pokeweed mitogen inhibited BPA production by mononuclear cells in a concentration-dependent manner. Since PHA and Con A can bind to and stimulate both monocytes/macrophages and T lymphocytes, some production of BPA by stimulated T cells in the presence of monocytes cannot be ruled out. Earlier studies demonstrated that T cells augment monocyte production of BPA. Thus, monocyte-T cell interactions, as well as activation of monocytes and perhaps lymphocytes, play an important role in regulation of BPA production in vitro.

Polymorphism in mitogenic effect of IgG1 monoclonal antibodies against T3 antigen on human T cells.

It has recently been described that monoclonal antibody OKT 3, that reacts with the T3 determinant on all peripheral T lymphocytes in man, is also mitogenic for T cells. We have produced two monoclonal antibodies (WT 31 and WT 32) which apparently react with the T3 antigen. We have now tested the mitogenic effect of these antibodies and compared it with the mitogenicity of three other anti-T3 monoclonal antibodies, OKT 3, UCHT 1 (ref. 4) and anti-Leu 4 (ref. 5). Two distinct patterns were observed. WT 32 and OKT 3, both of the IgG2a subclass, were mitogenic for human T cells in all cases studied. By contrast, WT 31, UCHT 1 and anti-Leu 4, all of the IgG1 subclass, were devoid of mitogenic effect in 30% of the individuals tested (non-responders). The mitogenicity of all five anti-T3 antibodies was fully dependent on the presence of monocytes. Addition of purified monocytes from a responder to purified lymphocytes from a non-responder induced responsiveness to both IgG1 and IgG2a anti-T3 antibodies. These results suggest that the polymorphism in the mitogenic effect of these IgG1 antibodies is caused by polymorphism in monocyte function, possibly at the level of the Fc receptor that reacts with mouse IgG1.

Human T lymphocyte proliferation induced by a pan-T monoclonal antibody (anti-Leu 4): heterogeneity of response is a function of monocytes.

Anti-Leu-4 is a murine monoclonal antibody that defines a molecule of 20,000 to 25,000 daltons present on all mature T lymphocytes in man. When cultured in the presence of 10 to 1000 ng/ml anti-Leu-4, the T cells of most individuals proliferate with peak responses on the third day of culture. T cells of both helper and suppressor lineages proliferate, but only in the presence of monocytes. Approximately 40% of individuals tested responded weakly or not at all to anti-Leu-4, despite normal responses to other stimuli. The variation in responsiveness between individuals could not be explained by differences in Leu-4 antigen density on the surface of T cells, differences in the rate of Leu-4 antigen modulation, or structural differences in the Leu-4 molecule as defined by the method of two-dimensional polyacrylamide gel electrophoresis. In the presence of monocytes from high responders, the T cells from low responders proliferated vigorously to anti-Leu-4, whereas monocytes from low responders failed to support proliferation by high responder T cells. On the other hand, low responder monocytes did not prevent T cells from proliferating in the presence of high responder monocytes. These results suggest that the failure of some individuals to respond to anti-Leu-4 is due to the absence or dysfunction of an essential monocyte population.

Distinction of two subtypes of human leukocyte interferon (IFN-alpha) on B cell activation. B cell proliferation by two subtypes of IFN-alpha.

We examined the effect of interferon (IFN), with particular emphasis on the effects of the two subtypes of IFN-alpha (IFN-alpha A and IFN-alpha B) on the B cell proliferation induced by Staphylococcus aureus Cowan I bacterium (SpA Col). An increase of SpA Col-induced proliferation was observed in the presence of 100 to 1000 U/ml of IFN-alpha, but a decrease of SpA Col-induced proliferation was observed in the presence of 1000 to 10,000 U/ml of IFN-beta. The two subtypes of IFN-alpha had different effects on cell proliferation; a significant enhancement was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha A, but inhibition was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha B. In the reconstitution test of the two subtypes of IFN-alpha, the boundary between enhancement and inhibition of SpA Col-induced proliferation was revealed when the proportion of IFN-alpha A and IFN-alpha B (IFN-alpha A:IFN-alpha B) ranged between 8:2 and 9:1. Toward the SpA Col-induced responses, the above IFN were all found to act on B cells directly, independent of the presence of T cells. Proliferative responses by IFN-alpha and IFN-alpha A, however, were shown to be slightly dependent on the presence of monocytes. The lymphocyte proliferation induced by other mitogens (phytohemagglutinin, concanavalin A, pokeweed mitogen, and protein A of S. aureus) were all inhibited by the above IFN.

The mitogenic activity of OKT3 and anti-Leu 4 monoclonal antibodies: A comparative study

The mitogenic properties of OKT3 (IgG 2a) and anti-Leu 4 (IgG 1), two monoclonal antibodies directed at the same surface antigen of human T cells, have been studied. Both antibodies, at subnanomolar concentrations, induced thymidine incorporation of comparable magnitude into human peripheral blood mononuclear cell cultures. Their mitogenicity reached peak values 3 days after stimulation, was totally inhibitable by monoclonal OKT11A antibody, and was strictly dependent on the presence of monocytes as auxiliary cells. Whereas mononuclear cells from all blood donors tested were proliferative to OKT3, cells from 12 of 30 donors (40%) were unresponsive to anti-Leu 4. The incidence of this unresponsiveness was significantly higher for cells from female than male donors, showing 60 and 20% nonresponders, respectively. Anti-Leu 4 unresponsiveness was found to be due to the inability of autologous monocytes to exert their auxiliary function; thus, lymphocytes from anti-Leu 4 nonresponders became mitogenically inducible by anti-Leu 4 after addition of monocytes from anti-Leu 4 responders, whereas lymphocytes from anti-Leu 4 responders failed to respond to anti-Leu 4 after supplementation of monocytes from anti-Leu 4 nonresponders. Considering the different immunoglobulin subtype of OKT3 (IgG 2a) and anti-Leu 4 (IgG 1), our results indirectly suggest that the auxiliary function of monocytes is mediated by their Fc receptors. Unresponsiveness to anti-Leu 4 could then be explained by the absence, paucity, or functional deficiency of monocytic Fc receptors for IgG 1.

Clastogenic action of tumor promoter phorbol-12-myristate-13 acetate in mixed human leukocyte cultures.

The tumor promoter phorbol-12-myristate-13-acetate (PMA) induces chromosomal aberrations in mitogen stimulated human lymphocyte cultures containing monocytes, polymorphonuclear cells (PMN) and platelets. Such cultures produce a diffusible clastogenic factor (CF) in response to PMA which causes aberrations in fresh blood cultures which have not been exposed to PMA. We have studied the contribution of monocytes, PMN and platelets to CF formation. 'Pure' lymphocyte cultures (containing no platelets and maximally 1% PMN and 2% monocytes) only produced CF when they were in contact with 1 to 1.8 X 10(6) monocytes attached to plastic during PMA treatment (18.5 +/- 5.3% mitosis with aberrations for CF produced in the presence of monocytes relative to 6.0 +/- 4% in their absence). They also produced CF of increasing potency upon addition of 0.25 to 5 X 10(6) PMN (20.5 +/- 5.9% mitosis with aberrations for CF produced in the presence of 5 X 10(6) PMN). Cultures containing 5-10 platelets/lymphocyte also formed CF upon PMA treatment. Cultures of purified monocytes and PMN were capable of producing CF in the absence of lymphocytes. The presence of bovine erythrocyte CuZn superoxide dismutase during PMA treatment decreased the activity of the resulting CF under all conditions. Catalase prevented CF production from PMN. It is concluded that the presence of monocytes, PMN or platelets is a prerequisite for CF formation by PMA. Neoplastic tissue is usually surrounded by inflammatory leukocytes. CF produced by these cells in response to tumor promoters such as PMA may induce chromosomal damage in the neighboring tumor cells.

The effect of monocytes and T cells pretreated with aggregated IgG on generation of concanavalin A induced suppressor T cells

The present studies showed that monocytes are necessary for the generation of Con A-induced suppressor T cells.Furthermore, the pokeweed mitogen response of B cells and mixid lymphocytes reaction were suppressed strongly by suppressor T cells which had been induced by Con A in the presence of monocytes at a concentration of 5-10%.The generation of Con A-induced suppressor T cells was blocked when these T cells were precultured with monocytes pretreated with aggregated IgG.In contrast, an enhancement of suppressor cell generation was observed when the T cells were treated with aggregated IgG before Con A activation.The monocytes of patients with systemic lupus erythematosus and rheumatoid arthritis contributed to the generation of Con A-induced suppressor T cells to a less degree.

Positive self regulation of cytotoxicity in human natural killer cells by production of interferon upon exposure to influenza and herpes viruses.

Augmentation of natural killer (NK) activity by influenza A/PC and HSV-1 viruses appears to be caused by the induction of interferon (IFN) within the NK cell population itself. These viruses induced high levels of IFN production by human large granular lymphocytes (LGL) that could be readily isolated from peripheral blood by Percoll density gradients. These LGL, which have been previously shown to account for and to be highly associated with endogenous NK activity, became augmented in their lytic function during the 18-h period that IFN was induced. Non-LGL helper cells did not appear to be required in the NK-IFN system (either T cells, B cells, or monocytes). Removal of these latter cell types by treatment with OKT3 plus complement, anti-IgM plus complement, or preincubation with silica or carrageenan had no effect on the ability of LGL to respond to the viruses. Production of IFN was also detected, albeit at lower levels, from monocytes cultured for 18 h with viruses, but no cytotoxic activity was induced. On the other hand, T cells, even in the presence of monocytes, showed neither property, and longer cultures, with virus up to 4 d, still did not alter the pattern. The IFN produced by both LGL and monocytes were predominantly IFN-alpha, as assessed by neutralization assays with antisera to IFN-alpha, -beta, and -gamma. In an individual with detectable serum antibodies to influenza A/PC, however, the IFN induced in LGL appeared to be gamma, presumably because of specific recognition of the virus. These data suggest an efficient positive self-regulatory mechanism in NK cells that may be readily switched on by viruses.

Modulation of human nk cells by interferon and prostaglandin E 2

Our studies have shown that stimulation of human natural killer (NK) cells by poly I: C does not depend on or require monocytes. In contrast, the presence of monocytes in a mixed population of mononuclear cells stimulated by poly I:C suppresses NK. activity. The suppression can be partially overcome if indomethacin (10 −6 M) is added to the culture during stimulation. Culture supernatants from poly I:C stimulated monocytes do not have detectable levels of anti-viral activity but contained appreciable amounts of PGE 2. Our results offer an explanation as to how NK cells may protect themselves from suppression by PGE 2. We have demonstrated that IFN-activated NK cells become resistant to PGE 2-mediated suppression; moreover the suppression does not require cells other than the large granular lymphocytes, the major effector cell type for NK. Taken together, the data suggest that stimulation of NK cells is dependent on and regulated by the relative levels of interferon produced by lymphocytes and PGE 2 produced by monocytes.

Identification of “active” T lymphocytes among effector cells in guinea pigs

Guinea pig T lymphocytes have receptors of different affinity for rabbit red blood cells (RRBC): those binding RRBC immediately are termed “active” T cells; the remainder, which bind RRBC only after longer incubation times, are “non-active” or “late-rosetting” T cells. We have found that these two subpopulations have different functional characteristics. Active T cells could not be stimulated effectively with phytohemagglutinin (PHA), and stimulation with concanavalin A (ConA) increased their DNA synthesis only at high concentrations. The non-active subpopulation responded better to PHA but poorly to ConA. Unseparated (total) T cells, however, responded well to both mitogens, suggesting a helper effect by the active T cells. The presence of monocytes in T-cell cultures further enhanced mitogen-induced DNA synthesis. Active T cells were not present in guinea pig bone marrow, whereas they constituted 10% of all lymphocytes in the thymus, 13% in the spleen, 29% in lymph nodes, and 32% in the peripheral blood. After administration of antigen in vivo, the number of active T cells in the regional lymph node increased, whereas the number of total T cells did not change. The similarity of the active T cell populations in guinea pigs and humans increases the usefulness of these animals for preclinical tests of potential new immunomodulating agents.

Lysozyme stimulates lymphocyte proliferation in monocyte-depleted mixed lymphocyte cultures.

Human LZM was found to enhance 3H-thy uptake and lymphoblast transformation in monocyte-depleted MLC. The effect was maximal (up to sixfold increase) in "low level" (less than 10,000 cpm) MLC. Maximal MLC enhancement was obtained with 250 micrograms/ml LZM; higher LZM concentrations appeared to be inhibitory. LZM enhancement of MLC could not be demonstrated in the presence of monocytes. LZM did not enhance lymphocyte responses to SKSD. Several observations support the possibility that LZM enhancement of MLC is due to LZM augmentation of stimulator cell antigenicity. LZM pretreatment of stimulator but not responder lymphocytes enhanced MLC. This enhancement was blocked by the LZM inhibitor tri-GlcNAc. LZM enhancement of MLC was maximum when the antigenic stimulus was minimized by either reducing the number of stimulator cells or utilizing DRw-matched donors in MLC. These data suggest that (1) LZM may enhance lymphocyte proliferative responses to allogeneic stimuli by altering stimulator cells' antigenicity and (2) monocyte-macrophage enhancement of lymphocyte proliferation in MLC may be partially mediated by LZM. (J Lab Clin Med 99:370, 1982.)

The temporal relationship between glomerular cell proliferation and monocyte infiltration in experimental glomerulonephritis.

During the course of an accelerated form of anti-glomerular basement membrane nephritis in rats, the authors determined the temporal relationships between (a) glomerular hypercellularity, (b) kinetics of cell proliferation and (c) monocyte infiltration in glomeruli. Although glomerular hypercellularity was not appreciable or mild by qualitative histology, mean glomerular cell counts rose between days two and six of nephritis by up to 30% over controls. Cell proliferation in glomeruli was assessed 2 h after pulse label with3H-thymidine by determining histoautoradiographic cell labeling indices (LI) and by measuring the total radioactivity of isolated glomeruli (TGA). On day one, mean LI of cells in Bowman’s capsule was increased by 356% over controls and fell subsequently. LI values of tuft cells rose more gradually, reaching a maximum increment of 405% on day four. TGA also increased on day one and two, and peaked on day four. In additional rats, studied in parallel,3H-thymidine incorporation was measured 48 h after the pulse in order to allow time for the labeling and glomerular influx of monocytes derived from dividing extra-renal precursors. In rats sacrificed on the first day of nephritis, the prolonged labeling time did not result in further gains of tuft LI or of TGA over values obtained 2 h after the pulse. On day four, by contrast, the 48-h labeling time was associated with markedly higher results for LI and TGA than the 2-h interval. These findings indicated that the glomerular influx of labeled blood-borne monocytes was not appreciable on day one but increased markedly between day two and day four. This was supported by histochemical studies for non-specific esterase which revealed a similar time course for the presence of monocytes in glomeruli by showing few esterase-positive monocytes in the early stage and many after day two. The results demonstrate that this model of glomerulonephritis is characterized by markedly enhanced replication of intrinsic glomerular cells, beginning on the first day of the injury at a time when the glomerular influx of monocytes is inconspicuous. Increases of glomerular cell prolifertion become most pronounced over the following days when they are associated with a sizeable monocytic infiltrate. The prominent cellular response in glomeruli is revealed by the applied quantitative methods in the absence of histologically impressive glomerular hypercellularity.

Regulation of human natural killing. I. The role of monocytes, interferon, and prostaglandins.

We have found that human endogenous natural killer activity as measured in a rapid 51Cr release assay is unaffected by the presence of monocytes. Moreover, stimulation of natural killer cells by poly I:C is independent of monocytes. In contrast, the presence of monocytes in a mixed population of mononuclear cells that have been stimulated by poly I:C suppresses NK activity. The suppression is shown to be partially reversible if indomethacin (10(-6) M) is added to the cultures during stimulation. Culture supernatants of monocytes stimulated with poly I:C are shown to contain PGE1 in a radioimmunoassay, and have NK inhibitory activity comparable to that obtained with exogenous PGE1 added to NK assays at a concentration range of 10(-7) to 10(-9) M. Culture supernatants from poly I:C-stimulated monocytes do not have detectable levels of antiviral activity. In contrast, plastic nonadherent cells stimulated with poly I:C produce significant levels of interferon (100 to 200 U/ml/2 x 10(6) cells) but almost undetectable amounts of PGE. Supernatants of nonadherent cells incubated with indomethacin (10(-6) M) alone augment NK activity. Taken together, the results suggest that stimulation of mononuclear cells with poly I:C is dependent on and regulated by the relative levels of interferon produced by lymphocytes and PGE produced by monocytes.