Nonadherent Population Research Articles

Presentation of Salmonella antigens by peritoneal cells of normal and Salmonella‐infected mice

A comparison of the ability of normal peritoneal cells (PC) and those harvested from mice 1-3 days after intraperitoneal immunization with live Salmonella enteritidis 11RX (11RX) to present antigen to 11RX-primed T cells was made using formalin-killed 11RX and a soluble 11RX antigen extract as antigens. Unfractionated PC and the adherent and non-adherent PC populations were analysed separately and the effects of the lysosomal function-impairing drug chloroquine and the fixative paraformaldehyde, used before or after antigen-pulsing, were also determined. The results presented indicate that immunization with live 11RX did not induce any detectable modulation of APC function which could account for the ability of live 11RX to induce cell-mediated immune responses involving Lyt 2+ T cells.

Induction of tumor cytotoxic immune cells using a protein from the bitter melon ( Momordica charantia)

The fruit and seeds of the bitter melon ( Momordica charantia) have been reported to have anti-leukemic and antiviral activities. This anti-leukemic and antiviral action was associated with an activation of murine lymphocytes. A partially purified protein factor from the bitter melon caused an infiltration and activation of peritoneal exudate cells in C57B1/6J, C3H/HeJ, and C3H/HeN mice. When the extract was injected twice a week at 8 μg of protein per ip injection for 0–4 weeks, the peritoneal exudate cells from the treated mice were cytotoxic in a long-term (18-hr) 51Cr-release assay against a range of labeled targets; L1210, P388, and MOLT-4 tumor cells. Cytotoxicity was also observed against YAC-1 targets in a short-term (4-hr) assay. Fractionation of the cytotoxic immune cells implicated a nonadherent cell population which was capable of killing an NK-sensitive cell line in a 4-hr 51Cr-release assay. Unit gravity sedimentation studies indicated that the cytotoxicity was due to either a neutrophil or a large lymphocyte. Antibody depletion experiments using antibody to asialo GM1, an NK cell-specific antibody, depleted cytotoxicity observed in nonadherent, Ficoll/Hypaque-separated PEC. This suggests that at least part of the anti-leukemic activity of the bitter melon extract is due to the activation of NK cells in the host mouse.

Sequential appearance of γ/δ‐ and α/β‐bearing T cells in the peritoneal cavity during an i.p. infection with Listeria monocytogenes

To search for a potential role of T cell antigen receptor (TcR) gamma/delta-bearing cells in host-defense against Listeria monocytogenes, we analyzed the sequential appearance of gamma/delta and alpha/beta T cell in the peritoneal exudate cells (PEC) during an i.p. infection with sublethal dose (2 X 10(3) of viable Listeria organisms in mice. The PEC on day 1 after the infection consisted of 48% macrophages and 50% lymphocytes, most of which were surface IgM+ (B) cells. The number of PEC increased to the maximal level by day 3. The PEC at this stage contained an appreciable number of CD3+ T cells in addition to a large number of macrophages. Of the CD3+ cells, the proportion of CD4- CD8- cells, most of which expressed no TcR alpha/beta, increased to the maximal level on day 3 after the infection. In correlation with an increased number of CD3+ CD4- CD8- TcR alpha/beta- cells, high level of TcR gamma/delta chain gene messages was detected in the nonadherent population of the PEC on this stage. On the other hand, the PEC on day 8 contained an increased number of CD4+ CD8- and CD4- CD8+ cells which expressed TcR alpha/beta chain on their surface. These results suggest that the gamma/delta T cells precede the alpha/beta T cells in appearance during listerial infection. The gamma/delta T cells may be involved at the first line of the host-defense against Listeria.

Monoclonal antibodies to ros 17/2.8 cells recognize antigens, some of which are restricted to osteoblasts and chondrocytes

We have raised a panel of 15 monoclonal antibodies (MAbs) recognizing cell surface antigens of the rat osteoblast-like cell line ROS 17/2.8. The MAbs were selected on the basis of preferential binding to ROS 17/2.8 cells compared to ROS 25/1 cells. Immunohistochemical studies of antigen localization on cryostat sections of rat calvaria, long bone, and soft tissues demonstrated that five of these MAbs, UBIM 1, 2, 3, 12, and 17, recognize antigens that are restricted to normal rat osteoblasts and chondrocytes. The antigens appear to be localized to the cell surface of the osteoblast, with no apparent staining of bone matrix in either undecalcified or decalcified sections. In vitro, these MAbs recognize cell surface antigens present on two additional cell lines, ROS 24/1 and Rat 2 cells, and on the adherent cell population cultured from rat long bone marrow. Of these MAbs, three (UBIM 1, 2, and 3) recognize high-molecular-weight antigens of Mr 200,000-225,000. This study has also identified cell surface antigens of ROS 17/2.8 cells that are not expressed by osteoblasts in vivo. MAbs UBIM 9 and 21 bind to marrow cells in long bone sections, to the 7-day-old nonadherent cell population from cultured marrow, and to lymphoid tissue in sections of spleen. Another four MAbs (UBIM 10, 11, 14, and 22) bind to a variety of cells and tissues both in vitro and in vivo. Studies of the interactions of this panel of MAbs with osteogenic tissues and cell lines may have an important impact on the understanding of osteoblast physiology.

Immunosuppressive effect of infectious pancreatic necrosis virus on rainbow trout (Oncorhynchus mykiss).

The infectivity of infectious pancreatic necrosis virus (IPNV) for rainbow trout (Oncorhynchus mykiss) mononuclear leukocyte subpopulations was investigated to determine the mechanisms of immunosuppression caused by the virus. IPNV was recovered from nylon wool-adherent, surface immunoglobulin (Ig)-positive leukocytes of head kidney, spleen and peripheral blood collected from virus-inoculated fish with higher titers than non-adherent, Ig-negative cells. Non-adherent cell population showed mitogenic response to phytohemagglutinin and concanavalin A but not to lipopolysaccharide. Conversely, the responses of adherent cells to these mitogens were weak. Mitogenic response and non-specific cytotoxicity of head kidney leukocytes significantly decreased by the inoculation of fish with the virus. These results suggest that the suppression of immune responses is involved in the establishment of carrier state in fish after infection with IPNV.

Efficacy of Avirulent Hemorrhagic Enteritis Virus Propagated in Turkey Leukocyte Cultures for Vaccination against Hemorrhagic Enteritis in Turkeys

Avirulent hemorrhagic enteritis virus (HEV-A) propagated in turkey leukocyte cell culture was tested as a vaccine to prevent hemorrhagic enteritis (HE) in turkeys in experimental and field trials. Immunization of turkeys with live HEV-A resulted in protection against a challenge with virulent HEV (HEV-V) as measured by the serological response and the absence of clinical disease and HEV antigen in spleens. In field trials, 19 out of 20 flocks seroconverted within 21 days after vaccination with live HEV-A distributed in the drinking water. The average immune response of the turkeys in these flocks was 96%. Most importantly, neither clinical HE nor other adverse effects caused by HEV-A vaccination were observed in any of the vaccinated flocks. Because maternal antibodies can interfere with the immune response to the vaccine, the optimum time for vaccination was determined. Using an established half-life value of 4.25 days for turkey antibody, and knowing the enzyme-linked immunosorbent assay titer of the maternal antibodies and age of the turkey, the time of vaccination could be calculated taking into account that maternal antibody titers should be lower than 40 to vaccinate the turkeys successfully and induce protection. In vivo tests with HEV-A preparations confirmed the replication of the virus in turkey leukocyte cultures and the potential to pass it in culture. Furthermore, in vivo analysis showed that most infectious virus was associated with preparations of the nonadherent cell population, confirming the results obtained by in vitro analysis. The potency of HEV-A preparations was dependent on the production method and varied from an average of 570 to 8135 doses per ml.

Comparison of T-cell responses in self-limiting versus progressive visceral Leishmania donovani infections in golden hamsters

Leishmania donovani infection in golden hamsters was studied as a model for human kala-azar. After intradermal inoculation of L. donovani amastigotes, hamsters developed positive skin reactions (delayed-type hypersensitivity [DTH]) to parasite antigens and lymphoid cells from these hamsters proliferated to parasite antigens in vitro and transferred DTH reactivity to normal recipients. In contrast, hamsters infected by the intracardial route developed progressive visceral infections and failed to respond to skin test antigens. Spleen cells, lymph node cells, and peripheral blood lymphocytes (PBLs) from these hamsters were unresponsive to parasite antigens in vitro, and spleen cells failed to transfer DTH to normal recipients. Spleen cells, but not PBLs, displayed depressed responses to T-cell mitogens and also suppressed the proliferative response of cells from hamsters inoculated intradermally. Removal of adherent cells restored the capacity of spleen cells, but not PBLs, to respond to parasite antigens. The nonadherent population of these spleen cells also transferred DTH to normal recipients. The adherent suppressor cells, which have the characteristics of macrophages, appear to be localized to the spleen and are apparently not responsible for the failure of peripheral lymphoid cells to respond to antigen. These studies suggest that hamsters with visceral infections develop a population of antigen-reactive cells and that in the absence of suppression these cells may express functional activities, including the capacity to elicit DTH responses.

Examination of macrophage cell surface antigen regulation by rIFN-γ and IFN-α/β utilizing digital imaging by a novel laser detection system: Anchored cell analysis station (ACAS) 470

The use of a computer-controlled scanning laser instrument, the ACAS 470, for the analysis of cell-surface antigens on adherent macrophages is described. The association of specific cell surface antigens with macrophage differentiation and the modulation of these markers by environmental signals has long been recognized. However, direct analysis of individual macrophages for cell-surface antigen expression by conventional methods has been fraught with difficulties. Primary macrophage cultures form highly adherent monolayers in vitro. To be analyzed by conventional flow cytometric methods (e.g., FACS analysis of non-adherent lymphoid populations), the adherent macrophages must be detached by enzymatic or mechanical methods which can result in damage to the cell membranes and/or strip off cell surface antigens. Other conventional techniques, e.g., antibody plus complement-mediated cytotoxicity or immunofluorescent microscopy, are semiquantitative at best. Surface antigen analysis using ELISA techniques provides a total population measure of changes in cell surface antigen expression, but fails to provide the number of antigen-positive cells or the density of antigen per cell. The ACAS 470 obviates all of these technical problems and allows for an analysis of individual adherent cells for the density and topography of antigen per cell, as well as expression of an antigen within a population. Using the ACAS 470, we have examined the expression of Ia antigens (class II major histocompatibility antigens) and the Mac-1 antigen (C3bi receptor) on thioglycollate-elicited murine macrophages, basally and after treatment with interferons. In this study, the density and distribution of these antigens per cell, as well as their expression within a population, are reported.

Regulation of MHC gene expression during the differentiation of bone marrow-derived macrophages

The ability of the macrophage to express class II MHC gene products appears to arise from both T-dependent and T-independent mechanisms. One mechanism by which macrophages express Ia-antigens in the absence of T-lymphocytes is postulated to be controlled by differentiation. By using a liquid bone marrow culture system, we have studied both class I and class II surface expression and mRNA accumulation during macrophage differentiation in vitro. The results demonstrated that Ia expression increased until 7 days in culture and then slowly declined. In contrast, class I expression appeared to steadily increase throughout the differentiation period. Northern blot analysis of RNA isolated from bone marrow-derived macrophages (BMDM) at various periods during culture, using Eα, Aα, and class I cDNA probes, correlated well with the results of Ia and H-2K surface expression. Further analysis demonstrated that the expression of Ia-antigens on BMDM was not the result of T-helper lymphocytes. This was determined by demonstrating (1) that bone marrow cultures were devoid of mature T-lymphocytes, (2) the absence of interferon (IFN)-γ transcripts in both adherent and nonadherent populations of bone marrow cells, and (3) that the addition of anti-IFN-γ monoclonal antibody (mAb) to the bone cultures did not alter the percentage of Ia-positive BMDM. Moreover, the addition of anti-tumor necrosis factor-α mAb to the bone marrow cultures had no effect on Ia expression by BMDM. Taken together, these results allow us to conclude that Ia expression by BMDM is probably not mediated via exogenous signals but rather results from an intrinsically controlled process.

Interleukin-2 augmentation of interleukin-1 and prostaglandin E2 production.

Some of the major side effects of interleukin-2 (IL-2) therapy in the treatment of malignancies may be related to increased interleukin-1 (IL-1) and/or prostaglandin E2 (PGE2) production. We examined the effect of recombinant (rIL-2) on the in vitro production of IL-1 beta and PGE2 by unstimulated and LPS-activated human blood mononuclear cells (PBMC). We also compared the effect of rIL-2 on IL-1 beta production by adherent and nonadherent blood mononuclear cell populations. Cultures of PBMC (5 x 10(6)/ml) were incubated for 24 hr in media only (control, 1,000 U/ml rIL-2, 2 micrograms/ml LPS, or both LPS and rIL-2. Supernatants obtained from these cultures were analyzed for levels of IL-1 beta and PGE2 by radioimmunoassays. The addition of rIL-2 caused an increase in IL-1 beta production in 13 of 13 control PBMC cultures and in 11 of 13 LPS-stimulated cultures, which were significant increases as determined by paired t tests. When PBMC were fractionated into plastic adherent and nonadherent populations, the rIL-2 induced increases in IL-1 beta production were more consistent in control (six of seven cases) and LPS (seven of seven cases) cultures of plastic nonadherent cells than in control (three of seven cases) and LPS (four of seven cases) cultures of plastic adherent cells. Recombinant IL-2 did not increase PGE2 production in control PBMC cultures (none of four cases), but did so in LPS-stimulated PBMC cultures (three of four cases]. These results suggest that rIL-2 may increase IL-1 production in vivo and thus possibly account for some of the side effects of this therapy.

Specific binding of human C-reactive protein to human monocytes in vitro.

The precise biologic function of C-reactive protein (CRP), a major acute phase protein in man, is unknown. The abilities of CRP to bind biologic substrates and to activate the C pathway, and its localization at sites of inflammation argue for an opsonic role for this protein. Such a role has been supported by recent reports of specific binding of CRP to neutrophils. Using highly purified radioiodinated human CRP, we have observed specific binding of this protein to human monocytes in vitro. The binding was reversible and rapid, with a t1/2 for the dissociation reaction of approximately 3 min. Binding was saturable at a CRP concentration of approximately 0.2 microM, with an estimated K from Scatchard analysis of 1.1 x 10(-7) M. Specific binding was calcium-dependent, with optimal binding occurring at calcium concentrations of more than 1.0 mM. No specific binding could be demonstrated to a non-adherent population of mononuclear cells (more than 80% lymphocytes). In other experiments, a 100-fold excess of human IgG failed to inhibit binding, although rabbit CRP produced competitive inhibition of binding which was quantitatively similar to human CRP. The binding was maximal at pH 7.4 and was sensitive to prior trypsin treatment of cells. These studies provide direct evidence for specific binding of soluble human CRP to human monocytes in vitro and thus provide further support for an important functional interaction of this acute phase protein with phagocytic cells in man.

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.

Generation and characterization of purified adherent lymphokine-activated killer cells in mice.

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.

Postprandial variations in the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents.

The diurnal variations in the specific activities of polysaccharide-degrading enzymes after feeding were monitored in adherent and non-adherent microbial populations separated from bovine rumen liquor and digesta solids. There were marked differences in the activity profiles of the enzymes within the subpopulations. Enzymes involved in the degradation of soluble carbohydrates were more active in the non-adherent populations, and in the liquor phase subpopulation activities increased in the 1-2 h post-feed period. The muralytic enzymes were most active in the adherent population. Specific activities increased by up to 20-fold over the 24 h period, with an initial five-fold increase occurring between 8 h and 12 h after feeding. Enzyme levels in the three non-adherent populations were similar at the end of the postprandial period. In the population recovered from the liquid associated with the digesta particles, however, the activities did not increase until the latter stages of the period, whereas in the non-adherent population from the digesta solids the activities varied little during the diurnal cycle. The numbers of micro-organisms associated with the digesta solids were similar at 2 h and 20 h after feeding; the variations in enzyme levels did not occur as a result of a population increase but were due to increased activities in an established population. The plant cell wall structural polysaccharides were degraded at different rates. There was no appreciable cellulose digestion during the first 8 h of the postprandial period and although hemicellulosic constituents were removed continuously the rate of loss of both polymers was increased in the later stages of the diurnal cycle when enzyme activities were maximal.

In-vitro processing of sperm with autoantibodies: analysis of sperm populations.

Ejaculates from 51 infertile men with significant levels of anti-sperm antibodies were submitted to processing in vitro in order to increase the number of antibody-free spermatozoa. Rapid removal and washing of spermatozoa from antibody-containing seminal fluid resulted in a variable decrease in IgG and/or IgA binding; only IgG binding was significantly reduced. Those spermatozoa were allowed to swim-up into an overlaying medium to select those with the best progression. Unexpectedly, in the post-migration samples (PM), the proportion of progressively motile spermatozoa coated with IgG and IgA antibodies increased significantly. An efficient selection of antibody-free spermatozoa was achieved prior to swim up migration by immuno-binding to polystyrene Petri plates coated with anti-human immunoglobulin antibodies. Non-adherent populations were depleted of 27-100% of IgG and IgA antibody-coated spermatozoa. After migration, the percentages of antibody-coated spermatozoa of those populations (PMP) remained low. The comparison between PM and PMP populations shows that immunobinding increases the number of motile antibody-free spermatozoa. The fertility potential of both sperm populations is currently under investigation.

The use of murine feeder cells in the cultivation of Plasmodium falciparum asexual blood stages.

Increased multiplication rates were observed in asexual erythrocytic stages of Plasmodium falciparum grown in the presence of a feeder-cell layer of mouse peritoneal wash cells (PWCs) using the candle-jar method of Trager and Jensen (1977). This held true for both new and established isolates of the parasite. When the PWC population was separated into adherent and non-adherent fractions, the adherent PWC population promoted an increase in parasite growth but the non-adherent population did not. An increase in parasite multiplication was not promoted by PWC-conditioned medium.

High cytotoxic cell activity in the marrow from patients with aplastic anemia.

The mechanism which produces marrow failure in idiopathic aplastic anemia is still unknown. Recent investigations have suggested the crucial role of NK cells in the regulation of normal hematopoiesis. In this study, the cytotoxic activity of mononuclear cells from human bone marrow and peripheral blood was examined against three NK-sensitive target cell lines in 15 patients with aplastic anemia as well as 21 normal subjects. Marrow mononuclear cells from aplastic anemia demonstrated a high cytotoxicity comparable to peripheral blood NK cells to these target cells. Neither large granular lymphocytes nor the cells expressing known NK cell surface phenotypes increased in aplastic marrow cell elements. The aplastic marrow cells showed strong killing activity rather than binding at single cell assay. They consisted of non-adherent and adherent cell population in plastic adherence and were unresponsive to IFN treatment. The existence of cytotoxic cells with high NK-like activity may be responsible for the mechanism of marrow failure in aplastic anemia.

Il-4 Production By T Depleted Cells From Nippostrongylus Brasiliensis Infected Mice

Interleukin-4 (IL-4) is known to be involved in both the in vivo IgE response and the elevated B cell IgE Fc receptor (Fc epsilon R11) expression seen after a parasite infection. To further analyze the relationship between Fc epsilon R11 expression and IL-4 production, purified B cells from uninfected, Nippostrongylus brasiliensis (Nbr) infected and from goat anti-mouse IgD (GaM delta) injected mice were isolated on various days post-treatment. The Fc episolon R11 levels on purified B cells from normal mice decreased after an overnight culture in media alone and addition of IL-4 to these cultures resulted in a 4 to 13-fold enhancement of Fc epsilon R11 levels. In contrast, the Fc epsilon R11 levels on B cells from Nbr infected mice were elevated after an overnight culture in media alone and addition of IL-4 did not further enhance the already upregulated Fc epsilon R11 levels. Overnight culture of purified B cell blasts from Nbr infected mice in the presence of an anti-IL-4 monoclonal antibody (11B11) caused the elevated Fc epsilon R11 levels to return to levels seen in normal mice, without affecting the Fc epsilon R11 levels on purified Go or B cell blasts from uninfected mice or Go B cells from Nbr infected mice. 11B11 also inhibited the elevated Fc epsilon R11 levels on highly purified B cells obtained by FACS sorting the non-adherent spleen cell population for class II+ cells. In contrast to Nbr infection, the Fc epsilon R11 levels on B cells were downregulated in the GaM delta injected mice. However, analogous to the Nbr system, the Fc epsilon R11 levels were unresponsive to the addition of exogenous IL-4. This study indicates that IL-4 production is seen in T depleted splenocytes and that this alternate source of IL-4 serves to maintain the elevated Fc epsilon R11 levels on B cells.

Influence of T cells on the expression of lymphokine-activated killer cell activity and in vivo tissue distribution.

Lymphokine-activated killer (LAK) cells are currently being evaluated in several cancer centers for the immunotherapy of patients with a variety of cancers. Understanding the in vivo distribution of LAK cells should help to optimize their antitumor efficacy. As a model system to examine this issue, nylon wool column-passed rat lymphocytes were cultured in the presence of rIL-2 for 1 and 2 days. The resulting cells were divided into two populations; one that adhered to the plastic flasks and the second which did not adhere. The adherent cells were found to be highly cytotoxic against NK-sensitive and NK-resistant targets, whereas the nonadherent cells were unable to kill NK-resistant targets unless T cells were removed from this population. These results indicate that T cells present in IL-2 activated bulk splenocytes may interfere with the activity of LAK cells. Adherent or nonadherent LAK cells were evaluated for their pattern of in vivo distribution after i.v. inoculation. These cells were found to display a restricted pattern of distribution, localizing mainly in the lungs at 2 h after i.v. injection but redistributing into the liver and the spleen by 24 h. LAK cells were rarely recovered from the lymphoid tissues, including the peripheral lymph nodes and the mesenteric lymph nodes. However, if T cells were not removed from the LAK cell population, some radioactivity was recovered from the peripheral and mesenteric lymph nodes. Fractionation of 2 day-activated, nonadherent population on discontinuous Percoll resulted in the enrichment of large granular lymphocyte (LGL)/LAK activity in low density fractions (42% and 45% Percoll), whereas high density fraction (70% Percoll) contained T cells which showed no cytolytic activity. Upon transfer into syngeneic rats, the 42% fraction showed typical LAK migration. In contrast, the 70% fraction showed typical T cell migration. What is more important, removal of the granulated cells resulted in a population which have no granules and resemble large agranular lymphocytes known to be pre-LGL/LAK cells. Large agranular lymphocytes showed a pattern of distribution different from both T and LGL/LAK cells.

Regulation of human natural cytotoxicity by enkephalins and selective opiate agonists

This study reports a bidirectional effect of the enkephalins and selective opiate receptor agonists on human natural killer (NK) cell activity. Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors and enriched for T lymphocytes and large granular lymphocytes (LGL) by passage over nylon wool columns. Nylon wool nonadherent cell populations were preincubated for 18 h in the presence of fetal bovine serum with and without methionine-enkephalin, leucine-enkephalin, dynorphin (fragment 1–3), [ d-Ser 2]-leucine-enkephalin-Thr (DSLET), and [ d-Ala 2, N Me-Phe 4, Gly-ol 5]-enkephalin (DAGO). NK activity was measured by a standard 51Cr release assay with radiolabeled K562 cells. The cytolytic capacity of low NK responder populations was enhanced by the endogenous opioids while the NK activity of high responder populations was suppressed. These results suggest an immunoregulatory action of opioid peptides on NK activity. This possibility was confirmed using a serum-free system in conjunction with recombinant interferon-α. In addition, the classic opioid receptor antagonist naloxone displayed both antagonist and direct immunomodulatory properties, which may indicate the presence of lymphocyte derived opioid peptides in the culture system.