Comparison of three strains of diabetic rats with respect to the rate at which retinopathy and tactile allodynia develop.

PURPOSE: We compared three rat strains to determine if different strains develop early-stage diabetic retinopathy or sensory neuropathy at different rates. METHODS: Sprague Dawley, Lewis, and Wistar rats were made diabetic with streptozotocin. Diabetic and nondiabetic animals had retinal vascular pathology measured at eight months of diabetes. The number of cells in the retinal ganglion cell layer (GCL), retinal function (using electroretinography [ERG]), and retinal levels of inducible nitric oxide synthase (iNOS), cyclooxygenase2 (COX2), and vascular endothelial growth factor (VEGF) were measured at four months of diabetes. Tactile allodynia was assessed in hind paws at two months of diabetes. RESULTS: Diabetes of eight months' duration resulted in a significant increase in retinal degenerate capillaries and pericyte ghosts in Lewis and Wistar rats, but not in Sprague Dawley rats. A significant loss of cells in the GCL occurred only in diabetic Lewis rats, whereas Wistar and Sprague Dawley rats showed little change. Diabetes-induced iNOS and VEGF were statistically significant in all strains. Cyclooxygenase 2 (COX2) was significantly elevated in the Sprague Dawley and Wistar strains. Lewis rats showed a similar trend, however, the results were not statistically significant. All strains tended to show diabetes-induced impairment of dark-adapted b-wave amplitude, but only Sprague Dawley and Lewis strains had a significant reduction in latency. All strains showed significant tactile allodynia in peripheral nerves. CONCLUSIONS: At the durations studied, Lewis rats showed accelerated loss of both retinal capillaries and ganglion cells in diabetes, whereas diabetic Wistar rats showed degeneration of the capillaries without significant neurodegeneration, and Sprague Dawley rats showed neither lesion. Identification of strains that develop retinal lesions at different rates should be of value in investigating the pathogenesis of retinopathy.

Apoptic renal carcinoma cells are better inducers of cross-presenting activity than their primary necrotic counterpart.

Vaccination with tumor-loaded dendritic cells (DC) is a promising treatment strategy for patients with renal cell carcinoma (RCC). Cells undergoing cell death proved useful as a source of tumor antigen for DC loading. Both apoptotic and necrotic tumor cells have been shown to efficiently load RCC-tumor antigens on DC. However, no direct comparison of these two kinds of death has been attempted in the same RCC. We compared DC pulsed with apoptotic cells, whole cell lysates or their supernatants of the cell line K1, derived from a patient with clear cell RCC, to determine their ability to activate T cells. Monocyte-derived DCs were pulsed with the different sources of tumor antigen, matured and co-cultured with autologouos peripheral blood lymphocytes. After three weekly re-stimulations with DCs, generation of cytotoxic T lymphocytes CTL was assessed by IFN-gamma release in an ELISpot assay in the presence of the sensitizing target. By comparison with lysate, apoptotic tumor cells induced a higher frequency of MHC class I-restricted IFN-gamma releasing lymphocytes. A higher CTL response was induced by pulsing DCs with cell lysate supernatant compared with whole cell lysate. These results indicate that, although necrotic death has been regarded as highly permissive when compared to apoptotic death, the immunogenicity of the death treatment may vary from one tumor to another.

Effect of prolactin on carcinoembryonic antigen-specific cytotoxic T lymphocyte response induced by dendritic cells.

The cytokine hormone prolactin (PRL) has been shown previously to modulate native cellular responses and maturation of antigen-presenting cells. Here we have addressed its effect on the antigen-specific response of cytotoxic T lymphocytes (CTL). CTL were generated from HLA-A2 lymphocytes after three rounds of stimulation with autologous dendritic cells loaded with HLA-A2-restricted carcinoembrionic antigen (CEA) Cap-1 (YLSGANLNL) peptide. Selected cultures were expanded on cytokine-supplemented feeder-layers, enriched for CD8+ lymphocytes and analysed for PRL-receptor (PRL-R) expression and PRL responsiveness. Resting CD8+ lymphocytes were negative for PRL-R, whereas antigen-activated CD8+ lymphocytes derived from long-term cultures were highly positive. Results of a 51Cr release assay showed CTL killing of CEA-loaded, but not unloaded, T2 cell line and the CEA-positive gastric carcinoma cell line KATO, but not of the CEA-negative T leukaemia cell line Jurkat. Interferon (IFN)-gamma release, evaluated in an ELISPOT assay against CEA-loaded T2, was enhanced (P < 0.05) by concentrations of PRL (12-25 ng/ml) very close to the physiological levels (6-20 ng/ml), but was decreased (P < 0.05) by high concentrations (200 ng/ml). Pre-incubation of the stimulators with the anti-MHC class I MoAb W6.32 induced a 40-60% decrease of the PRL-boosted IFN-gamma release, thus proving the MHC restriction of the lymphocyte response. Cytotoxicity against CEA-loaded T2 and KATO cell lines was also increased by 12-25 ng (P < 0.05) and decreased (P < 0.05) by 200 ng PRL. Pre-incubation of CTL with an antibody specific for the PRL-R almost completely abrogated this effect.

Effect of prolactin on the antigen presenting function of monocyte-derived dendritic cells.

Monocyte derived macrophages (Mphi) and dendritic cells (DC) play critical roles at the interface between innate and adaptive immunity. Both types of cells can effectively phagocytose exogenous antigens, whereas only DC can process and present them efficiently to antigen-specific T lymphocytes. The hormone PRL is also produced by immune cells and is regarded as a key component of the neuroendocrine--immune loop and a local regulator of lymphocyte response. Its main feature is cooperation with cytokines and hemopoietins. Triggering of monocyte PRL receptors with physiological-to-supraphysiological concentrations of PRL up-regulates the GM-CSF receptors, resulting in synergistic PRL-GM-CSF induced maturation of immature (i)DC. Further incubation induces increased antigen-presenting activity at the highest PRL concentrations studied (200 ng/ml). IFN-gamma, release by allogeneic lymphocytes is dependent on T cell-triggered IL-12 release by PRL-preincubated iDC. This, in turn, may be secondary to increased DC expression of CD40 or IFN-gamma. The permissive action of high PRL concentrations in the antigen presenting process may be of significance in initiation of the response against major histocompatibility complex (MHC)-presented self-antigens and may explain the association of hyperprolactinemia with autoimmune diseases.

Ex vivo host response to gastrointestinal cancer cells presented by autologous dendritic cells.

BACKGROUND: Dendritic cells (DCs) capture apoptotic tumors and cross-present their antigens in the MHC class I and class II pathways for recognition by CD4+ and CD8+ T lymphocytes. Here we have tested the ability of fresh surgically resected colon and gastric cancer tumors to specifically activate host T lymphocytes when presented by autologous DCs. METHODS: DCs derived from adherent blood mononuclear cells of five patients, after a 7-day culture with GM-CSF and IL-4, were exposed to apoptotic autologous tumor (AAT) or apoptotic autologous peritumor normal (AAN) cells and cultured 24 h with monocyte-conditioned medium to achieve full DC maturation. Tumor-specific response was evaluated as single-cell cytokine release in an enzyme-linked immunospot (ELISPOT) and as cytotoxicity in a cold target inhibition (51)Cr-release assay. RESULTS: AAT-DCs induced specific IFN-gamma by T lymphocytes of two patients (rectal and gastric cancer), whereas in another two patients (rectal and gastric cancer) this response was depressed with a similar tumor-specific pattern and in one patient (rectal cancer) there was no response. Activation of IFN-gamma release was accompanied by tumor cytotoxicity and both responses were enhanced by IL-12, indicating the functional integrity of patients' lymphocytes. CONCLUSION: These data show that T-cell memory against rectal/gastric carcinoma antigens can be triggered by tumor-loaded autologous DCs. However, escape mechanisms may exist among tumors of the same histological origin that can inhibit this host response. A DC-based antitumor immunological monitoring assay with autologous tumor biopsies may allow patients to be screened to determine those who are suitable candidates for immune-based immunotherapy.

Prolactin in autoimmunity and antitumor defence.

Prolactin (PRL) enhances inflammatory and antitumor responses in vitro and thus exhibits Th1-type cytokine-like effects. Evidence from experimental models indicates that inhibition of PRL release by bromocriptine downregulates immune reactions and ameliorates autoimmune diseases in which Th1 responses are predominant. A direct effect of locally produced PRL in some Th1 diseases, such as rheumatoid arthritis, supports this concept. Paradoxically, however, hyperprolactinemia can also be associated with conditions such as pregnancy, where remission of Th1-mediated diseases is known to occur in the context of a Th2-dominated milieu. This reversal of the Th1-promoting effect of PRL may be due to major changes in the levels of other hormones that can annul and/or override the PRL-mediated proinflammatory state. Nevertheless, PRL, as an immunopotentiating agent, may have a powerful therapeutic role in cancer and other immunocompromised patients.

Individual and combined effect of granulocyte-macrophage colony-stimulating factor and prolactin on maturation of dendritic cells from blood monocytes under serum-free conditions.

Prolactin (PRL) shares structural and functional features with haemopoietic factors and cytokine peptides. Dendritic cells (DC) are involved in both initiating the primary and boosting the secondary host immune response and can be differentiated in vitro from precursors under the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus other factors. Because PRL has been shown to functionally interact with GM-CSF, we have addressed its role on GM-CSF-driven differentiation of DC. Monocytic DC precursors from peripheral blood mononuclear cells (PBMC) were enriched either by adhesion to a plastic surface or CD14-positive selection and cultured for 7 days in serum-free medium containing GM-CSF, interleukin (IL)-4 and PRL, alone or in combination. Cells with large, veiled cytoplasm, expressing major histocompatibility complex (MHC) class II and the costimulatory molecules CD80, CD86 and CD40 and lacking the monocyte marker CD14, were considered as having the phenotype of cytokine-generated DC. Functional maturation was assessed by proliferation and interferon-gamma (IFN-gamma) release of allogeneic T lymphocytes. Physiological (10-20 ng/ml) concentrations of PRL interacted synergistically with GM-CSF and the effect was similar to that induced by IL-4 on GM-CSF-driven DC maturation. When used alone, the physiological concentrations of PRL were inhibitory, whereas higher concentrations (80 ng/ml) were stimulatory. The synergistic effect of PRL may in part be caused by its ability to counteract the down-modulation of the GM-CSF receptor observed in serum-free conditions. These data provide further evidence of the significance of PRL in the process of T lymphocyte activation.

Association between elevated prolactin levels and circulating erythroid precursors in dialyzed patients.

The prolactin (PRL) receptor (R), a member of the cytokine hemopoietin receptor superfamily, has been shown to activate early differentiation steps along the erythroid pathway. In particular PRL, a product of bone marrow stroma, induces functional erythropoietin (EPO)-R on CD34+ hemopoietic progenitors. In this study, expression of EPO-R mRNA and responsiveness to EPO were assessed on enriched hemopoietic progenitor cells (HPC) from seven hyperprolactinemic and three normoprolactinemic patients and two normal subjects. Expression of EPO-R mRNA by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was found in HPC of four out of seven hyperprolactinemic patients but not in normoprolactinemic patients or normal donors. Development of EPO-dependent Colony Forming Unit-Erythroid (CFU-E) colonies in semi-solid medium was observed only in hyperprolactinemic patients (six out of seven). A much higher number of CFU-E colonies was observed in the four patients with a positive EPO-R message. We conclude from these data that abnormally high levels of PRL may increase the number of EPO-responsive hemopoietic precursors in vivo as they do in vitro. Since hyperprolactinemia associates in these patients with depressed EPO production, it may be regarded as a compensatory mechanism for the reduced availability of the hemopoietic factor.

Cooperation of pituitary hormone prolactin with interleukin-2 and interleukin-12 on production of interferon-gamma by natural killer and T cells.

The pituitary hormone prolactin (PRL) is also produced by cells of the immune system and participates in early and late T cell activating events. We have previously shown a modulatory role of PRL during maturation of dendritic cells (DC). Production of IL-12 by T cell receptor (TCR)-activated DC is necessary for T cells to acquire the Th1 cytokine (i.e., IFN-gamma secreting) profile, which is associated with activation of cellular response. In a separate work, PRL has been shown to increase IFN-gamma synthesis by natural killer (NK) cells. We have extended that study by exploring the ability of PRL to induce IFN-gamma production by T and NK cells in the presence of the specific stimuli IL-12 and IL-2. The individual effect of PRL, IL-12, and IL-2 was specific for NK cells, and IL-2 and IL-12 were much more efficient than PRL. Cooperation of IL-2 and PRL was observed on NK cells. IL-2-induced synthesis of IFN-gamma was increased by physiological concentrations of PRL but was unaffected or inhibited by high concentrations. By contrast, optimal enhancement of IL-12-induced IFN-gamma release was observed with T cells but not with NK cells. Unexpectedly, interaction between PRL and IL-12 occurred only at high concentrations of PRL. These data indicate a complex role of PRL in the cytokine network and point to a revaluation of the proposed immunosuppression by stress-related hyperprolactinemia.

Expression of prolactin and prolactin receptors by non-Hodgkin's lymphoma cells.

Prolactin (PRL) interacts with lymphocyte-signaling molecules and cytokines. Previous work has shown independent and synergistic effects of PRL on the generation of IL-2-driven anti-tumor lymphokine activated killer (LAK) activity by peripheral blood mononuclear cells (PBMC). The potential importance of PRL as a biological immunomodifier, however, is challenged by its ability to influence normal lymphocyte mitogenesis and hence lymphoid tumor growth. Since non-Hodgkin's lymphoma (NHL) cell lines were efficiently killed by LAK generated with native (n) or recombinant (r) human PRL combined with low, per se ineffective doses of IL-2, we have addressed here the question of whether PRL acts as a growth factor for LAK targets. NHL cells were analyzed for: 1. expression of the PRL receptor (PRL-R); 2. responsiveness to nPRL or rPRL; 3. constitutive expression and release of PRL; 4. existence of a PRL autocrine loop. PRL-R, defined by multiple antibodies, was detected in 3 of 12 NHL cell lines. However, nPRL or rPRL, in a wide range of concentrations (0.75-50 ng/ml), were not mitogenic for growth-arrested, PRL-R positive NHL cell lines. PRL mRNA was detected by RT-PCR in 10 of the 12 cell lines examined with a higher frequency among AIDS-related NHL cell lines. PRL protein in the immunoprecipitate of (35)S-methionine-labeled cell lysates and supernatants paralleled mRNA expression, and Western blotting analysis showed the presence of the pituitary/lymphocyte non-glycosylated (23.5 kDa) and glycosylated (25 kDa) isoforms. Experiments with blocking antibodies showed the independence from endogenous PRL for NHL cell growth.

Up-modulation of interferon-gamma mediates the enhancement of spontanous cytotoxicity in prolactin-activated natural killer cells.

Prolactin (PRL) has been shown to participate in lymphocyte activation. In particular, the constitutive natural killer (NK) and the lymphokine-activated killer (LAK) cytotoxicity of CD56+ CD16+ cells is increased by its physiological to supraphysiological concentrations. As PRL has been shown to up-regulate the production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells, we studied its effect on IFN-gamma production by NK cells as a possible mechanism of autocrine activation of cytotoxicity. Released and intracellular IFN-gamma, as well as IFN-gamma mRNA expression, were increased by pituitary and recombinant human PRL, which stimulated optimal NK and LAK cytotoxicity. Treatment with blocking anti-IFN-gamma monoclonal antibody (mAb) selectively affected PRL-increased killing of K562 targets, demonstrating that PRL-mediated enhancement of spontaneous cytotoxicity depends, at least in part, on up-regulation of IFN-gamma.

[Tumor samples as a tool for large-scale monitoring of antigen-specific lymphocytes in gastric and colorectal neoplasms]. / L'utilizzo di campioni di tessuto tumorale come mezzo per il monitoraggio su larga scala dei linfociti antigene specifici nelle neoplasie gastriche e colorettali.

The characterization of tumor-associated antigens has enabled to direct the host immune response towards the autologous tumor through appropriate loading and presentation of the antigen. In vivo conditions that generate large numbers of tumor antigens would be an important step in vaccine strategies. In this study we have therefore tested the ability of freshly isolated gastric and colorectal cancer cells to induce a specific anti-tumor response in autologous T lymphocytes. Because dendritic cells (DC) are critically involved in both initiating and boosting host immune responses, they have been used to present apoptotic bodies generated by irradiated tumor cells. Results show that these native antigens stimulate T cytotoxic response against tumor, but not peritumor normal tissues. Induction of IFN-gamma secreting cell activity, which is a standard readout in current cancer vaccine protocols, was also demonstrated by Elispot single-cells assay. These data show the antigenicity of gastric and colorectal tumor cells and open new perspectives in immunotherapy.

A role for growth hormone and prolactin in leukaemia and lymphoma?

Growth hormone (GH) and prolactin (PRL) quality as lymphohaemopoietic growth and differentiation factors, and so does insulin-like growth factor (IGF)-I, which mediates many of GH activities. Although there is only limited evidence that endocrine, paracrine or autocrine GH or PRL play a role in human leukaemia and lymphoma, the expression of these factors or their receptors may have diagnostic or therapeutic implications. Indeed, the participation of GH, PRL or IGF-I in the development or progression of certain haematological malignancies or to the antitumour immune response has been documented. Examples discussed in this review include a rat lymphoma in which the PRL receptor acts as an oncogene; the rat Nb2 lymphoma, which is dependent on PRL for growth; and experiments showing that PRL stimulates natural killer cell activity and the development of lymphokine-activated killer cells.

Prolactin is an autocrine growth factor for the Jurkat human T-leukemic cell line.

Despite convincing evidence of cooperation between IL-2 and endogenous prolactin (PRL) during T cell activation, the individual role of PRL as a T-cell lineage cytokine remains to be defined. We have examined the production and function of PRL on the Jurkat human T-leukemic cell line, which does not constitutively produce IL-2. The majority of Jurkat cells expressed PRL receptor (R) under standard culture conditions, whereas appearance of the alpha chain of the IL-2-R required PHA-PMA stimulation, as did IL-2 synthesis. Western blotting revealed a predominant band at 23.5 kDa and a weaker band at 25.5 kDa in both Jurkat cell lysates and human (h) pituitary PRL. Metabolic labeling of the cell lysates with 35S-methionine and immunoprecipitation with an antiserum against hPRL showed that both forms of PRL are actively synthesized by the Jurkat cell line. PRL released in the medium was biologically active in the rat Nb2 lymphoma mitogenic assay. Depletion of medium PRL with two polyclonal anti-hPRL antisera inhibited the growth of Jurkat cells in a dose-dependent manner, as evaluated by cell number and 3H-TdR uptake. Purified pituitary or recombinant hPRL at a wide range of concentrations had no significant effect on their growth, but reversed the blocking activity of the anti-hPRL antibody. Recombinant IL-2 had no effect on the antibody-induced growth inhibition. Taken as a whole, these results demonstrate that PRL can act as an autocrine T cell growth factor independently of IL-2 and are the first evidence of its involvement in human leukemic growth and possibly in leukemic transformation.

Bone marrow stroma-derived prolactin is involved in basal and platelet-activating factor-stimulated in vitro erythropoiesis.

Cooperation between in vitro exogenous prolactin (PRL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) at an early step of in vitro erythroid differentiation has been shown in a previous study. To gain more insight into the role of PRL in in vivo hematopoiesis, we have now addressed the involvement of endogenous PRL in the growth of hematopoietic progenitors in a bone marrow (BM) stroma environment. The possible modulation of local PRL production by the inflammatory mediator platelet-activating factor (PAF), which is known to be produced by BM cells and to regulate pituitary PRL release, has also been evaluated. Development of burst-forming unit-erythroid (BFU-E) colonies from CD34+ hematopoietic progenitors cultured on a BM stroma cells (BMSC) layer was slightly, but significantly, reduced in the presence of an anti-human PRL antibody. Pretreatment of BMSC with PAF increased the BFU-E colony efficiency of cocultured CD34+ cells, and this effect was completely abrogated by the antiserum. PAF-modulated release of PRL by BMSC was confirmed by an enzyme-linked-immunospot (Elispot) technique. In addition, immunoprecipitation and Western blotting experiments showed two immunoreactive products in the BMSC culture medium. These corresponded to the nonglycosylated (23 kD) and glycosylated (25.5 kD) forms of pituitary PRL that are also expressed by the B-lymphoblastoid cell line IM9-P3. Specific increase of the nonglycosylated form and decrease of the glycosylated form was observed after PAF treatment. Polymerase chain reaction (PCR) amplification of reverse transcribed RNA using PRL-specific primers showed the presence of PRL message in BMSC and IM9-P3 cells. In situ hybridization experiments with a rat PRL cDNA probe cross-reacting with human PRL mRNA confirmed its presence in a small fraction of unstimulated BMSC and in the majority of PAF-stimulated BMSC. The enhancing effect of PAF on PRL-mediated colony formation, PRL release, and mRNA activation was counteracted by pretreating BMSC with the PAF-receptor (R) antagonist WEB 2170. Lastly, responsiveness of BMSC to PAF was substantiated by the presence of the PAF-R mRNA on these cells.

Action of pituitary and lymphocyte prolactin.

In vivo and in vitro data combined show that prolactin (PRL) can mimic or interact with known lymphocyte cytokines and that these, in turn, can regulate PRL synthesis at the site of immune response. In contrast, pituitary PRL is under the control of both immune system products (non-cognitive stimuli) and signals to the CNS (cognitive stimuli). The role of PRL as a cytokine and as an endocrine hormone is discussed. In particular, assignment of PRL to the T helper 1 phenotype is proposed, based on its ability to enhance NK cell function, activate the interferon-regulated factor (IRF-1) transcription factor and to interact with or generate IL-2 and IFN gamma. Since hyperprolactinemia and hypoprolactinemia are both immunosuppressive, physiological levels of circulating PRL must be necessary to maintain normal immunocompetence. Moderate increases in PRL during immune stimulation of the hypothalamic-pituitary axis may counteract glucocorticoid inhibition, whereas inappropriate prolongation of PRL synthesis could lead to autoimmune diseases. Increased release of PRL by the pituitary during stress may inhibit NK cell antitumor cytotoxicity. The variety of PRL isotypes, the existence of multiple receptor subunits, and the complexity of their intracellular signaling may explain the specificity of PRL action on different target cells.

Specific increase in p85alpha expression in response to dexamethasone is associated with inhibition of insulin-like growth factor-I stimulated phosphatidylinositol 3-kinase activity in cultured muscle cells.

The stimulation of phosphatidylinositol (PI) 3-kinase by insulin-like growth factor I (IGF-I) in L6 cultured skeletal muscle cells is inhibited by the glucocorticoid dexamethasone. The objective of this study was to investigate the mechanism of dexamethasone action by determining its effects on the expression of the p85alpha and p85beta regulatory subunit isoforms of PI 3-kinase, their coupling with the p110 catalytic subunit, and their association with insulin receptor substrate 1 (IRS-1) in response to IGF-I stimulation. Dexamethasone induced a 300% increase in p85alpha protein content in the L6 cultured myoblast cell line, whereas it increased p110 content by only 38% and had no effect on p85beta. The increase in p85alpha protein was associated with a coordinate increase in p85alpha mRNA. Stimulation with IGF-I induced the association of p85alpha and p85beta with IRS-1, and this was accompanied by increased amounts of the p110 catalytic subunit and markedly increased PI 3-kinase activity in IRS-1 immunoprecipitates. In cells treated with dexamethasone, greater amounts of p85alpha and lower amounts of p85beta, respectively, were found in IRS-1 immunoprecipitates, such that the alpha/beta ratio was markedly higher than in control cells. In spite of the increase in both total and IRS-1-associated p85alpha following dexamethasone treatment, IRS-1-associated p110 catalytic subunit and PI 3-kinase activity were decreased by approximately 50%. Thus, dexamethasone induces a specific increase in expression of the p85alpha regulatory subunit that is not associated with a coordinate increase in the p110 catalytic subunit of PI 3-kinase. As a consequence, in dexamethasone-treated cells, p85alpha that is not coupled with p110 competes with both p85alpha.p110 and p85beta.p110 complexes for association with IRS-1, leading to increased p85alpha but decreased p85beta, p110, and PI 3-kinase activity in IRS-1 immunoprecipitates.

Role of prolactin in the in vitro development of interleukin-2-driven anti-tumoural lymphokine-activated killer cells.

Exogenous prolactin (PRL) has been shown to synergize with low-dose interleukin-2 (IL-2) and induce the proliferation and lymphokine-activated killer (LAK) maturation of natural killer (NK) cells. PRL itself can also generate LAK activity. Here we show that its local production occurs during, and is necessary for, LAK development. IL-2-stimulated peripheral blood mononuclear cells (PBMC) and purified NK cells were exposed to anti-human (h)PRL antiserum, and residual LAK activity was measured on day 7 against the promyelocytic leukaemia cell line HL-60. Inhibition of LAK activity was much more evident in PBMC compared with NK cell cultures (47% decrease. P - 0.013 and 18.5% decrease. P = 0.048, respectively). Up-modulation of a 32S-methionine-labelled 27,000 MW protein was detected in the lysates and supernatants of IL-2-stimulated PBMC immunoprecipitated with an anti-PRL antiserum. By contrast, the cytoplasmic PRL immunoreactivity observed in freshly isolated NK cells and in IL-2-stimulated, but not unstimulated, NK cell cultures was not associated with PRL gene activation, and can thus be referred to internalized PRL. Preferential re-uptake of externally derived PRL by IL-2-stimulated NK cells was also indicated by up-modulation of the PRL receptor. These data, as a whole, indicate that the PRL promotion of LAK differentiation is mainly mediated by paracrine secretion, with a minor contribution from internalized PRL.