The role of neutrophils in antiviral defense--in vitro studies on the mechanism of antiviral inhibition.

Highly enriched populations of bovine neutrophils were added, in the presence of antiviral antibody, to herpesvirus-infected bovine cell cultures. A cell dose-dependent reduction in virus-induced cytopathology was observed. The mechanism of inhibition was presumed to be mediated by a subcellular neutrophil product and not the result of either direct cytotoxicity or antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, inhibition of comparable magnitude was observed when neutrophils and virus-infected cells were separated by cell impermeable membranes. In addition, plaque reduction occurred when antiviral immunoglobulins or fragments unable to mediate ADCC were used in the assays. Killed neutrophils and sonicates were unable to mediate plaque inhibition. Speculations were made as to the origin of the virus-inhibitory substances and the role that neutrophils might assume in mediating recovery from virus infection.     

Activation of granulocyte cytotoxic function by purified mouse colony-stimulating factors

Highly purified mouse colony-stimulating factors (CSF) were tested for their effect on neutrophil cytotoxic function in a homologous antibody-dependent cell-mediated cytotoxicity (ADCC) assay in which TNP-coupled mouse thymoma cells coated with mouse anti-TNP antibodies were used as targets, and purified normal mouse bone marrow neutrophils or induced peritoneal neutrophils were used as effector cells. Biochemically pure granulocyte-macrophage (GM)- and granulocyte (G)-CSF enhanced the cytotoxic activity of neutrophils obtained from both sources, allowing them to kill target cells at low antibody concentrations. Furthermore, GM- and G-CSF showed an additive effect, suggesting either the presence of separate receptors for GM- and G-CSF or of separate subsets of neutrophils. Induced peritoneal neutrophils showed a higher level of basal cytotoxic activity than did bone marrow neutrophils, suggesting neutrophil activation in vivo, but both reached similar levels of cytotoxicity upon maximal stimulation with CSF. In addition, CSF was found to be cross-reactive between mouse and human species in their enhancement of neutrophil cytotoxicity. By testing purified mouse CSF on human neutrophils, it could be shown that G-CSF and GM-CSF are functionally distinct molecules, because only G-CSF enhanced ADCC by human neutrophils. These experiments show that the purified factors that control the production of neutrophils by progenitor cells in vitro also activate differentiated neutrophils to carry out their cytotoxic activity in a more effective manner.     

Effects of leukocyte inhibitory factor (LIF) on neutrophil mediated antibody-dependent cellular cytotoxicity

The human lymphokine, leukocyte inhibitory factor (LIF), was investigated for its effect on neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) for K562 targets. Highly purified LIF (0.5 to 2 U/ml) induced a significant dose-dependent potentiation of neutrophil ADCC by up to 54.9% (p less than 0.001). Higher concentrations of LIF inhibited cytotoxicity. The degree of cytotoxicity was found to correlate (r = 0.99) with the increased secretion of superoxide after neutrophil-target cell interaction. Anaerobic conditions inhibited cytotoxicity mediated by both control and LIF-treated neutrophils. The latter observation lends support to the concept that enhanced ADCC was mediated through increased superoxide production and not through the induction of a separate pathway. Increased superoxide production may have resulted from an upregulation of the transduction mechanism leading to neutrophil stimulation through the Fc receptor. In addition, we demonstrated an increased capacity of the neutrophil to adhere to its target (average 3.3:1 effector:target ratio in untreated cells to 4.8:1 after treatment with LIF), and this may also have been responsible for the increase in the respiratory burst and subsequent enhanced ADCC. These observations provide potential support for an in vivo role for LIF in tumor immunity.     

Effects of leukocyte inhibitory factor (LIF) on neutrophil phagocytosis and bactericidal activity

Human leukocyte inhibitory factor (LIF) is a lymphokine initially defined by its ability to inhibit the random migration of neutrophils. We have recently demonstrated that LIF also potentiates a number of f-met-leu-phe-mediated functions as well as enhancing one Fc receptor-mediated function (antibody-dependent cellular cytotoxicity). In this paper, we have extended our studies involving the effects of LIF on the neutrophil, specifically its effect on phagocytosis and bactericidal activity. We demonstrate that LIF (2 U/ml) potentiates phagocytosis of opsonized heat-killed Staphylococcus aureus (up to 57.2%) and sheep erythrocytes (124.4%) as well as unopsonized latex particles (59.9%). Phagocytosis of opsonized sheep erythrocytes was inhibited by an anti-neutrophil Fc receptor antibody with control PMN but not using the LIF-treated PMN. LIF (1/2 to 1 U) also potentiates the killing of S. aureus by up to 51.6%. Higher concentrations of LIF (greater than or equal to 4 U) inhibits killing. These effects were shown not to be associated with an increase in Fc receptor availability. It is therefore possible that potentiation of these neutrophil activities by LIF may occur either as a result of increased receptor turnover or, more likely, secondary to an increase in nonspecific neutrophil adherence. These studies further support the concept that LIF may have an important role in vivo in inflammation and immunity.     

Lymphokine production in the cell-mediated immune response (author's transl)

In response to tuberculin, lymphocytes derived from BCG-sensitized guinea pigs elaborated soluble materials capable of inducing some reactions in vitro which correlated with the cell-mediated immune response in vivo. The lymphokine activity of these soluble materials was demonstrated by the following biological effects: macrophage migration inhibition, macrophage aggregation, cytotoxicity to HeLa cells and skin reaction.     

A lymphokine distinct from interferon-gamma that activates human monocytes to kill Leishmania donovani in vitro

Human interferon-gamma (IFN-gamma), a T cell lymphokine (LK), activates monocytes to kill many intra- and extracellular pathogens. In fact, previous reports assert that all activity in LK for macrophage activation is due to IFN-gamma. To test this assertion, we examined monocyte interactions with amastigotes of Leishmania donovani after treatment with recombinant DNA or affinity-purified leukocyte IFN-gamma and IFN-gamma containing LK. Cells treated with at least 200 IU/ml IFN-gamma were microbicidal for L. donovani. Analysis of IFN-gamma dose responses for induction of microbicidal activity by recombinant IFN-gamma (r-IFN-gamma) and LK, however, documented a striking difference: LK was 25-fold more efficient than r-IFN-gamma at equivalent IFN-gamma titers. This large difference suggested that monocyte activation factor(s) in LK may not be IFN-gamma. Rabbit anti-IFN-gamma completely inhibited antiviral activity in LK but did not abrogate the ability to induce monocyte cytotoxicity against leishmania. Furthermore, removal of IFN-gamma from LK by monoclonal anti-IFN-gamma affinity chromatography or by treatment with anti-IFN-gamma followed by staphylococcal protein A chromatography also did not inhibit LK activity. Fractionation of LK on Sephadex G-100 revealed two activity peaks: one in the 50,000 to 60,000 m.w. range coincident with IFN-gamma, and the other at 25,000 to 30,000 daltons with no IFN-gamma. These studies document LK physicochemically and antigenically distinct from IFN-gamma that activate monocytes to kill L. donovani. Such novel factors may have broad import for the study of macrophage-mediated host defenses and for development of immunotherapeutic regimens.     

Development and comparison of in vivo and in vitro models for endometritis in cows and mares

In order to investigate pathogenic mechanisms of acute endometritis in cows and mares, we established an in vivo model in both species. Based on the results of an in vitro transmigration system, human recombinant interleukin-8 (rhIL-8; 1.25 microg per mare and 5 microg per cow in 50 ml phosphate-buffered saline) was used to attract polymorphonuclear neutrophil granulocytes (PMNs) into the uteri. Peak numbers of uterine neutrophils were attracted after 6h, in both cows and mares. On average, mares responded more sensitively than cows, with 15 times higher numbers of rhIL-8-attracted uterine neutrophils (72+/-8 x 10(7)cells). In contrast to in vitro studies, in vivo migrated neutrophils (uterine neutrophils) of both species displayed a significantly reduced MHC class I expression. Expression of the CD11a molecule was significantly enhanced on equine uterine neutrophils but downregulated on bovine cells. Compared with untreated autologous peripheral neutrophils, both uterine and in vitro migrated neutrophils showed no alteration of phagocytic capacity. The ability to generate reactive oxygen species (ROS) was significantly upregulated in bovine and equine uterine neutrophils. This was also observed after in vitro migration of equine neutrophils, whereas ROS generation by bovine neutrophils was significantly depressed. In summary, the concept of inducing endometritis directly by local application of human interleukin-8 has been reliably successful in cows and mares. The model permits the analysis of PMN migration into the uterus under defined and controlled conditions. The observed differences between cows and mares with respect to phenotypical and functional characteristics of in vivo attracted uterine cells point to species-related features of neutrophil migration. In vitro transmigrated bovine and equine cells partially differ in phenotype and function from uterine neutrophils. Therefore, the in vitro transmigration assay cannot completely represent the in vivo endometritis model described here.     

Turnover and fate of I-Ak antigen on the murine macrophage cell surface

The macrophage plasma membrane is a major site of the cell's activities, including phagocytosis, antibody-dependent cellular cytotoxicity, and antigen presentation. To present antigen, the expression by the macrophage of immune region-associated (Ia) antigen is required. The turnover and fate of this cell surface constituent was studied in macrophages cultured with lymphokine or recombinant interferon-gamma. Surface-labeled subregion I-Ak antigen was lost from the cell surface at a rapid rate, with a half-life of approximately 24 hours. However, the shedding of I-A antigen into the culture fluid was not detected. Therefore, the loss of I-A antigen from the macrophage surface is most likely by its degradation. Upon removal of lymphokine or interferon from macrophage cultures, I-A antigen expression declined, with an apparent half-life of 2 days.     

Tumour necrosis factor-alpha and interleukin-8 inhibit neutrophil migration in vitro and in vivo

Pretreatment of human neutrophils with recombinant tumour necrosis factor-alpha (rTNF-alpha) and/or interleukin-8 (rIL-8), but not with either transforming growth factor-beta, interleukin-6 or interferon-gamma, rendered these cells less responsive to FMLP, in microchemotaxis assays. This inhibitory effect was dose dependent and more powerful when neutrophils were pretreated with a mixture of both cytokines. Intravenous injection of human rIL-8 (hrIL-8) and/or murine rTNF-alpha (mrTNF-alpha) also significantly reduced in vivo neutrophil migration into peritoneal cavities of rats stimulated with carrageenan. These data suggest that the defect in neutrophil migration during septicaemia or endotoxaemia may be the result of the continuous release of IL-8 and TNF-alpha into the circulation. Thus, either the selective control or blockade of releasing of these cytokines as well as of its effects on neutrophils may be clinically useful in reestablishing the cell defence mechanisms.     

Abrogation of species specificity for activation of tumoricidal properties in macrophages by recombinant mouse or human interferon-gamma encapsulated in liposomes

Highly purified human blood monocytes, isolated by continuous Percoll density gradients under endotoxin-free conditions, and mouse peritoneal exudate macrophages (PEM) were activated in vitro by the combination of muramyl dipeptide (MDP) and recombinant interferon-gamma (r-IFN-gamma) to become tumoricidal against their respective tumorigenic target cells. The activation of human monocytes or mouse PEM by free unencapsulated r-IFN-gamma and MDP was species specific: human r-IFN-gamma activated human blood monocytes to lyse allogeneic melanoma cells, but did not activate mouse PEM. Mouse r-IFN-gamma activated mouse PEM to lyse syngeneic melanoma cells, but did not activate cytotoxic properties in human monocytes. The encapsulation of either mouse or human r-IFN-gamma with MDP within the same liposome preparation produced synergistic activation of cytotoxic properties in both PEM and monocytes without apparent species specificity. The activation of tumoricidal properties in macrophages by r-IFN-gamma and MDP occurred as a consequence of intracellular interaction. We base this conclusion on the data showing that whereas free r-IFN-gamma and MDP did not activate macrophages pretreated with pronase, liposome-encapsulated r-IFN-gamma and MDP did. Moreover, the i.v. injection of liposomes containing human or mouse r-IFN-gamma and MDP produced in vivo activation of mouse alveolar macrophages. These data suggest that in contrast to activation with free r-IFN-gamma, which requires binding to macrophage surface receptors, the intracellular interaction of r-IFN-gamma, which produces tumoricidal activity in macrophages, is not species specific.     

Recombinant human interleukin-8, but not human interleukin-1beta, induces bovine neutrophil migration in an in vitro co-culture system

A co-culture system was established by culturing a bovine mammary epithelial cell line (MAC-T) and a bovine aortic endothelial cell line on calf tail collagen pre-coated inserts. This system allowed us to study bovine neutrophil migration across endothelium, extracellular matrix (ECM), and epithelium in the correct sequence and direction in vitro. The effect of recombinant interleukin-1beta (rHIL-1beta) and interleukin-8 (rHIL-8) on bovine neutrophil migration was investigated using this system. rHIL-8 stimulated bovine neutrophil migration in a dose-dependent fashion. The level of migrating bovine neutrophils increased up to approximately 25% when 100 ng/ml of rHIL-8 was used. On the other hand, rHIL-1beta at concentrations up to 100 ng/ml did not directly induce bovine neutrophil migration. Furthermore, pre-incubation with 5 ng/ml of rHIL-1beta in the co-culture system for 4 or 24 h failed to have any effect. These results suggest that IL-8 plays an important role in neutrophil migration into bovine mammary glands during mastitis.     

An acidic lymphokine distinct from interferon-gamma inhibits the replication of herpes simplex virus in human pulmonary macrophages

Supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells were fractionated by gel filtration and isoelectric focusing. A fraction with an isoelectric point of 2.2-3.3 containing macrophage migration inhibition factor activity inhibited the replication of herpes simplex virus type 1 in human pulmonary macrophages and U937 cells. This fraction did not inhibit the replication of herpes simplex virus in human fibroblasts. Moreover, the ability of this lymphokine fraction to inhibit viral growth in macrophages was not neutralized by antibody against interferon-gamma. These findings identify a macrophage specific antiviral lymphokine which is distinct biochemically and immunologically from interferon-gamma.     

Cytotoxic activity and interferon production by lymphocytes from patients with multiple sclerosis

Peripheral blood lymphocytes from MS patients and from healthy control donors were compared for their ability to mediate spontaneous and antibody-dependent cell-mediated cytotoxicity. They were also compared for their ability to respond to infection with various strains of measles and sSPE viruses with interferon production and enhanced NK activity. Neither SLMC nor ADCC against several different target cells was found to be impaired in the MS population. Furthermore, no defect was detected in the response of patients' lymphocytes to virus challenge in vitro in terms of both activation of NK cells and interferon production. Enhanced NK activity was also induced by an exogenous interferon preparation and by Poly I:C to the same extent in patients and controls.     

RECOMBINANT HUMAN INTERLEUKIN-8, BUT NOT HUMAN INTERLEUKIN-1β, INDUCES BOVINE NEUTROPHIL MIGRATION IN AN IN VITRO CO-CULTURE SYSTEM

A co-culture system was established by culturing a bovine mammary epithelial cell line (MAC-T) and a bovine aortic endothelial cell line on calf tail collagen pre-coated inserts. This system allowed us to study bovine neutrophil migration across endothelium, extracellular matrix (ECM), and epithelium in the correct sequence and direction in vitro. The effect of recombinant interleukin-1β (rHIL-1β) and interleukin-8 (rHIL-8) on bovine neutrophil migration was investigated using this system. rHIL-8 stimulated bovine neutrophil migration in a dose-dependent fashion. The level of migrating bovine neutrophils increased up to approximately 25% when 100 ng/ml of rHIL-8 was used. On the other hand, rHIL-1β at concentrations up to 100 ng/ml did not directly induce bovine neutrophil migration. Furthermore, pre-incubation with 5 ng/ml of rHIL-1β in the co-culture system for 4 or 24 h failed to have any effect. These results suggest that IL-8 plays an important role in neutrophil migration into bovine mammary glands during mastitis.     

Effects of C-reactive protein on human lymphocyte responsiveness

C-reactive protein (CRP), a trace serum protein that increases markedly in concentration during inflammatory reactions, was recently shown to bind to a subset of human IgG-FcR-bearing peripheral blood lymphocytes in the presence of a ligand such as pneumococcal C-polysaccharide (CPS). CRP has also been detected on a small percentage of PBL that are associated with NK activity. In the present study, we assessed the effects of CRP and CRP-CPS complexes on a variety of human lymphocyte functions in vitro. CRP and CRP complexes significantly enhanced (generally two to threefold) cell-mediated cytotoxicity, minimally enhanced the MLC reaction, and induced a small but regularly detectable blastogenic response in resting PBL. CRP or CRP-CPS complexes had no effect on mitogen-induced blastogenesis, PWM-induced generation of IgM plaque-forming cells, E-rosette formation, antibody-dependent cell-mediated cytotoxicity, or NK activity. The basis for the preferential ability of CRP to enhance cytotoxicity responses in vitro is under further investigation.     

Ethanol inhibition of cell-mediated lysis of antibody-sensitized target cells at a calcium-dependent step

Ethanol inhibits antibody-dependent cell-mediated cytotoxicity in a dose-dependent manner. The inhibitory effect of ethanol was reversed by the addition of excess calcium or calcium ionophore A23187. Excess calcium at 4-8 mM concentrations was required to reverse 50% of the inhibition caused by ethanol. In seven of nine experiments, 16 mM excess calcium completely reversed the inhibition and produced greater lysis than the control. Excess calcium in the absence of ethanol induced a dose-dependent increase in lytic activity by the spleen cells. However, the reversal of inhibition by ethanol could not be attributed to a simple additive effect resulting from the increased cytolytic capacity of the lymphocytes in the presence of excess Ca2+. Ionophore A23187 at 1 microM also partially reversed the inhibitory effect caused by ethanol. Ionophore alone did not potentiate lytic activity. When target cells were not sensitized with antibody, excess calcium had no effect on the lysis of target cells in the presence of ethanol-treated or untreated lymphocytes. These data suggest that ethanol inhibits antibody-dependent cell-mediated cytotoxicity at a calcium-dependent step.     

Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors

Recombinant human interferon-gamma (rHuIFN-gamma) and natural human tumor necrosis factor beta (nHuTNF-beta) (previously called lymphotoxin), purified to homogeneity, were used to assess their effects on certain functions of human polymorphonuclear neutrophils (PMN) in vitro. The treatment of PMN with 100 U of either rHuIFN-gamma or nHuTNF-beta for 20 min significantly increased their ability to phagocytize 1.5-microns latex beads as detected by flow cytometry. Preparations of recombinant human TNF-beta (rHuTNF-beta) showed activities similar to those of its natural counterpart in activating phagocytosis. In addition, a significant enhancement in PMN-mediated antibody-dependent cellular cytotoxicity was observed after treatment for 2 hr with IFN gamma and both TNF-alpha and TNF-beta. The enhancement by treatment with a combination of rHuIFN-gamma and nHuTNF-beta exceeded the enhancement caused by either agent alone. We also show that although lipopolysaccharide (LPS) is a potent stimulator of PMN function, polymyxin B can block LPS-induced but not lymphokine-induced activation. These data demonstrate new activities for both TNF-alpha and TNF-beta in augmenting the phagocytic and cytotoxic activities of PMN.     

Priming of normal human neutrophils by tachykinins: tuftsin-like inhibition of in vitro chemotaxis stimulated by formylpeptide or interleukin-8.

Tachykinins have priming effects on polymorphonuclear neutrophils, since they may activate the neutrophils to exhibit an exaggerated inflammatory response to phlogistic mediators. In order to investigate mechanisms involved in this action, we determined the influence of substance P and neurokinin A on chemotaxis of human neutrophils towards gradients of formymethionyl-leucyl-phenylalanine or recombinant human interleukin-8. As seen with other neutrophil-priming agents such as tumor necrosis factor-alpha, exposure of neutrophils to substance P or neurokinin A had an inhibitory effect upon a stimulated migration, with effective concentrations being in the nanomolar range. Tuftsin, a known neutrophil activating peptide, similarly inhibited stimulated migration. Analysis of structure-activity relationships revealed that activity of tachykinins is located in amino-terminal, tuftsin-like sequences. The inhibition of stimulated migration was partly reversed by (Pro1)-tuftsin, a partial tuftsin receptor antagonist, which suggests that the effects of amino-terminal tachykinins involves activation of tuftsin receptors of neutrophils.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Comparative activation requirements of human peripheral blood, spleen, and lymph node B cells

Human peripheral blood, spleen, and lymph node B cells were stimulated with Cowan I Staphylococcus aureus (SA) or F(ab')2 fragments of anti-mu antibody (anti-mu) and various lymphokines and were analyzed for proliferation and generation of Ig-secreting cells (ISC). SA alone but not anti-mu stimulated minimal proliferation of each population. Recombinant IL 2 (r-IL 2) effectively promoted proliferation of SA-stimulated blood and spleen B cells, but supported less vigorous responses of lymph node B cells. By contrast, r-IL 2 enhanced DNA synthesis of all anti-mu-stimulated B cells early in culture, but did not promote sustained proliferation of anti-mu-stimulated lymph node B cells and only promoted ongoing DNA synthesis of some anti-mu-activated blood (eight out of 17) and spleen (five out of 14) B cell preparations. Recombinant interferon-gamma (r-IFN-gamma) and a commercial preparation of B cell growth factor (BCGF) also augmented DNA synthesis of all three B cell populations stimulated with SA or anti-mu early in culture, but neither alone was able to sustain maximal proliferation. Markedly enhanced sustained proliferation of all three anti-mu- and SA-stimulated B cell populations was noted when cultures were supported by the combination of r-IL 2 and BCGF, or to a lesser extent by r-IL 2 and r-IFN-gamma. The generation of ISC from SA-stimulated blood or spleen but not lymph node B cells was effectively supported by r-IL 2 alone. Differentiation of lymph node B cells required the combination of r-IL 2 and BCGF. These studies emphasize the importance of both the activation stimulus and the origin of the B cells in determining the lymphokine requirements of human B cell responsiveness.