HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Heterogeneity of helper/inducer T lymphocytes. I. Lymphokine production and lymphokine responsiveness.

Antigen-specific, Ia-restricted helper/inducer T lymphocytes consist of subsets that can be distinguished by lymphokine secretion. One, called Th1, secretes IL-2 and the other, termed Th2, produces BSF-1/IL-4 in response to stimulation by lectin or antigen receptor signals, and each uses the respective lymphokine as its autocrine growth factor. Cloned lines representing Th2 cells proliferate in response to both IL-2 and their autocrine lymphokine, BSF-1/IL-4, but this proliferation is dependent on the synergistic costimulator activity of the monokine, IL-1. In contrast, Th1 clones proliferate only in response to IL-2, are unresponsive to BSF-1/IL-4, and their growth is unaffected by IL-1. These response patterns are not attributable to variations in culture conditions but apparently reflect intrinsic properties of the two T cell subsets. Moreover, the unresponsiveness of Th1 cells to BSF-1/IL-4 may be related to lower levels of expression of surface receptors for this lymphokine. These results may explain the observed heterogeneity among bulk populations of T cells in terms of lymphokine responsiveness and requirement for accessory factors (costimulators). In addition, our findings suggest that IL-2, unlike BSF-1/IL-4, is a fully competent growth factor that is potentially involved in antigen-independent expansion of bystander T cells present at sites of immune stimulation.

Impact of toll-like receptor 4 on the severity of acute pancreatitis and pancreatitis-associated lung injury in mice.

BACKGROUND AND AIMS: Acute pancreatitis is an inflammatory disease involving acinar cell injury, and the rapid production and release of inflammatory cytokines, which play a dominant role in local pancreatic inflammation and systemic complications. Toll-like receptor 4 (TLR4) initiates a complex signalling pathway when it interacts with lipopolysaccharide (LPS), which ultimately results in a proinflammatory response. We hypothesised that TLR4 is important in the pathophysiology of acute pancreatitis, independently of LPS. Using two different models of acute pancreatitis, we investigated how genetic deletion of TLR4 or its co-receptor CD14 effects its progression and severity. METHODS: We induced acute pancreatitis by administering either caerulein or L-arginine to wild-type, TLR4(-/-), and CD14(-/-) mice. Control mice received normal saline injections. The severity of acute pancreatitis was determined by measuring serum amylase activity, quantifying myeloperoxidase (MPO) activity in the pancreatic tissue, and histologically assessing acinar cell injury. RESULTS: It was found that administering caerulein and L-arginine to wild-type mice resulted in acute pancreatitis (as assessed by hyperamylasaemia, oedema, increased pancreatic MPO activity, and pancreatic necrosis) and associated lung injury. The same treatment to TLR4(-/-) or CD14(-/-) mice resulted in significantly less severe acute pancreatitis, and reduced lung injury. We found no evidence of either bacteria or LPS in the blood or in pancreatic tissue. CONCLUSIONS: The severity of acute pancreatitis is ameliorated in mice that lack either TLR4 or CD14 receptors. Furthermore, these results indicate that TLR4 plays a significant pro-inflammatory role independently of LPS in the progression of acute pancreatitis.

Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.

A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection. Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery with adenovirus vectors.

Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide.

Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with Gi/Go heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPI-anchored receptor and the intracellular signaling molecules with which it is physically associated.

Novel strategies for targeting innate immune responses to influenza.

We previously reported that TLR4(-/-) mice are refractory to mouse-adapted A/PR/8/34 (PR8) influenza-induced lethality and that therapeutic administration of the TLR4 antagonist Eritoran blocked PR8-induced lethality and acute lung injury (ALI) when given starting 2 days post infection. Herein we extend these findings: anti-TLR4- or -TLR2-specific IgG therapy also conferred significant protection of wild-type (WT) mice from lethal PR8 infection. If treatment is initiated 3 h before PR8 infection and continued daily for 4 days, Eritoran failed to protect WT and TLR4(-/-) mice, implying that Eritoran must block a virus-induced, non-TLR4 signal that is required for protection. Mechanistically, we determined that (i) Eritoran blocks high-mobility group B1 (HMGB1)-mediated, TLR4-dependent signaling in vitro and circulating HMGB1 in vivo, and an HMGB1 inhibitor protects against PR8; (ii) Eritoran inhibits pulmonary lung edema associated with ALI; (iii) interleukin (IL)-1ß contributes significantly to PR8-induced lethality, as evidenced by partial protection by IL-1 receptor antagonist (IL-1Ra) therapy. Synergistic protection against PR8-induced lethality was achieved when Eritoran and the antiviral drug oseltamivir were administered starting 4 days post infection. Eritoran treatment does not prevent development of an adaptive immune response to subsequent PR8 challenge. Overall, our data support the potential of a host-targeted therapeutic approach to influenza infection.

Membrane interleukin 1 induction on human endothelial cells and dermal fibroblasts.

Human endothelial cells and dermal fibroblasts both expressed a membrane-associated interleukin 1 (IL-1) activity when stimulated with either recombinant tumor necrosis factor (TNF) or recombinant lymphotoxin but stimulated endothelial cells expressed significantly more membrane IL-1 per cell than did fibroblasts. Lipopolysaccharide induced membrane IL-1 activity on endothelial cells but not fibroblasts. Interferon-gamma treatment of endothelial cells and fibroblasts had no direct effect on membrane IL-1 expression and little effect when used as a pretreatment for TNF or lipopolysaccharide stimulation. Endothelial cell membrane IL-1 activity was induced within 24 hr of culture with TNF or lipopolysaccharide, and increased up to 72 hr of incubation. Antibodies raised against human monocyte-derived IL-1 species neutralized the membrane IL-1 activity of TNF-stimulated endothelial cells. Both absorption studies and neutralization with specific sera indicated that endothelial cell membrane IL-1 is structurally related to IL-1 alpha. Endothelial cells expressed both IL-1 beta mRNA in response to TNF, lymphotoxin, and recombinant IL-1 species, as detected by Northern blot analysis. These studies demonstrate that endothelial cells can be activated to express a cell-surface IL-1 activity which is structurally, as well as functionally, related to the secreted form of IL-1.

In vivo and in vitro expression of macrophage membrane interleukin 1 in response to soluble and particulate stimuli.

We examined the expression of a membrane form of interleukin 1 (IL 1) by macrophages. Murine peritoneal macrophages fixed immediately after harvesting, in suspension, did not show membrane IL 1. Membrane IL 1 was expressed after a 3-hr culture on plastic dishes. These findings allowed us to examine some conditions in vivo that may trigger the expression of this protein; that is, by fixing the macrophages in suspension we could determine if a given stimulus had an effect. We found that membrane IL 1 was expressed briefly after administration of live or dead Listeria monocytogenes or endotoxin. Serum proteins were ineffective. At the time of maximal activation of macrophages by live Listeria, membrane IL 1 was not expressed. Analysis of in vitro conditions indicated that expression of membrane IL 1 and Ia molecules could be dissociated. In culture, recombinant interferon-gamma induced Ia but no membrane IL 1. Uptake of dead Listeria organisms had no effect on Ia but triggered membrane IL 1. The stimulation of membrane IL 1 was not caused by phagocytosis per se: latex particles were ineffective. Opsonized red cells stimulated membrane IL 1 on macrophages that were activated in vivo by inflammatory stimuli.

B-cell stimulatory factor 1 and not interleukin 2 is the autocrine growth factor for some helper T lymphocytes.

Clonal expansion of T lymphocytes of the helper/inducer class is generally thought to be mediated by an interleukin 2 (IL-2)-dependent autocrine mechanism. Thus, T cells stimulated by antigens or mitogenic lectins secrete IL-2 and, under appropriate conditions, express membrane receptors for IL-2, and the specific hormone-receptor interaction induces cellular proliferation. Recent studies indicate that B-cell stimulatory factor 1 (BSF-1) is secreted by T cells and is capable of stimulating T-cell proliferation. We now report that BSF-1 and not IL-2 is the sole autocrine growth factor for certain cloned lines of inducer T lymphocytes. On stimulation by the lectin concanavalin A, anti-receptor antibody, or specific antigen with antigen-presenting cells, such clones secrete a lymphokine that stimulates DNA synthesis by the "IL-2 indicator line," HT2, but is identified as BSF-1 by specific inhibition with monoclonal antibodies. The proliferative response of such BSF-1-secreting clones to receptor-mediated signals is dependent on BSF-1 and not IL-2. These results demonstrate a function of BSF-1 and confirm the existence of a previously unknown autocrine pathway of T-cell activation.

Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells.

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

Interleukin-1 corrects the defective autologous mixed lymphocyte response in multiple sclerosis.

Patients with chronic progressive multiple sclerosis (MS) have alterations of T cell regulation that can be measured by in vitro assays and include decreases of the autologous mixed lymphocyte reaction (AMLR). Whether a defect in cytokine secretion was involved in the altered AMLR was investigated in 29 MS patients and 13 age- and sex-matched controls. The response of CD4+ T cell populations to irradiated non-T cells was decreased in MS as compared to control subjects. As previously reported, decreases in the AMLR were similarly observed with whole T cells of MS subjects as compared to controls. The addition of recombinant interleukin (IL)-1 to cultures of either whole T cells or CD4+ T cells with irradiated non-T cells in the AMLR corrected the immune defect in subjects with MS but had no effect on the AMLR in control subjects. In contrast, addition of rIL-2 or rIL-4 to the AMLR did not correct the decreased AMLR in MS patients as compared to controls. The lymphokines IFN-gamma and TGF-beta 2 both decreased the AMLR in MS patients and controls while TNF had no effect. Further, the magnitude of the AMLR response corresponded to IL-1 secretion induced by LPS in the non-T cell population. These studies indicate that defects in IL-1 may be related to immune defects of suppression in MS patients. Selective correction of immunoregulatory defects using lymphokines or their inducers in subjects with autoimmune diseases such as MS may be possible.

Identification of a membrane-associated interleukin 1 in macrophages.

We have found a surface membrane-associated interleukin 1 (IL-1) with potent thymocyte and T-cell stimulatory activity on peptone-elicited peritoneal macrophages. The IL-1 activity was demonstrated on both fixed macrophage monolayers and on isolated membranes from unfixed macrophages. Membrane IL-1 was induced by adherence and/or by adding heat-killed Listeria monocytogenes to macrophage cultures. The macrophage membrane IL-1 was similar functionally and antigenically to soluble IL-1, but its expression could be temporally dissociated from IL-1 secretion; membrane IL-1 was induced earlier and persisted longer than IL-1 secretion during in vitro macrophage culture. Moreover, when cultured macrophages that had ceased both secretion and membrane expression of IL-1 were restimulated by adding heat-killed Listeria, substantial membrane IL-1 was induced in the absence of detectable IL-1 secretion. Membrane IL-1 appears to be an integral membrane protein since it was solubilized by detergent but was not eluted by EDTA, high salt, or low pH treatment of the membranes.

Immune complex effects on murine macrophages. II. Immune complex effects on activated macrophages cytotoxicity, membrane IL 1, and antigen presentation.

We investigated the effects of immune complexes on macrophage functions in vitro. Immune complexes inhibit lymphokine induction of both I-Ak expression and cytotoxic activity by fetal calf serum elicited macrophages during long-term (7 days) culture. In addition, induction of antigen presentation was significantly inhibited by immune complexes. Expression of membrane interleukin 1 (IL-1) (a membrane-bound bound form of the T cell mitogen required for antigen presentation by fixed cells) was minimally inhibited by immune complexes. Therefore, inhibition of antigen presentation was primarily due to effects on Ia expression rather than membrane IL 1 expression. The inhibitory effect of immune complexes was not found during short-term culture (4 to 48 hr) when activated macrophages (bearing high levels of Ia) from mice infected with Listeria monocytogenes were examined. Immune complexes maintained or even increased levels of both I-Ak and cytotoxicity in activated macrophages. The implications of these findings for immune complex modulation of the immune response are discussed.

Endothelial cell activation and high interleukin-1 secretion in the pathogenesis of acute Kawasaki disease.

To investigate the pathogenesis of Kawasaki disease, the effects of intravenous gammaglobulin treatment on circulating cytotoxic antibodies against endothelial cells, in-situ endothelial cell activation, and cytokine production and action were examined. Gammaglobulin treatment did not reduce cytotoxic antibody activity against endothelial cells in six patients tested. Expression of endothelial cell activation antigens (endothelial-leucocyte adhesion molecule-1 [ELAM-1] and intercellular adhesion molecule-1) was detected by means of immunoperoxidase staining in skin biopsy samples from five patients before treatment. Samples were obtained immediately after treatment from six patients; there was no endothelial cell activation in four and the two with persistent activation had persistent fever and mucocutaneous symptoms. Peripheral blood mononuclear cells from ten of sixteen acute Kawasaki disease patients spontaneously secreted high levels of interleukin-1 (IL-1). IL-1 secretion remained high in four gammaglobulin-treated patients in whom coronary artery abnormalities developed but fell to normal in six treated patients who had no coronary artery abnormalities. In cell culture, gamma globulin did not inhibit endothelial cell expression of ELAM-1 in response to IL-1 or tumour necrosis factor. The association between improvement of clinical symptoms and the reduction of cytokine secretion and reversal of endothelial cell activation supports a role for immune-mediated injury to cytokine-induced endothelial cell antigens in the pathogenesis of this disorder.

The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice.

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.

Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.

The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.