Complexes of platelet factor 4 and heparin activate Toll-like receptor 4.

BACKGROUND: In some patients, the anticoagulant heparin elicits formation of antibodies that can cause the life and/or limb-threatening syndrome known as heparin-induced thrombocytopenia (HIT). HIT antibodies target complexes formed at specific molar ratios of heparin and platelet factor 4 (PF4). The unpredictable occurrence and the mechanism of this atypical immune response to PF4:heparin complexes are poorly understood. OBJECTIVE: We investigated whether complexes formed at specific PF4:heparin ratios (PHRs) might resemble molecular patterns associated with host defense responses. METHODS: We used an in vitro cytokine release assay to determine whether defined PHRs caused cytokine release from human whole blood. Lipopolysaccharide (LPS) was used as a positive assay control, and some experiments included antibodies to block Toll-like receptor 4 (TLR4). RESULTS: PF4:heparin complexes caused release of the biomarker interleukin 8 in whole blood, and the level of response varied with the stoichiometric ratio of PF4 to heparin. The profile of response to LPS and to PF4:heparin complexes varied among blood donors, and the interleukin 8 response to both LPS and PF4:heparin was inhibited by TLR4-blocking antibodies. CONCLUSIONS: Specific PF4-heparin complexes can elicit a TLR4-mediated response, suggesting that these complexes can mimic a pathogen-associated molecular pattern, and supporting the suggestion that the HIT immune response represents a misdirected host defense mechanism.

Effect of anti-platelet factor-4/heparin antibody induction on early saphenous vein graft occlusion after coronary artery bypass surgery.

BACKGROUND: Antibodies to complexes of heparin and platelet factor 4 (PF4) are capable of causing heparin-induced thrombocytopenia (HIT). Recent evidence suggests that anti-PF4/heparin antibodies may be prothrombogenic even in the absence of thrombocytopenia and clinically-recognized HIT. OBJECTIVES: To determine if induction of anti-PF4/heparin antibodies is an independent risk factor for early saphenous vein graft (SVG) occlusion or adverse clinical outcome after coronary artery bypass graft (CABG) surgery. PATIENTS/METHODS: Anti-PF4/heparin antibody titers were measured in 368 patients prior to and then 4 days, 6 weeks and 6 months after CABG surgery. Serotonin release assay (SRA) and antibody isotype analysis were also performed on 6-week samples. SVG patency was determined in 297 patients 6 months after surgery by multidetector computed tomography coronary angiography. RESULTS: Six weeks after surgery, 52% of patients were anti-PF4/heparin seropositive and 9% were SRA positive. Six months after surgery, neither the percentage of occluded SVG (19% vs. 20%, P = NS), the percentage of patients with an occluded SVG (33% vs. 33%, P = NS) nor the incidence of adverse clinical events (21% vs. 24%, P = NS) differed between seropositive and seronegative groups. Neither IgG isotype nor SRA positivity was additionally predictive of SVG occlusion or adverse clinical outcome. CONCLUSION: Induction of anti-PF4/heparin antibodies, even those capable of heparin-dependent platelet activation, is not independently associated with early SVG occlusion or adverse clinical outcomes after CABG surgery.

Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin.

The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.

In vivo indomethacin treatment causes microglial activation in adult mice.

The current study was undertaken to study the role of prostaglandins in regulating microglial activation. Mice were treated with indomethacin (2 microg/ml) in their drinking water to selectively inhibit cyclooxygenase activity. After 4-8 days, the effect of inhibiting prostaglandin synthesis on microglial activity was evaluated. This was accomplished by analyzing microglial expression of Mac-1 (C3 complement receptor) as an indicator of activation. Mac-1 expression was assessed by immunohistochemistry of fixed brain cryosections, and by flow cytometric analysis of immunostained single cell suspensions. Both methods demonstrated that compared to age-matched, untreated controls, brains of indomethacin-treated mice had increased levels of Mac-1 expression, suggesting an increase in the state of microglial activation. These results demonstrate the importance of prostaglandins in down regulating microglial activity, and that inhibition of prostaglandin synthesis with indomethacin may act to increase the reactivity of the brain's immune system.

Pinealocyte ultrastructure in mutant mice that lack sympathetic innervation to the pineal gland.

Pinealocyte synaptic ribbons (SR) and dense-cored vesicles (DCV) were quantitated in mice that have developmental deficiencies in the sympathetic innervation of the pineal gland due to a null mutation for the p75 low-affinity NGF receptor (NGFR). SR exhibited a significant increase whereas there was a significant decline in the frequency of DCV in mutant mice. These findings support the hypothesis that pinealocyte SR and DCV are regulated by neural mechanisms associated with sympathetic system. Ultrastructural characteristics of pinealocytes in mutants included degenerative changes that culminated in the breakdown of cellular components and the accumulation of flocculent-containing vesicles within the cytoplasm. Ribosomal lamellar complexes were also commonly seen in pinealocytes of mutant rats. Although the mutant pineal gland exhibited signs of metabolic imbalances, the cytoarchitecture of the gland (e.g., vascular compartment) and differentiation of the cells were generally unaffected by developmental deficiencies in the gland's innervation.

Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34+ natural suppressor cells.

Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown to contain CD34+ natural suppressor cells that inhibit the activity of intratumoral T-cells. The present study evaluated whether GM-CSF production and the presence of CD34+ cells within primary HNSCC would translate into increased recurrence, metastasis or cancer-related death during the 2 years following surgical excision. Freshly excised primary HNSCC of 20 patients that subsequently developed disease, and of 17 patients that remained with no evidence of disease were analyzed for production of GM-CSF and for CD34+ cell content. The cancers of patients that subsequently developed recurrences or metastatic disease produced almost 4-fold the levels of GM-CSF and had approximately 2.5-fold the number of CD34+ cells as did cancers of patients that remained disease-free. In a second method of analysis, the prognostic significance of high vs. low GM-CSF and CD34+ cell values was evaluated. These analyses showed that patients whose cancers produced high GM-CSF levels or had a high CD34+ cell content had a disproportionately high incidence of recurrence or metastatic disease (94% and 100%, respectively), while the majority of patients whose primary cancers produced low levels of GM-CSF or had a low CD34+ cell content remained disease-free (16% and 19%, respectively). Our results indicate that the presence of CD34+ cells in GM-CSF-producing HNSCC is associated with a poorer prognosis for the cancer patients and suggest the utility of these parameters as prognostic indicators of outcome. Mechanistically, our results suggest that the presence of immune suppressive CD34+ cells in GM-CSF-producing HNSCC leads to increased tumor recurrence or metastasis.

Increased interleukin-6 production by cerebral cortical tissue of adult versus young mice.

The cytokine interleukin-6 (IL-6) has been implicated as a contributor to injury in several neurological disorders. The amounts of IL-6 released by the cerebral cortical tissue of mice of varying maturational age groups were measured and found to increase with age. Specifically, the basal level of IL-6 released from the tissue of infant (less than 2 weeks old) mice was low, although the tissue could be readily stimulated to secrete high levels of IL-6. Higher levels of IL-6 were released from young adult (2 month old) mice. Adult mice that were either 6 or 9 months of age secreted significantly higher levels of IL-6 compared to the tissue of either the young adult or infant animals, although the difference between levels secreted by the 6- or 9-month old groups was minimal. IL-6 production by adult cerebral cortical tissue could be further stimulated, but it was less readily achieved compared to the tissue of infants. In response to the negative regulator transforming growth factor-beta (TGF-beta), the levels of IL-6 released by stimulated cerebral cortical tissue of infants, as well as by unstimulated and stimulated tissue of adults, were reduced to the low basal levels of IL-6 produced by infant tissue. These results suggest that normal development and aging are correlated with an increase in IL-6 production that may be due to shifts in levels of stimulatory or inhibitory regulatory controls, but not to an inability of young tissue to produce IL-6 or to a lack of responsiveness of adult tissue to negative regulatory control by TGF-beta.

Vitamin D3 treatment of tumor bearers can stimulate immune competence and reduce tumor growth when treatment coincides with a heightened presence of natural suppressor cells.

By secreting granulocyte-macrophage colony-stimulating factor (GM-CSF), Lewis lung carcinoma tumors induce immune suppressive granulocyte-macrophage progenitor cells. Treating mice having established tumors and high levels of suppressor activity with vitamin D3 eliminated suppressor activity, increased anti-tumor immunity, induced an immune stimulatory cell population, and reduced tumor growth. When instead, the vitamin D3 treatment was initiated earlier, when implanted tumors first became detectable and when natural suppressor activity was less prominent, the treatment had no effect. Thus, vitamin D3 treatment can stimulate the immune competence of tumor bearers when treatment is targeted to coincide with a heightened presence of GM-CSF-induced suppressor cells.

Mechanisms of immune suppression in patients with head and neck cancer: influence on the immune infiltrate of the cancer.

Freshly excised human head and neck cancers (219 primary cancers; 64 metastatic lymph node cancers) were analyzed for the immune inhibitory mediators released from the cancer tissues and the immune infiltrate within the tumor. Significant levels of the immune inhibitory mediators transforming growth factor-beta (TGF-beta), prostaglandin E2 (PGE2) and interleukin-10 (IL-10) were released from these cancers. Also released was granulocyte-macrophage colony-stimulating factor (GM-CSF), whose secretion was associated with an intratumoral presence of CD34+ cells. We have previously shown that CD34+ cells within human head and neck cancers are immune inhibitory granulocyte-macrophage progenitor cells. The presence of TGF-beta, PGE2 and IL-10 was associated with a reduced content of CD8+ T-cells within the cancers. The CD4+ cell content appeared to be less affected by these immune inhibitory mediators. Instead, parameters indicative of CD4+ cell function (p55 IL-2 receptor expression, release of IL-2 and IFN-gamma) were diminished in cancers that released higher levels of TGF-beta, IL-10 and GM-CSF and had a higher CD34+ cell content. Furthermore, metastatic cancers released higher levels of the soluble immune inhibitory mediators and lower levels of IFN-gamma and IL-2 than did primary cancers, although CD34+ cells were similarly present in both primary and metastatic cancers. Our results show that human head and neck cancers have a multiplicity of non-mutually exclusive mechanisms of immune suppression that are most prominently associated with reduced CD8+ cell influx and reduced influx and altered function of intratumoral CD4+ cells.

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells.

Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D3 eliminated natural suppressor activity. In mice that were first treated with vitamin D3 and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D3/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon gamma (IFN gamma), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D3/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D3 to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFN gamma production and cytolysis by regional lymph node cells of autologous tumor.

Effect of a new aminopeptidase P inhibitor, apstatin, on bradykinin degradation in the rat lung.

Bradykinin (Bk), a potent vasoactive and cardioprotective peptide hormone, is almost completely inactivated during a single circulation through the rat lung. It has been hypothesized that membrane-bound aminopeptidase P, which can hydrolyze the Arg1-Pro2 bond of Bk, and angiotensin-converting enzyme (ACE) act in concert to degrade Bk in the pulmonary circulation. To test this hypothesis, an inhibitor of aminopeptidase P was designed and synthesized. N-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-prolyl-L-prolyl-L - alaninamide (apstatin) inhibited purified rat lung membrane-bound aminopeptidase P with a Ki value of 2.6 microM (linear mixed-type inhibition, alpha = 5.1, beta = 0). Apstatin did not inhibit ACE or other known Bk-degrading enzymes. Apstatin and an ACE inhibitor, ramiprilat, were tested for their ability to inhibit Bk degradation in the isolated perfused rat lung. [2,3-Proly-3,4-3H(N)]-bradykinin ([3H]-Bk) was perfused through the isolated lung in the presence or absence of inhibitors. The perfusate was then subjected to HPLC to identify and quantitate radiolabeled fragments. In the absence of inhibitors, no intact [3H]-Bk was found in the perfusate. In the presence of ramiprilat (0.5 microM), only 22% +/- 6% of the radioactivity in the perfusate was intact [3H]-Bk, and the remaining radioactivity indicated cleavage of the Arg1-Pro2 bond. When apstatin (40 microM) was perfused along with ramiprilat, degradation of [3H]-Bk was almost completely blocked (92% +/- 4% intact [3H]-Bk in the perfusate). The results indicate that the Bk-degrading activity in the rat pulmonary vascular bed can be fully accounted for by aminopeptidase P (30%) and ACE (70%).

Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression, and reduces tumor metastasis and recurrence.

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colony-stimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1 alpha,25-dihydroxyvitamin D3 reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D3 reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D3 treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D3 after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D3 treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.

Ineffective immune enhancement by IL-12 in tumor-bearing mice whose immune depression is mediated by suppressive granulocyte-macrophage progenitor cells.

Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and induce immunosuppressive granulocyte-macrophage (GM)-progenitor cells. Treating mice having palpable tumors with IL-12 enhanced the frequency of GM-progenitors and did not diminish GM-suppressor activity. Proliferation of splenic T-cells of tumor-bearers to Con-A or to anti-CD3 plus IL-2 was suppressed; this was not enhanced by IL-12 treatment. Also not stimulated was T-cell secretion of IL-2 in response to autologous tumor, or the intratumoral T-cell content. IL-12 slightly increased splenic IFN-gamma secretion, and increased cytotoxicity of lymph node (but not spleen) cells toward autologous tumor. In these tumor-bearing mice that were immune depressed as a result of GM-suppressor cells, immune modulatory effects of IL-12 were marginal and did not affect tumor size or metastasis.

Circannual analysis of pineal gland indoles and vasotocin-like immunoreactivity in male rats kept on constant photoschedules and temperature.

Levels of vasotocin-like immunoreactivity (iVT) and the indoles serotonin (5HT), 5-hydroxyindole acetic acid, N-acetyl-serotonin (NAS) and melatonin were measured over 24-hour periods at monthly intervals in the pineal gland of male rats kept under constant laboratory conditions of photoperiod and temperature. The 8- to 10-fold increase in pineal iVT, which occurred during mid-August, was not related to variations over the 24-hour light:dark (L:D) cycle. Tryptophan and 5HT exhibited seasonal variations over the 24 h L:D cycle with a single peak during the winter months. Twenty-four-hour levels of NAS tended to increase during the fall and spring. Seasonal effects on indole metabolism were further suggested by a marked decline during the fall in the correlation between NAS versus melatonin in individual pineal glands. Positive correlations between 5HT versus NAS and 5HT versus melatonin tended to be significant only during the summer months. These data suggest that environmental cues other than photo-period and temperature can regulate pineal neuroendocrine functions.

Magnetic resonance imaging (MRI) effects on rat pineal neuroendocrine function.

This study investigated the effects of MRI on receptor-mediated activation of pineal gland indole biosynthesis. Exposure of rats to MRI reduced the effects of isoproterenol on pineal serotonin and N-acetylserotonin levels suggesting that strong magnetic fields and/or radio-frequency pulsing used in MRI inhibited beta-adrenergic activation of the gland. There was no effect of MRI on saline controls.

A seasonal pineal peptide rhythm persists in superior cervical ganglionectomized rats.

Neurohypophyseal peptide hormone activity is present in the pineal gland of mammals, and varies over a seasonal cycle. Pineal peptide levels, measured by arginine vasotocin (AVT) radioimmunoassay, increase dramatically for a brief time during August each year. The manner in which this cycle is regulated is as yet unknown. Input to the pineal from sympathetic axons arising in the superior cervical ganglia (SCG) is essential for the generation and regulation of the circadian rhythm in melatonin synthesis, and is the only pathway known to regulate pineal biochemical processes. It was of interest then to determine the impact of the SCG on the seasonal peptide cycle. Levels of pineal arginine vasotocin immunoactivity (iAVT) were monitored during August, 1984, in rats which had been superior cervical ganglionectomized (SCGX), in sham-operated and intact controls (L:D 12:12), and in rats subjected to L:D 22:2. The results indicate that SCGX does not abolish the seasonal cycle, but may influence the timing of the iAVT peak. Inhibition of pineal melatonin synthesis by exposure of rats to L:D 22:2 did not mimic the phase delay seen with SCGX, but did cause a significant increase in the amplitude of the August iAVT activity peak.

Effects of isoproterenol on synaptic ribbons in pinealocytes of the rat and C57BL/6J mouse.

Synaptic ribbons (SR) in melatonin-deficient pinealocytes of the C57BL/6J mouse were quantitatively compared to SR in pinealocytes of the rat after beta-adrenergic receptor activation by isoproterenol. Two populations of SR comprising synaptic spherules (SRsp) and synaptic rods (SRr) were described in both the mouse and the rat, but species differences existed in the ratio of SRr to SRsp. Isoproterenol caused a significant increase in frequency of SR of the rat but had little or no effect on SR populations in the mouse. It is unlikely that beta-adrenergic receptors are absent on mouse pinealocytes or were not activated since isoproterenol elevated plasma renin concentrations indicating activation of beta-adrenergic receptors. Furthermore the pineal of both species receives heavy sympathetic input. These findings indicate that the role and regulation of pinealocyte SR are complex and are functionally linked to beta-adrenergic receptors as well as other mechanisms related to the production of melatonin.

Influence of age on August levels of pineal immunoreactive arginine vasotocin in rats.

The present study was undertaken to evaluate carefully the influence of age on physiological levels of arginine vasotocin-like peptide in rat pineal glands. Glands were collected from male and female rats aged 13, 33, 53, and 73 days on August 4, 12, and 19, 1984. Individual extracts were assayed for arginine vasotocin (AVT) by radioimmunoassay. The results confirm our previous observation that rat pineal AVT immunoactivity (iAVT) increases significantly during August each year; and in this study, each group of rats reached the same peak level of iAVT (700-750 pg/gland) regardless of age or gender. Thus we do not confirm a previously reported decrease in AVT activity with age. In our studies thus far, season of the year is the physiological variable with the most significant influence on pineal AVT activity levels.

Correlations of serotonin and its metabolites in individual rat pineal glands over light:dark cycles and after acute light exposure.

Profiles of pineal indolealkylamines were estimated by high performance liquid chromatography and were correlated in individual glands of male rats sacrificed over several light:dark cycles and after acute exposure to light at night. A significant and positive correlation of 5HIAA vs 5HT in individual glands over both normal and experimental lighting conditions suggested that oxidative deamination is not a major factor in photic regulation of pineal 5HT levels and that the formation of 5HIAA is dependent on substrate availability. Regression analysis of other indole constituents revealed that there was a positive and significant correlation between 5HT vs N-acetylserotonin, but not between 5HT vs melatonin and N-acetylserotonin vs melatonin in individual glands during the dark phase of a light:dark cycle. We propose that this effect may be related to a pulsatile release of melatonin into the blood stream and is the result of sampling glands at different stages in the storage/release of melatonin.

Pineal ultrastructure and indole profiles spanning the summer rise in arginine vasotocin immunoactivity.

A correlative radioimmunological-biochemical-ultrastructural study of the rat pineal gland was undertaken during the summer months when pineal arginine vasotocin (AVT) immunoactivity increases up to 200-fold. RIA confirmed a rapid rise in AVT activity during mid-August regardless of the time of day sampled. Pineal indoles were separated by HPLC and measured using electrochemical detection. Serotonin (5-HT) and 5-hydroxyindoleacetic acid levels were consistently elevated in daytime samples, and there was a significant trend for increased day and nighttime levels of 5-HT from July to September. Mid-dark levels of melatonin also exhibited a significant increase over the sample period. Nighttime levels of N-acetylserotonin mirrored fluctuations in 5-HT in the preceding photoperiod. Ultrastructural components implicated in peptide/protein and/or indole biosynthesis were quantified by stereological morphometry. The greatest amounts of rough endoplasmic reticulum stacks, lipid droplets, and annulate lamellae-like bodies coincided with peak AVT activity. Dense-cored vesicles and synaptic ribbons were consistently more frequent during the dark period. The number of dense-cored vesicles and nucleolar size tended to be greatest before and after the peak in AVT immunoactivity. These observations are consistent with the hypotheses that endoplasmic reticulum and lipid are functionally related to the synthesis and/or storage of peptide/protein factors and that numerical changes in synaptic ribbons and dense-cored vesicles are more closely related to day/night differences in indole metabolism.