Human Thoracic Duct Research Articles

Positive chronotropic action of HCN channel antagonism in human collecting lymphatic vessels.

Spontaneous action potentials precede phasic contractile activity in human collecting lymphatic vessels. In this study, we investigated the expression of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels in human collecting lymphatics and by pharmacological inhibition ex vivo tested their potential role in controlling contractile function. Spontaneous and agonist‐evoked tension changes of isolated thoracic duct and mesenteric lymphatic vessels—obtained from surgical patients with informed consent—were investigated by isometric myography, and ivabradine, ZD7288 or cesium were used to inhibit HCN. Analysis of HCN isoforms by RT‐PCR and immunofluorescence revealed HCN2 to be the predominantly expressed mRNA isoform in human thoracic duct and mesenteric lymphatic vessels and HCN2‐immunoreactivity confirmed protein expression in both vessel types. However, in functional experiments ex vivo the HCN inhibitors ivabradine, ZD7288, and cesium failed to lower contraction frequency: conversely, all three antagonists induced a positive chronotropic effect with concurrent negative inotropic action, though these effects first occurred at concentrations regarded as supramaximal for HCN inhibition. Based on these results, we conclude that human collecting vessels express HCN channel proteins but under the ex vivo experimental conditions described here HCN channels have little involvement in regulating contraction frequency in human collecting lymphatic vessels. Furthermore, HCN antagonists can produce concentration‐dependent positive chronotropic and negative inotropic effects, which are apparently unrelated to HCN antagonism.

Spontaneous contractions of the human thoracic duct—Important for securing lymphatic return during positive pressure ventilation?

The thoracic duct is responsible for the circulatory return of most lymphatic fluid. The return is a well‐timed synergy between the pressure in the thoracic duct, venous pressure at the thoracic duct outlet, and intrathoracic pressures during respiration. However, little is known about the forces determining thoracic duct pressure and how these respond to mechanical ventilation. We aimed to assess human thoracic duct pressure and identify elements affecting it during positive pressure ventilation and a brief ventilatory pause. The study examined pressures of 35 patients with severe congenital heart defects undergoing lymphatic interventions. Thoracic duct pressure and central venous pressure were measured in 25 patients during mechanical ventilation and in ten patients during both ventilation and a short pause in ventilation. TD contractions, mechanical ventilation, and arterial pulsations influenced the thoracic duct pressure. The mean pressure of the thoracic duct was 16 ± 5 mmHg. The frequency of the contractions was 5 ± 1 min−1 resulting in an average increase in pressure of 4 ± 4 mmHg. During mechanical ventilation, the thoracic duct pressure correlated closely to the central venous pressure. TD contractions were able to increase thoracic duct pressure by 25%. With thoracic duct pressure correlating closely to the central venous pressure, this intrinsic force may be an important factor in securing a successful return of lymphatic fluid. Future studies are needed to examine the return of lymphatic fluid and the function of the thoracic duct in the absence of both lymphatic complications and mechanical ventilation.

T follicular helper cells in human efferent lymph retain lymphoid characteristics.

T follicular helper cells (Tfh), a subset of CD4+ T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of CXCR5+ and PD-1+ cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1-bright (CXCR5BrPD-1Br) Tfh population in thoracic duct lymph (TDL). These CXCR5BrPD-1Br TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in CXCR5BrPD-1Br TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the CXCR5BrPD-1Br TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue.

Acidosis inhibits rhythmic contractions of human thoracic ducts.

Lymph vessels counteract edema by transporting interstitial fluid from peripheral tissues to the large veins and serve as conduits for immune cells, cancer cells, and pathogens. Because edema during inflammation and malignancies is frequently associated with acidosis, we tested the hypothesis that acid‐base disturbances affect human thoracic duct contractions. We studied, by isometric and isobaric myography, the contractile function of human thoracic duct segments harvested with written informed consent from patients undergoing esophageal cancer surgery. Human thoracic ducts produce complex contractile patterns consisting of tonic rises in tension (isometric myography) or decreases in diameter (isobaric myography) with superimposed phasic contractions. Active tone development decreases substantially (~90% at 30 vs. 7 mmHg) at elevated transmural pressure. Acidosis inhibits spontaneous as well as noradrenaline‐ and serotonin‐induced phasic contractions of human thoracic ducts by 70–90% at extracellular pH 6.8 compared to 7.4 with less pronounced effects observed at pH 7.1. Mean tension responses to noradrenaline and serotonin – averaged over the entire period of agonist exposure – decrease by ~50% at extracellular pH 6.8. Elevating extracellular [K+] from the normal resting level around 4 mmol/L increases overall tension development but reduces phasic activity to a level that is no different between human thoracic duct segments investigated at normal and low extracellular pH. In conclusion, we show that extracellular acidosis inhibits human thoracic duct contractions with more pronounced effects on phasic than tonic contractions. We propose that reduced phasic activity of lymph vessels at low pH attenuates lymph propulsion and increases the risk of edema formation.

Spontaneous and α-adrenoceptor-induced contractility in human collecting lymphatic vessels require chloride.

Human lymphatic vessels are myogenically active and respond to sympathetic stimulation. The role of various cations in this behavior has recently been investigated, but whether the anion Cl- is essential is unclear. With ethical approval and informed consent, human thoracic duct and mesenteric lymphatic vessels were obtained from surgical patients. Spontaneous or norepinephrine-induced isometric force production from isolated vessels was measured by wire myography; the transmembrane Cl- gradient and Cl- channels were investigated by substitution of extracellular Cl- with the impermeant anion aspartate and inhibition of Cl- transport and channels with the clinical diuretics furosemide and bendroflumethiazide as well as DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The molecular expression of Ca2+-activated Cl- channels was investigated by RT-PCR, and proteins were localized using immunoreactivity. Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl- substitution with aspartate. About 100-300 µM DIDS or 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200 µM DIDS. Furosemide lowered only spontaneous constrictions, whereas bendroflumethiazide had nonspecific inhibitory effects. Consistent expression of transmembrane member 16A [TMEM16A (anoctamin-1)] was found in both the thoracic duct and mesenteric lymphatic vessels, and immunoreactivity with different antibodies localized TMEM16A to lymphatic smooth muscle cells and interstitial cells. The significant change in contractile function observed with inhibitors and anion substitution suggests that Cl- movement over the plasma membrane of lymphatic myocytes is integral for spontaneous and α-adrenoceptor-evoked contractility in human collecting lymphatic vessels. Consistent detection and localization of TMEM16A to myocytes suggests that this channel could play a major functional role. NEW & NOTEWORTHY In this study, we report the first observations of Cl- being a critical ionic component of spontaneous and agonist-evoked contractility in human lymphatics. The most consistently expressed Ca2+-activated Cl- channel gene in the human thoracic duct and mesenteric lymphatic vessels appears to be transmembrane member 16A, suggesting that this channel plays a major role.

Fine structure of human thoracic duct as revealed by light and scanning electron microscopy.

Little information has been available regarding microanatomy of human thoracic duct in spite of the importance for an understanding of pathophysiology in clinical medicine. The present study demonstrated a fine structure of human thoracic duct system by light and scanning electron microscopy. A number of longitudinal or spiral ridges and grooves were formed on luminal surfaces of the lymphangia and lymph sac, it likely facilitating fluent lymph flow. The endothelial cells displayed various cell shapes in compliance with their distributed regions. The lymph sac joining large vein composed a peculiar multiple valve structure presumably ensuring lymph storage and prevention of lymph backflow. The longitudinal muscle sheet in the tunica intima and circular muscle bundles in the tunica media constructed an integrated power unit probably eliciting spontaneous lymph propulsion. Furthermore, the thoracic duct was richly supplied with blood vessels not only in the tunica externa, but also just beneath the endothelium. The present findings provide a morphological basis for investigation of human thoracic duct in basic and clinical medicine.

Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine.

Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4±4.9% of control in the presence of 20nmoll(-1) nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the CaV1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo.

The contribution of K(+) channels to human thoracic duct contractility.

In smooth muscle cells, K(+) permeability is high, and this highly influences the resting membrane potential. Lymph propulsion is dependent on phasic contractions generated by smooth muscle cells of lymphatic vessels, and it is likely that K(+) channels play a critical role in regulating contractility in this tissue. The aim of this study was to investigate the contribution of distinct K(+) channels to human lymphatic vessel contractility. Thoracic ducts were harvested from 43 patients and mounted in a wire myograph for isometric force measurements or membrane potential recordings with an intracellular microelectrode. Using K(+) channel blockers and activators, we demonstrate a functional contribution to human lymphatic vessel contractility from all the major classes of K(+) channels [ATP-sensitive K(+) (KATP), Ca(2+)-activated K(+), inward rectifier K(+), and voltage-dependent K(+) channels], and this was confirmed at the mRNA level. Contraction amplitude, frequency, and baseline tension were altered depending on which channel was blocked or activated. Microelectrode impalements of lymphatic vessels determined an average resting membrane potential of -43.1 ± 3.7 mV. We observed that membrane potential changes of <5 mV could have large functional effects with contraction frequencies increasing threefold. In general, KATP channels appeared to be constitutively open since incubation with glibenclamide increased contraction frequency in spontaneously active vessels and depolarized and initiated contractions in previously quiescent vessels. The largest change in membrane voltage was observed with the KATP opener pinacidil, which caused 24 ± 3 mV hyperpolarization. We conclude that K(+) channels are important modulators of human lymphatic contractility.

The human thoracic duct is functionally innervated by adrenergic nerves

Lymphatic vessels from animals have been shown to be innervated. While morphological studies have confirmed human lymphatic vessels are innervated, functional studies supporting this are lacking. The present study demonstrates a functional innervation of the human thoracic duct (TD) that is predominantly adrenergic. TDs harvested from 51 patients undergoing esophageal and cardia cancer surgery were either fixed for structural investigations or maintained in vitro for the functional assessment of innervation by isometric force measurements and electrical field stimulation (EFS). Electron microscopy and immunohistochemistry suggested scarce diffuse distribution of nerves in the entire vessel wall, but nerve-mediated contractions could be induced with EFS and were sensitive to the muscarinic receptor blocker atropine and the α-adrenoceptor blocker phentolamine. The combination of phentolamine and atropine resulted in a near-complete abolishment of EFS-induced contractions. The presence of sympathetic nerves was further confirmed by contractions induced by the sympathomimetic and catecholamine-releasing agent tyramine. Reactivity to the neurotransmitters norepinephrine, substance P, neuropeptide Y, acetylcholine, and methacholine was demonstrated by exogenous application to human TD ring segments. Norepinephrine provided the most consistent responses, whereas responses to the other agonists varied. We conclude that the human TD is functionally innervated with both cholinergic and adrenergic components, with the latter of the two dominating.

Identification of Interstitial Cajal-Like Cells in the Human Thoracic Duct

Interstitial Cajal-like cells (ICLCs) are speculated to be pacemakers in smooth muscle tissues. While the human thoracic duct (TD) is spontaneously active, the origin of this activity is unknown. We hypothesized that ICLCs could be present in the TD and using histological techniques, immunohistochemistry and immunofluorescence we have investigated the presence of ICLCs, protein markers for ICLCs and the cellular morphology of the human TD. Transmission electron microscopy was employed to investigate ultrastructure. Methylene blue staining, calcium-dependent fluorophores and confocal microscopy were used to identify ICLCs in live tissue. Methylene blue stained cells with morphology suggestive of ICLCs in the TD. Immunoreactivity localized the ICLC protein markers c-kit, CD34 and vimentin to many cells and processes associated with smooth muscle cells (SMCs): coexpression of c-kit with vimentin or CD34 was observed in some cells. Electron microscopy analysis confirmed ICLCs as a major cell type of the human TD. Lymphatic ICLCs possess caveolae, dense bands, a patchy basal lamina, intermediate filaments and specific junctions to SMCs. ICLCs were ultrastructurally differentiable from other interstitial cells observed: fibroblasts, mast cells, macrophages and pericytes. Lymphatic ICLCs were localized to the subendothelial region of the wall as well as in intimate association with smooth muscle bundles throughout the media. ICLCs were morphologically distinct with multiple processes and also spindle shapes. Confocal imaging with calcium-dependent fluorophores corroborated cell morphology and localization observed in fixed tissues. Lymphatic ICLCs thus constitute a significant cell type of the human TD and physically interact with lymphatic SMCs.

Human thoracic duct in vitro: diameter-tension properties, spontaneous and evoked contractile activity

The current study characterizes the mechanical properties of the human thoracic duct and demonstrates a role for adrenoceptors, thromboxane, and endothelin receptors in human lymph vessel function. With ethical permission and informed consent, portions of the thoracic duct (2-5 cm) were resected and retrieved at T(7)-T(9) during esophageal and cardia cancer surgery. Ring segments (2 mm long) were mounted in a myograph for isometric tension (N/m) measurement. The diameter-tension relationship was established using ducts from 10 individuals. Peak active tension of 6.24 +/- 0.75 N/m was observed with a corresponding passive tension of 3.11 +/- 0.67 N/m and average internal diameter of 2.21 mm. The equivalent active and passive transmural pressures by LaPlace's law were 47.3 +/- 4.7 and 20.6 +/- 3.2 mmHg, respectively. Subsequently, pharmacology was performed on rings from 15 ducts that were normalized by stretching them until an equivalent pressure of 21 mmHg was calculable from the wall tension. At low concentrations, norepinephrine, endothelin-1, and the thromboxane-A(2) analog U-46619 evoked phasic contractions (analogous to lymphatic pumping), whereas at higher contractions they induced tonic activity (maximum tension values of 4.46 +/- 0.63, 5.90 +/- 1.4, and 6.78 +/- 1.4 N/m, respectively). Spontaneous activity was observed in 44% of ducts while 51% of all the segments produced phasic contractions after agonist application. Acetylcholine and bradykinin relaxed norepinephrine preconstrictions by approximately 20% and approximately 40%, respectively. These results demonstrate that the human thoracic duct can develop wall tensions that permit contractility to be maintained across a wide range of transmural pressures and that isolated ducts contract in response to important vasoactive agents.

Hepatolymphomania

After three years of penning Landmarks in Hepatology, it is a unique pleasure to have the occasion to respond to a letter to the editor from an avid fan, albeit one who is a little miffed by her perception that lymphology was given short shrift in a couple of articles. Although the writer's letter would probably not pass muster for inclusion in a collection of letters to the editor of The Times,1 like the curate's egg it was good in parts. I am grateful that Dr. Witte has offered the opportunity for greater recognition of the role of the lymphatic system in cirrhosis than was possible in “My Cup Runneth Over,” the main focus of which was intended to be The Overflow Hypothesis, proposed by Telfer Reynolds, as a landmark in hepatology from an historical perspective. Perforce, such a format undoubtedly may overlook competing hypotheses, and scholarly scientific reviews should be consulted for a more balanced account of any given topic. Of course, as the writer reminds us, ascites only accumulates if there is an imbalance between the rate of formation and absorption of visceral lymph, as described so elegantly in the cited reference 2 by her late illustrious husband, Charles L. Witte, herself, and Allan Dumont. Finally, Gaspar Asellius did indeed rediscover the lymphatic system in the postprandial dog by seeing the glistening chyliferous vessels that Hippocrates before him saw as “white blood,” Aristotle described as vessels containing a colorless fluid, and both Herophilus and Erasistratus noticed too. The eminent Danish physician, naturalist, physiologist, and anatomist Thomas Bartholin's contribution was to confirm the existence of the human thoracic duct, in the cadavers of two criminals (donated by the king), following Jean Pecquet's discovery of this structure in dogs. Adrian Reuben M.B.B.S., F.R.C.P.*, * Division of GI/Hepatology, Department of Medicine, Medical University of South Carolina, Charleston, SC.

Immunohistochemical demonstration of lymphatic vessels in human dental pulp

The existence of lymphatic vessels in dental pulp has been a matter of continuing controversy because of the difficulty of discriminating them in ordinary stained tissue sections. Recently, we have succeeded in establishing a new identification method for lymphatic vessels in human frozen sections by using a commercial monoclonal antibody specific for the human thoracic duct and anti-human laminin antiserum. The present study aimed to examine the lymphatic vessels in human dental pulp using the new immunostaining method, and compared the results with those in human small intestine. The study clearly demonstrated the distribution of lymphatic vessels in human dental pulp. Large lymphatic vessels are located in the central part of the pulp and there are small lymphatic vessels in the periphery of the pulp. This suggests that lymphatic drainage of the human dental pulp starts from the periphery of the pulp and collects in the central part of the pulp. A notable difference between the small intestine and dental pulp was found in the immunoreactivity of lymphatic vessels to anti-human laminin antiserum. In small intestine, immunoreactivity was significantly weaker than that of the blood vessels, whereas in dental pulp, that of lymphatic vessels was almost the same as blood vessels, except for some lymphatic vessels showing very weak reactivity. These findings suggest that the development of the basement membrane in both the lymphatic and blood vessels of human dental pulp is not as marked as in other tissue.

Phenotypical characterization of cells in the thoracic duct of patients with and without systemic inflammatory response syndrome and multiple organ failure.

The subset composition and recirculation properties of the migrating lymphocyte pool in humans is largely unknown. The present study was conducted in order to phenotypically characterize cells in human thoracic duct lymph of patients under non-inflammatory and inflammatory conditions. These data were compared with data from peripheral blood, with special emphasis on those cells homing to the gut. Thoracic duct lymph and peripheral blood contained comparable proportions of B and T lymphocytes and CD8+ cells. Thoracic duct lymph contained proportionally more CD4+ cells, more CD4+CD45RO+ that express alpha 4 beta 7 cells and more CD8+CD45RO+ that express alpha 4 beta 7, as compared to peripheral blood. These data suggest an equal recirculation rate of B and T lymphocytes; a more active recirculation of CD4+ cells compared to CD8+ cells; and a more active recirculation of memory cells to the gut as compared to other extra-lymphoid sites in patients under non-inflammatory conditions. Data were also obtained in patients with the system inflammatory response syndrome and multiple organ failure. Although it is generally assumed that granulocytes and monocytes do not recirculate, lymph of multiple organ failure patients contained significantly more granulocytes than monocytes, indicating that in severe generalized inflammatory states these cells re-enter the circulation through the thoracic duct. Furthermore, no increased activation of cells homing to the gut was found in these patients.

Human thoracic duct lymph inhibits lipopolysaccharide-induced release of cytokines

Oral starvation causes gut atrophy and breakdown of barrier function, which can lead to transport of lipopolysaccharide (LPS) across the intestinal mucosa. Since triglyceride-rich lipoproteins can inhibit lipopolysaccharide-induced cytokine release, it can be hypothesized that enteral feeding protects the body against translocated LPS at the level of the thoracic duct by increasing the levels of triglycerides in thoracic duct lymph. We sought to determine the LPS-neutralizing capacity of human chyle by measuring LPS-induced cytokine production in vitro in the presence or absence of thoracic duct lymph. Moreover, we assessed whether enteral administration of triglyceride-rich lipoproteins changed the ability of lymph to influence LPS activity. Indeed, the presence of 10% and 100% triglyceride-poor and triglyceride-rich lymph induced a significant reduction in the TNF and IL-6 production elicited by LPS. However, there was no difference in the extent of inhibition of cytokine-release by triglyceride-poor and triglyceride-rich lymph. This study shows that lymph can inhibit LPS activity. This is not affected by prior enteral administration of triglycerides.

Identification of Lymphatic Vessels by Using a Monoclonal Antibody Specific for the Human Thoracic Duct

Light microscopic identification of lymphatic vessels of the human small intestine was studied immuno-histochemically with a monoclonal antibody specific for human thoracic duct (mAb-D) and rabbit anti-human laminin antibody (anti-L). Two types of vessels were observed with anti-L; one strongly reacted with regularly outlined vessels and the other weakly reacted with irregularly outlined vessels and thin walls. Immunoreactivity to mAb-D was strong in the weakly reacting vessels, and the strongly reacting vessels did not react to mAb-D. Immunoreactivity of lymphatic vessels to anti-L has been reported to be weaker than that of the blood vessels; only irregularly outlined vessels which were weakly immunostained with anti-L reacted to mAb-D and no cross-reaction to mAb-D was observed in the regularly outlined vessels. This indicates that the vessels strongly reacting with anti-L are blood vessels and the vessels immunostained with mAb-D must be lymphatics. The study shows that it is possible to identify lymphatic vessels light microscopically by mAb-D. This method will be useful in studying the fine distribution of lymphatic vessels in normal tissue and also in pathological tissue such as metastasis of malignant tumors.

Tissue culture of human and canine thoracic duct endothelium

Endothelial cells from the canine or human thoracic duct were harvested using 0.2% collagenase digestion and grown in Media 199 supplemented with fetal bovine serum. The canine endothelial cells grew to confluence (4.4 to 12 X 10(4) cells/cm2) in 6 to 10 d; doubling times ranged from 1.5 to 2.8 d. There was a minimum critical density for cell growth between 5000 and 10 000 cells/cm2. The canine endothelial cells have been maintained in culture for periods up to 11 mo. The human thoracic duct endothelial cells are more difficult to grow and maintain. Endothelial cells were isolated from 5 out of 35 human thoracic ducts and grew for periods of up to 2 wk before degenerating. Both human and canine endothelial cells were Factor VIII positive. It has thus been demonstrated that it is possible to grow canine and, less easily, human thoracic duct endothelium in tissue culture.

Histamine-releasing activity: IV. Molecular heterogeneity of the activity from stimulated human thoracic duct lymphocytes

Previous studies with the lymphokine, histamine-releasing activity (HRA), showed that HRA consisted of a heterogeneous group of molecules. The possibility of using thoracic duct lymphocytes (TDL) as a source of large quantities of HRA has been investigated. Antigen-stimulated TDL synthesize and release HRA in quantities similar to an equivalent number of peripheral blood lymphocytes (PBL). Streptokinase (SK) antigen routinely caused TDL to produce HRA ~ 15,000 Da. In contrast, staphylococcus enterotoxin B (SEB) induced the formation of a heterogeneous mixture of HRAs with apparent molecular weights of 50,000 and 15,000. Two peaks of activity (HRA I and II) were recovered when the supernatant from SK-stimulated TDL was subjected to ion-exchange chromatography. Interestingly, basophil chemotactic activity (BCA) was also eluted in these two peaks. Although interferon (IFN) is also released by antigen-stimulated TDL, the nonidentity of IFN and HRA was established by fundamental differences in Chromatographic properties and specific antisera to IFN. In contrast, these studies suggest that HRA and BCA may be present on the same molecular entity.

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages.

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.

Transfer of human lymph chylomicron constituents to other lipoprotein density fractions during in vitro lipolysis

To ascertain whether chylomicron constituents would be transferred to low density lipoprotein (LDL, d 1.019-1.063 g/ml) and high density lipoprotein (HDL, d 1.063-1.21 g/ml) density fractions during lipolysis in the absence of other lipoproteins, the in vitro effect of bovine milk lipoprotein lipase on human thoracic duct lymph chylomicrons in the presence of albumin was examined. In incubations without lipase, over 90% of chylomicron constituents remained in the 1.006 g/ml supernate, and large particles ranging in diameter mainly from 750-6000 A were observed by electron microscopy. After the addition of lipase, lipolysis ranged from 69.0-94.6% and numerous collapsed particles with redundant surface were seen, as well as smaller particles within the LDL and HDL density region. With lipolysis, the majority of chylomicron cholesterol and phospholipid mass was transferred to LDL and HDL, while chylomicron apolipoprotein (apo) A-I, A-II, and C-II mass was transferred mainly to HDL. Utilizing either radioiodinated apoA-I and apoA-II reassociated with chylomicrons or radiolabeled chylomicrons, a similar redistribution of apoA-I and apoA-II radioactivity was noted with lipolysis. In contrast, chylomicron apoB (mainly B-48) radioactivity was transferred predominantly to LDL with lipolysis. These data are consistent with the concept that during lymph chylomicron triglyceride hydrolysis, chylomicron apolipoproteins, cholesterol, and phospholipid can be transferred to the LDL and HDL density regions in the absence of acceptor particles.