Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Tel-Aviv University

Pharmacologic modulation of the perioperative physiologic stress response, using the beta-blocker propranolol, combined with the COX-2 inhibitor etodolac, has been shown to reduce metastatic spread and increase survival rates following surgery for primary tumor excision in rodents. Prior to implantation of this pharmacological approach in clinical trials in patients with colon cancer, the safety of this technique has to be evaluated. This study assessed the effects of these drugs on the healing of colonic anastomosis in rats.

Effect of beta blocker combined with COX-2 inhibitor on colonic anastomosis in rats.

Stress responses are known to modulate leukocyte trafficking. In the skin, stress was reported both to enhance and reduce skin immunity, and the chronicity of stress exposure was suggested as a key determining factor. We here propose a dual-stage hypothesis, suggesting that stress, of any duration, reduces skin immunity during its course, while its cessation is potentially followed by a period of enhanced skin immunity. To start testing this hypothesis, rats were subcutaneously implanted with sterile surgical sponges for four-hours, during or after exposure to one of several acute stress paradigms, or to a chronic stress paradigm. Our findings, in both males and females, indicate that numbers of sponge-infiltrating leukocytes, and their specific subsets, were reduced during acute or chronic stress, and increased after stress cessation. Studying potential mediating mechanisms of the reduction in leukocyte numbers during acute stress, we found that neither adrenalectomy nor the administration of beta-adrenergic or glucocorticoid antagonists prevented this reduction. Additionally, administration of corticosterone or epinephrine to adrenalectomized rats did not impact skin leukocyte numbers, whereas, in the blood, these treatments did affect numbers of leukocytes and their specific subsets, as was also reported previously. Overall, our findings support the proposed dual-stage hypothesis, which can be evolutionally rationalized and accounts for most of the apparent inconsistencies in the literature regarding stress and skin immunity. Other aspects of the hypothesis should be tested, also using additional methodologies, and its predictions may bear clinical significance in treatment of skin disorders related to hyper- or hypo-immune function.

Stress and skin leukocyte trafficking as a dual-stage process.

To investigate early predictors of sleep patterns in pre-school age children. Specifically, we were interested in exploring whether infant sleep patterns and parenting factors assessed at 12 months would predict sleep in four year-old children.

Infant sleep and early parental sleep-related cognitions predict sleep in pre-school children.

Young adults often encounter sleep deprivation and stressful events. Both have been separately reported to modulate immunity, and occasionally they occur simultaneously. We assessed the combined effects of these conditions on immune competence in healthy students.

Resilience of the immune system in healthy young students to 30-hour sleep deprivation with psychological stress.

To test whether marginating-pulmonary (MP) leukocytes in mice have a unique potential to identify and destroy aberrant circulating cells, we compared MP to circulating leukocytes with respect to natural killer (NK) cytotoxicity, proinflammatory characteristics, molecular determinants of activation, and response to IL-12 immunostimulation. Cytotoxicity was assessed employing the YAC-1, B16F10, and 3LL target lines. C57BL/6 mice were injected with either saline or murine IL-12 (0.1 or 0.5 µg/mouse), either once or three times 48-h apart. Twenty-four hours after last injection, cardiac blood was withdrawn and MP leukocytes were collected by forced lung perfusion. NK cytotoxicity, cellular composition, and surface molecular markers were studied. MP leukocytes exhibited greater NK cytotoxicity than circulating leukocytes against the syngeneic B16F10 and 3LL tumor lines, but not against the allogeneic YAC-1 line. NKG2D and IL-12 receptor expression predicted NK cytotoxicity in circulating leukocytes, but not in MP leukocytes. IFNγ-receptor, IL-12-receptor, CD69, CD11a, and CD11b showed different patterns of expression in the two leukocyte populations, suggesting pro-inflammatory characteristics of the MP compartment. IL-12 stimulation caused differential effects on these markers and also elevated cytotoxicity in both compartments, but in different effector: target ratio-dependent patterns. MP leukocytes may play a critical role in eliminating aberrant circulating cells due to their enhanced NK cytotoxicity and given their strategic location in the lungs vasculature, which forces physical interactions with all circulating aberrant cells. MP-NK cells are unique in their cytotoxic mechanisms against syngeneic targets and in their activation profile and response to immunostimulatory agents.

The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.

Extensive oncological surgeries were previously suggested to increase cancer recurrence rates. We herein studied the impact of different surgical procedures and sex on colorectal cancer liver metastasis, employing several tumor inoculation approaches in BALB/c mice.

Impact of surgical extent and sex on the hepatic metastasis of colon cancer.

Most in vitro and ex-vivo studies indicate a profound suppression of NK cell cytotoxicity (NKCC) by glucocorticoids; while catecholamines and prostaglandins were reported both to suppress and to enhance NKCC. However, methodological considerations hinder our ability to deduce from these findings to the impact of endogenous release of these factors on in vivo levels of NKCC and their implications to NK-dependent resistance to pathologies in living humans or animals. Here we used an in vivo approach that sensitively and specifically reflects NKCC in living F344 rats, based on lung clearance of NK-sensitive tumor cells (MADB106), and based on comparing effects between NK-intact and NK-depleted rats. To study the role of corticosterone, epinephrine, and prostaglandins, we administered these factors to rats, or antagonized their endogenous release following different stress paradigms or surgery. The results indicated that endogenous or exogenous elevated corticosterone levels can suppress in vivo NKCC levels, but only under some conditions, and mostly secondarily to the NK-suppressing impact of epinephrine. Specifically, corticosterone-induced NKCC suppression occurred (i) only under prolonged, but not short exposure to stress, and mainly in males; (ii) was smaller than the prominent impact of epinephrine; (iii) was mostly ascribed to corticosterone-induced potentiation of the effects of epinephrine or/and prostaglandins; and (iv) was completely abolished through antagonizing epinephrine or/and prostaglandins. Overall, these findings markedly limit the significance of stress/surgery-induced corticosterone release in the in vivo suppression of NKCC, and highlight the blockade of epinephrine or/and prostaglandins as effective and clinically feasible approaches to overcome such immuno-suppressive effects.

In vivo suppression of NK cell cytotoxicity by stress and surgery: glucocorticoids have a minor role compared to catecholamines and prostaglandins.

IL-12 is a prominent Th1 differentiator and leukocyte activator. Ample studies showed suppression of IL-12 production by numerous stress factors, including prostaglandins, catecholamines, glucocorticoids, and opioids, but did so in vitro and in the context of artificial leukocyte activation, not simulating the in vivo setting. In a recent study we reported in vivo suppression of plasma IL-12 levels by behavioral stress and surgery. The current study aims to elucidate neuroendocrine mechanisms underlying this phenomenon in naïve F344 rats. To this end, both adrenalectomy and administration of specific antagonists were used, targeting the aforementioned stress factors. The results indicated that corticosterone and prostaglandins are prominent mediators of the IL-12-suppressing effects of stress and surgery, apparently through directly suppressing leukocyte IL-12 production. Following surgery, endogenous prostaglandins exerted their effects mainly through elevating corticosterone levels. Importantly, stress-induced release of epinephrine or opioids had no impact on plasma IL-12 levels, while pharmacological administration of epinephrine reduced plasma IL-12 levels by elevating corticosterone levels. Last, a whole blood in vitro study indicated that prostaglandins and corticosterone, but not epinephrine, suppressed IL-12 production in non-stimulated leukocytes, and only corticosterone did so in the context of CpG-C-induced IL-12 production. Overall, the findings reiterate the notion that results from in vitro or pharmacological in vivo studies cannot indicate the effects of endogenously released stress hormones under stress/surgery conditions. Herein, corticosterone and prostaglandins, but not catecholamines or opioids, were key mediators of the suppressive effect of stress and surgery on in vivo plasma IL-12 levels in otherwise naïve animals.

Plasma IL-12 levels are suppressed in vivo by stress and surgery through endogenous release of glucocorticoids and prostaglandins but not catecholamines or opioids.

In vitro and ex vivo studies assessing the impact of stress hormones on immune competence commonly replace the natural milieu of leukocytes with an artificial medium, excluding plasma factors, hormones, and cytokines. Given prevalent inconsistencies between in vitro, ex vivo, and in vivo findings, we studied whether such procedures could yield misleading outcomes regarding the impact of stress hormones on NK cell cytotoxicity (NKCC), using fresh human whole blood samples. We found that in the presence of plasma 10-30-fold higher concentrations of cortisol, epinephrine, and prostaglandin-E2 (PGE2) were required to reach suppression levels evident in the context of artificial medium. Importantly, whereas the NK suppressive effects of PGE2 occurred immediately and remained stable upon prolonged exposure, the suppressive effects of cortisol slowly increased over time. Last, to simulate the exclusion of stress factors in the ex vivo approach, we subjected whole blood to stress hormones (as occurs in vivo), and abruptly removed them. We found that the effects of epinephrine and PGE2 quickly disappeared, while the effects of cortisol persisted. Overall, these findings demonstrate the potential misleading nature of in vitro and ex vivo procedures, and specifically suggest that (i) the common in vitro findings of profound suppression of NKCC by stress hormones are overestimation of their direct effects expected in vivo; and (ii) the common ex vivo approach cannot reflect the direct in vivo suppressive effects of epinephrine and PGE2 on NKCC, while inflating the effects of glucocorticoids. Some of these fallacies may be circumvented by using non-delayed whole blood NKCC assays in humans.

The misleading nature of in vitro and ex vivo findings in studying the impact of stress hormones on NK cell cytotoxicity.

The use of TLR agonists as an anti-cancer treatment is gaining momentum given their capacity to activate various host cellular responses through the secretion of inflammatory cytokines and type-I interferons. It is now also recognized that the perioperative period is a window of opportunity for various interventions aiming at reducing the risk of cancer metastases-the major cause of cancer related death. However, immune-stimulatory approach has not been used perioperatively given several contraindications to surgery. To overcome these obstacles, in this study, we used the newly introduced, fully synthetic TLR-4 agonist, Glucopyranosyl Lipid-A (GLA-SE), in various models of cancer metastases, and in the context of acute stress or surgery. Without exerting evident adverse effects, a single systemic administration of GLA-SE rapidly and dose dependently elevated both innate and adaptive immunity in the circulation, lungs and the lymphatic system. Importantly, GLA-SE treatment led to reduced metastatic development of a mammary adenocarcinoma and a colon carcinoma by approximately 40-75% in F344 rats and BALB/c mice, respectively, at least partly through elevating marginating-pulmonary NK cell cytotoxicity. GLA-SE is safe and well tolerated in humans, and currently is used as an adjuvant in phase-II clinical trials. Given that the TLR-4 receptor and its signaling cascade is highly conserved throughout evolution, our current results suggest that GLA-SE may be a promising immune stimulatory agent in the context of oncological surgeries, aiming to reduce long-term cancer recurrence.

Perioperative treatment with the new synthetic TLR-4 agonist GLA-SE reduces cancer metastasis without adverse effects.

Liver metastases are a major cause of colorectal cancer death, and the perioperative period is believed to critically affect the metastatic process. Here we tested whether blocking excess release of catecholamines and prostaglandins during surgical procedures of different extent can reduce experimental liver metastasis of the syngeneic CT26 colon cancer in female and male BALB/c mice. Animals were either treated with the beta-blocker, propranolol, the COX-2 inhibitor, etodolac, both drugs, or vehicle. The role of NK cells in controlling CT26 hepatic metastasis and in mediating the effect of the drugs was assessed by in vivo depletion or stimulation of NK cells, using anti-asialo GM1 or CpG-C, respectively. Surgical extent was manipulated by adding laparotomy to small incision, extending surgical duration, and enabling hypothermia. The results indicated that combined administration of propranolol and etodolac, but neither drug alone, significantly improved host resistance to metastasis. These beneficial effects occurred in both minor and extensive surgeries, in both sexes, and in two tumor inoculation approaches. NK cell-mediated anti-CT26 activity is involved in mediating the beneficial effects of the drugs. Specifically, CpG-C treatment, known to profoundly activate mice marginating-hepatic NK cytotoxicity, reduced CT26 hepatic metastases; and NK-depletion increased metastases and prevented the beneficial effects of the drugs. Overall, given prevalent perioperative psychological and physiological stress responses in patients, and ample prostaglandin release by colorectal tumors and injured tissue, propranolol and etodolac could be tested clinically in laparoscopic and open colorectal surgeries, attempting to reduce patients' metastatic disease.

Lee Shaashua

Sagol School of Neuroscience & School of Psychological Sciences

Tel-Aviv University

Selective Harvesting of Marginating-pulmonary Leukocytes

Selective Harvesting of Marginating-hepatic Leukocytes

Lee Shaashua

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Sagol School of Neuroscience & School of Psychological Sciences

Tel-Aviv University

Selective Harvesting of Marginating-pulmonary Leukocytes

Selective Harvesting of Marginating-hepatic Leukocytes

Sagol School of Neuroscience & School of Psychological Sciences