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Platelet-Derived Growth Factor BB-Mediated Normalization of Dermal Interstitial Fluid Pressure After Mast Cell Degranulation Depends on β3 but Not β1 Integrins

Interstitial fluid pressure (PIF) is one of the determinants of transcapillary fluid flux and thereby interstitial fluid volume. Cell-mediated control of PIF regulates fluid content in the loose interstitial connective tissues that surround the capillary bed. To maintain a normal PIF in dermis, &bgr;1 integrins mediate the tensile strength applied by connective tissue cells on the extracellular matrix. Platelet-derived growth factor (PDGF)-BB normalizes anaphylaxis-induced reduction of PIF. Anti–&bgr;3 integrin IgG and a cyclic RGD peptide that inhibits the &agr;V&bgr;3 integrin blocked the ability of PDGF-BB to normalize the lowered PIF resulting from mast cell degranulation. PDGF-BB was unable to normalize PIF lowered as a result of mast cell degranulation in &bgr;3-negative mice. Monoclonal anti–&bgr;3 integrin IgG had no effect on PIF in normal mouse dermis. In contrast, administration of anti–&bgr;1 integrin IgM lowered PIF in normal dermis but had no effect on PDGF-BB–induced normalization of PIF after anaphylaxis. Furthermore, collagen gel contraction mediated by wild-type mouse embryonal fibroblasts were only marginally affected by function-blocking anti–&bgr;1 integrin antibodies, especially in the presence of PDGF-BB. In contrast, contraction mediated by &agr;V-negative mouse embryonic fibroblasts was completely blocked by anti–&bgr;1 integrin antibodies, even after stimulation with PDGF-BB. These results show a previously unrecognized in vivo function for the &agr;V&bgr;3 integrin, as a participant in the control of PIF during inflammatory reactions. Furthermore, our data demonstrate that PDGF-BB induces connective tissue cells to generate tensile forces via &agr;V&bgr;3 during such reactions.

Different serotypes of endotoxin (lipopolysaccharide) cause different increases in albumin extravasation in rats.

Endotoxin (lipopolysaccharide [LPS] from cell membranes of gram-negative bacteria) is frequently used in experimental models of septic shock that are characterised by hypotension, peripheral vasodilation, and edema formation, as well as greatly enhanced flux of macromolecules and fluid from plasma to tissues. The edema formation and increased albumin extravasation (Ealb) could be caused by increased permeability and/or increased capillary net filtration pressure. We have measured interstitial fluid pressure (Pif) and Ealb after i.v. injection of two different serotypes of LPS in female Wistar Møller rats (200–250 g) in pentobarbital anaesthesia. Two experimental groups and one control group were studied (n = 8 in each group). Group 1, serotype 0111:B4, received 3 mg/kg LPS, Group 2, serotype 0127:B8, received 1.5 mg/kg LPS, and controls received saline vehicle (0.4 mL). Five minutes after injection of LPS or saline vehicle, human serum albumin labelled with 125I (125I-HAS; 0.05 MBq) was injected i.v. and was followed 55 min later by 131I-HSA (0.05 MBq). Five minutes thereafter the rats were killed and tissue samples were obtained from skin, muscle, and small intestine. Ealb was estimated as the difference between the plasma equivalent distribution volumes of 125I-HSA and 131I-HSA. The pattern of extravasation between the groups was the same in all the tissues studied. Group 1 serotype (0111:B4) and controls had much lower Ealb than Group 2 serotype (0127:B8;P < 0.05). Ealb differed among the tissues both in relative and absolute numbers, being largest in the intestine and smallest in skeletal muscle. We previously demonstrated a lowering of Pif after LPS injection using serotype (0127:B8). The present results demonstrate that the same serotype of LPS also causes a significant increase of Ealb, and is therefore likely caused by the lowering of Pif.

Differential cytokine response in interstitial fluid in skin and serum during experimental inflammation in rats.

Tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) are important mediators produced during inflammation. We hypothesized that the pro‐inflammatory cytokine response in the interstitial fluid (IF) is different from that in serum, and we aimed at quantifying the amount of TNF‐α and IL‐1β in the IF. By centrifugation of rat skin at < 424 g pure IF is extracted. Using ELISA such fluid was analysed for cytokines in back and/or paw skin of pentobarbital‐anaesthetized rats, after either induction of endotoxaemia or ischaemia–reperfusion (I/R) injury. During endotoxaemia, TNF‐α increased in the IF from 0 in control to 640 ± 100 pg ml−1 (mean ±s.e.m.) after 90 min, with the serum concentration being 5–10 times higher at all time points. The response pattern of IL‐1β after lipopolysaccharide (LPS) challenge differed greatly from that of TNF‐α with a large increase in IF from 390 ± 90 to 28 000 ± 1500 pg ml−1 after 210 min, and a significantly smaller increase in serum (600 ± 45 pg ml−1). During reperfusion of the hind paw after 2 h of ischaemia, there was a gradual increase of TNF‐α in both IF of the paw skin and serum after 3 min of reperfusion. Both declined after 20 min. The pattern for IL‐1β differed, increasing significantly less in serum (25 ± 15 pg ml−1 after 20 min of reperfusion) than in the IF (1100 ± 200 pg ml−1). Immunostaining of the inflamed tissues showed increased expression of the two cytokines in cells of both epidermis and dermis compared to controls. Subdermal injections of TNF‐α and IL‐1β at the same concentrations found in IF after LPS infusion affected interstitial fluid pressure significantly. Local TNF‐α production dominates after I/R injury, whereas in endotoxaemia systemic production predominates. For IL‐1β local production dominates in both conditions. Thus, there is a differential pattern of cytokine production and the current method allows the study of the role of cytokines in IF during different inflammatory reactions.

A novel function of insulin in rat dermis

In this study we present a novel function of insulin in rat dermis. We investigated local effects of insulin on interstitial fluid pressure (Pif), and capillary albumin leakage and pro‐inflammatory cytokine production in skin and serum after intravenous lipopolysaccharide (LPS), tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) challenge treated with a glucose–insulin–potassium regimen (GIK). The main objective for this study was to investigate anti‐inflammatory effects of insulin. Work by others shows that insulin stimulates cell adhesion, and that this effect is dependent upon phosphatidylinositol 3‐kinase (PI3K) activity. Cytokines like platelet‐derived growth factor BB (PDGF‐BB) attenuate lowering of Pif, possibly via PI3K. LPS and pro‐inflammatory cytokines contribute to oedema development during acute inflammation by lowering the Pif. Intravenous injection of LPS, TNF‐α or IL‐1β to Wistar Møller rats caused a lowering of Pif, but after local injection of insulin in the paw, Pif increased back to control values. IL‐1β caused a lowering in control from −0.5 ± 0.2 mmHg to −3.0 ± 0.2 mmHg after 20 min (mean ±s.e.m.) (P < 0.05). Within 50 min after insulin injection the pressure was increased to −0.6 ± 0.2 mmHg (P > 0.05 compared with control). Insulin was given together with a PI3K inhibitor (wortmannin) locally in the skin, almost abolishing the effect of insulin on Pif. A GIK regimen was given as a continuous intravenous infusion, significantly attenuating the oedema formation after LPS or TNF‐α/IL‐1β challenge. The same GIK regimen caused a significant reduction in pro‐inflammatory cytokines in serum and in interstitial fluid in skin of endotoxaemic rats. These experiments show a possible role for insulin in the interstitium during inflammation induced by LPS and TNF‐α/IL‐1β. Insulin can attenuate a lowering of Pif possibly via PI3K, and it has an anti‐inflammatory effect by inhibiting production of pro‐inflammatory cytokines.

Effect of α-trinositol on interstitial fluid pressure, edema generation, and albumin extravasation after ischemia-reperfusion injury in rat hind limb

Reperfusion of ischemic tissue often leads to an acute inflammatory response, which acts directly to aggravate the injury in the reperfused zone, characterized by adhesion and subsequent infiltration of inflammatory cells that injure the tissue through the generation of oxygen-derived free radicals and release of various inflammatory mediators. The rapid edema formation associated with reperfusion injury is induced by increased microvascular permeability to plasma proteins and/or increased net filtration pressure across the microvascular wall, and the latter is at least in part induced by lowering of the interstitial fluid pressure (Pif). We investigated the anti-inflammatory effect of &agr;-trinositol (D-myo-inositol-1,2,6-trisphosphate) on edema formation, microvascular protein leakage, and Pif in rat hind limb after ischemia-reperfusion (I/R) injury. There was significant increase of both albumin extravasation from 0.02 ± 0.02 to 0.41 ± 0.21 mL g dry weight−1 (P < 0.05) and total tissue water from 1.08 ± 0.07 to 1.65 ± 0.55 mL g dry weight−1(P < 0.05) in the skin of paws undergoing I/R injury. Pif was significantly lowered from −0.51 ± 0.34 to −5.00 ± 1.53 mmHg (P < 0.05) concomitant with substantial edema formation. The edema formation, and lowering of Pif during I/R injury was significantly lowered and nearly totally abolished in the animals treated with &agr;-trinositol 30 min before reperfusion. We conclude that &agr;-trinositol limits the increased vascular permeability and edema formation by preventing the decrease in Pif as well as acting protective on the microvascular wall.

Effect of tumor necrosis factor-α, IL-1β, and IL-6 on interstitial fluid pressure in rat skin

Interstitial fluid pressure (Pif) decreases in several experimental models of acute inflammation, enhancing edema formation. The present study was designed to determine the effect of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β as well as lipopolysaccharides (LPS) on Pif in a model of gram-negative sepsis. Pif was measured in the paw skin of anesthetized rats (pentobarbital sodium, 50 mg/kg ip) using micropipettes (3-7 μm) and servo-controlled counterpressure technique. Test substances were injected intra-arterially (ia), intravenously (iv), or subdermally (sd). After intra-arterial or intravenous administration, the test substances were circulated for 1 min before circulatory arrest was induced with an intravenous injection of KCl while the rats were under pentobarbital anesthesia. Circulatory arrest was induced to avoid edema formation, which would raise interstitial fluid volume to cause a more positive Pif. Administration of 0.5 ml of LPS (5 mg/ml ia) lowered Pif significantly from control values of -0.2 ± 0.3 to -2.0 ± 0.3 mmHg ( P < 0.05) within 1 h. Corresponding values for TNF-α (500 ng/ml iv) were -0.4 ± 0.2 to -2.3 ± 0.1 mmHg ( P < 0.05). Administration of 5 μl (5 mg/ml sd) of LPS did not affect Pif significantly ( P > 0.05), but TNF-α, IL-1β, and IL-6 had a significant effect on Pif when given subdermally. IL-6 (50 ng/ml) caused a decrease in Pif from control values of -1.2 ± 0.3 to -2.8 ± 0.5 mmHg ( P < 0.05) within 1 h. The experiments demonstrate that LPS, TNF-α, IL-1β, and IL-6 induce lowering of Pif when given intravenously or intra-arterially, whereas only TNF-α, IL-1β, and IL-6 induce lowering of Pif when given subdermally. We therefore suggest that the lowering of Pif in this experimental model of sepsis is related to the release of and a local effect in skin of TNF-α, IL-1β, and IL-6.Interstitial fluid pressure (P(if)) decreases in several experimental models of acute inflammation, enhancing edema formation. The present study was designed to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-1beta as well as lipopolysaccharides (LPS) on P(if) in a model of gram-negative sepsis. P(if) was measured in the paw skin of anesthetized rats (pentobarbital sodium, 50 mg/kg ip) using micropipettes (3-7 micrometer) and servo-controlled counterpressure technique. Test substances were injected intra-arterially (ia), intravenously (iv), or subdermally (sd). After intra-arterial or intravenous administration, the test substances were circulated for 1 min before circulatory arrest was induced with an intravenous injection of KCl while the rats were under pentobarbital anesthesia. Circulatory arrest was induced to avoid edema formation, which would raise interstitial fluid volume to cause a more positive P(if). Administration of 0.5 ml of LPS (5 mg/ml ia) lowered P(if) significantly from control values of -0.2 +/- 0.3 to -2.0 +/- 0.3 mmHg (P < 0.05) within 1 h. Corresponding values for TNF-alpha (500 ng/ml iv) were -0.4 +/- 0.2 to -2.3 +/- 0.1 mmHg (P < 0.05). Administration of 5 microliter (5 mg/ml sd) of LPS did not affect P(if) significantly (P > 0.05), but TNF-alpha, IL-1beta, and IL-6 had a significant effect on P(if) when given subdermally. IL-6 (50 ng/ml) caused a decrease in P(if) from control values of -1.2 +/- 0.3 to -2. 8 +/- 0.5 mmHg (P < 0.05) within 1 h. The experiments demonstrate that LPS, TNF-alpha, IL-1beta, and IL-6 induce lowering of P(if) when given intravenously or intra-arterially, whereas only TNF-alpha, IL-1beta, and IL-6 induce lowering of P(if) when given subdermally. We therefore suggest that the lowering of P(if) in this experimental model of sepsis is related to the release of and a local effect in skin of TNF-alpha, IL-1beta, and IL-6.

Integrin αvβ3 acts downstream of insulin in normalization of interstitial fluid pressure in sepsis and in cell-mediated collagen gel contraction

The administration of insulin is recommended to patients with severe sepsis and hyperglycemia. Previously, we demonstrated that insulin may have direct anti-inflammatory properties and counteracted fluid losses from the circulation by normalizing the interstitial fluid pressure (P(IF)). P(IF) is one of the Starling forces determining fluid flux over the capillary wall, and a lowered P(IF) is one of the driving forces in early edema formation in inflammatory reactions. Here we demonstrate that insulin restores a lipopolysaccharide (LPS)-lowered P(IF) via a mechanism involving integrin alpha(v)beta(3). In C57 black mice (n = 6), LPS lowered P(IF) from -0.2 +/- 0.2 to -1.6 +/- 0.3 (P < 0.05) and after insulin averaged -0.8 +/- 0.2 mmHg (P = 0.098 compared with after LPS). Corresponding values in wild-type BALB/c mice (n = 5) were -0.8 +/- 0.1, -2.1 +/- 0.3 (P < 0.05), and -0.8 +/- 0.3 mmHg (P < 0.05 compared with LPS) after insulin administration. In BALB/c integrin beta(3)-deficient (beta(3)(-/-)) mice (n = 6), LPS lowered P(IF) from -0.1 +/- 0.2 to -1.5 +/- 0.3 mmHg (P < 0.05). Insulin did not, however, restore P(IF) in these mice (averaged -1.7 +/- 0.3 mmHg after insulin administration). Cell-mediated collagen gel contraction can serve as an in vitro model for in vivo measurements of P(IF). Insulin induced alpha(v)beta(3)-integrin-dependent collagen gel contraction mediated by C2C12 cells. Our findings suggest a beneficiary effect of insulin for patients with sepsis with regard to the fluid balance, and this effect may in part be due to a normalization of P(IF) by a mechanism involving the integrin alpha(v)beta(3).

Effect of Topical Anaesthetics on Interstitial Colloid Osmotic Pressure in Human Subcutaneous Tissue Sampled by Wick Technique

Objectives To measure colloid osmotic pressure in interstitial fluid (COPi) from human subcutaneous tissue with the modified wick technique in order to determine influence of topical application of anaesthetics, dry vs. wet wick and implantation time on COPi. Material and Methods In 50 healthy volunteers interstitial fluid (IF) was collected by subcutaneous implantation of multi-filamentous nylon wicks. Study subjects were allocated to two groups; one for comparing COPi obtained from dry and saline soaked wicks, and one for comparing COPi from unanaesthetized skin, and skin after application of a eutectic mixture of local anaesthetic (EMLA®, Astra Zeneca) cream. IF was sampled from the skin of the shoulders, and implantation time was 30, 60, 75, 90 and 120 min. Colloid osmotic pressure was measured with a colloid osmometer. Pain assessment during the procedure was compared for EMLA cream and no topical anaesthesia using a visual analogue scale (VAS) in a subgroup of 10 subjects. Results There were no significant differences between COPi obtained from dry compared to wet wicks, except that the values after 75 and 90 min. were somewhat higher for the dry wicks. Topical anaesthesia with EMLA cream did not affect COPi values. COPi decreased from 30 to 75 min. of implantation (23.2±4.4 mmHg to 19.6±2.9 mmHg, p = 0.008) and subsequently tended to increase until 120 min. EMLA cream resulted in significant lower VAS score for the procedure. Conclusion COPi from subcutaneous tissue was easily obtained and fluid harvesting was well tolerated when topical anaesthetic was used. The difference in COPi assessed by dry and wet wicks between 75 min. and 90 min. of implantation was in accordance with previous reports. The use of topical analgesia did not influence COPi and topical analgesia may make the wick technique more acceptable for subjects who dislike technical procedures, including children. Trial Registration ClinicalTrials.gov NCT01044979

Interstitial Fluid Colloid Osmotic Pressure in Healthy Children

Objective The colloid osmotic pressure (COP) of plasma and interstitial fluid play important roles in transvascular fluid exchange. COP values for monitoring fluid balance in healthy and sick children have not been established. This study set out to determine reference values of COP in healthy children. Materials and Methods COP in plasma and interstitial fluid harvested from nylon wicks was measured in 99 healthy children from 2 to 10 years of age. Nylon wicks were implanted subcutaneously in arm and leg while patients were sedated and intubated during a minor surgical procedure. COP was analyzed in a colloid osmometer designed for small fluid samples. Results The mean plasma COP in all children was 25.6 ± 3.3 mmHg. Arbitrary division of children in four different age groups, showed no significant difference in plasma or interstitial fluid COP values for patients less than 8 years, whereas patients of 8-10 years had significant higher COP both in plasma and interstitial fluid. There were no gender difference or correlation between COP in interstitial fluid sampled from arm and leg and no significant effect on interstitial COP of gravity. Prolonged implantation time did not affect interstitial COP. Conclusion Plasma and interstitial COP in healthy children are comparable to adults and COP seems to increase with age in children. Knowledge of the interaction between colloid osmotic forces can be helpful in diseases associated with fluid imbalance and may be crucial in deciding different fluid treatment options. Trial Registration ClinicalTrials.gov NCT01044641

PO-0027 Interstitial Fluid Colloid Osmotic Pressure In Healthy Children

Background and aims Colloid osmotic pressure (COP) of plasma and interstitial fluid play important roles in transvascular fluid exchange were small pressure alterations may result in fluid shifts into or out of the capillaries optimising homeostasis. This study was conducted to determine reference values of COP in healthy children, and to evaluate the methodology of harvesting interstitial fluid. Methods COP in plasma and interstitial fluid isolated from nylon wicks implanted subcutaneously was measured in 99 healthy children from 2 to 10 years of age. Patients were sedated and intubated during wick implantation in arm and leg, and COP was analysed in a colloid osmometer. Results Mean plasma COP in all children was 25.6 ± 3.3 mmHg. Arbitrary division of children in age groups, showed no significant difference in plasma or interstitial fluid COP values for patients less than 8 years, whereas patients of 8–10 years had significant higher COP both in plasma and interstitial fluid. There was no gender difference or correlation between COP in interstitial fluid sampled from arm and leg and no significant effect on interstitial COP of gravity. Prolonged implantation time did not affect interstitial COP. Abstract PO-0027 Figure 1 Colloid osmotic pressure in plasma and interstitium. Colloid osmotic pressure in plasma (p) and interstitium (i) (arm and leg merged) related to age. There was significant difference in pressure beween 2–3 years and 8–10 years for plasma (p < 0.05, *) and between first three age groups and 8–10 years (p < 0.01, **) in interstitial fluid Abstract PO-0027 Figure 2 Colloid osmotic pressure in arm and leg. Colloid osmotic pressure from wicks after implantation in arm vs. leg related to age. There was no significant difference in the pressures obtained in arm and leg Conclusions This study justifies the presumption that plasma and interstitial COP in healthy children are similar to adults. Children between 8 and 10 years had higher values than younger age groups. Knowledge of interaction between colloid osmotic forces in health and disease can be helpful in diseases associated with fluid imbalance and may be crucial in deciding different fluid treatment options.