Vidya Sarma

In vivo suppression of NF-kappa B and preservation of I kappa B alpha by interleukin-10 and interleukin-13.

IL-10 and IL-13 have powerful antiinflammatory activities in vitro and in vivo. In the IgG immune complex model of lung injury in rats, exogenously administered IL-10 or IL-13 have recently been shown to suppress neutrophil recruitment and ensuing lung injury by greatly depressing pulmonary production of TNF alpha. Transcriptional control of the TNF alpha gene is regulated by the nuclear factor kappa B (NF-kappa B). Activation of NF-kappa B involves the degradation of its cytoplasmic inhibitor I kappa B alpha, allowing the nuclear translocation of NF-kappa B, with ensuing transcriptional activation. In this study, we sought to determine whether the protective effects of IL-10 and IL-13 in IgG immune complex-induced lung injury were mediated by inhibition of NF-kappa B activation. Electrophoretic mobility shift analysis of nuclear extracts from alveolar macrophages and whole lung tissues demonstrated that both IL-10 and IL-13 suppressed nuclear localization of NF-kappa B after in vivo deposition of IgG immune complexes. Western blot analysis indicated that these effects were due to preserved protein expression of I kappa B alpha in both alveolar macrophages and whole lungs. Northern blot analysis of lung mRNA showed that, in the presence of IgG immune complexes, IL-10 and IL-13 augmented I kappa B alpha mRNA expression. These findings suggest that in vivo, IL-10 and IL-13 may operate by suppressing NF-kappa B activation through preservation of I kappa B alpha.

Marked synergism between tumor necrosis factor-alpha and interferon-gamma in regulation of keratinocyte-derived adhesion molecules and chemotactic factors.

T lymphocytes and mononuclear cells preferentially accumulate in the epidermis in inflammatory skin disease. To determine the role of keratinocytes in both the chemotaxis and adhesion of these cells to the epidermis, cultured keratinocytes were incubated with IFN-gamma and tumor necrosis factor-alpha (TNF-alpha), and mRNA detected and quantitated for IL-8, monocyte chemotaxis and activating factor, and intercellular adhesion molecule-1. Whereas induction of these mRNAs was either absent, or relatively weak and transient, to either IFN-gamma or TNF-alpha alone, when administered in combination there was a dramatic increase and persistence in the induction of all three genes. Pretreatment of the keratinocytes with cycloheximide failed to eliminate transcription, implying that all three are primary response genes. Transforming growth factor-beta, which modulates other keratinocyte functions (not related to adhesion or chemotaxis of inflammatory cells) failed to induce any of the genes. These novel findings potentially explain the selective recruitment of T cells and monocytes observed in inflammatory skin disease, because IFN-gamma and TNF-alpha can co-ordinately regulate keratinocyte-derived chemoattractants and adhesion molecule production.

Increased C5a receptor expression in sepsis

Excessive production of the complement activation product C5a appears to be harmful during the development of sepsis in rodents. Little is known about the role of the C5a receptor (C5aR) and its presence in different organs during sepsis. Using the cecal ligation/puncture (CLP) model in mice, we show here that C5aR immunoreactivity was strikingly increased in lung, liver, kidney, and heart early in sepsis in both control and neutrophil-depleted mice. C5aR mRNA expression in these organs was also significantly increased during sepsis. Immunohistochemical analysis revealed patterns of increased C5aR expression in parenchymal cells in all four organs following CLP. Mice injected at the start of CLP with a blocking IgG to C5aR (alphaC5aR) showed dramatically improved survival when compared with animals receiving nonspecific IgG, as did mice injected with alphaC5a. In alphaC5aR-treated mice, serum levels of IL-6 and TNF-alpha and bacterial counts in various organs were significantly reduced during CLP when compared with control CLP animals. These studies demonstrate for the first time that C5aR is upregulated in lung, liver, kidney, and heart during the early phases of sepsis and that blockade of C5aR is highly protective from the lethal outcome of sepsis.

Role of C5a in Multiorgan Failure During Sepsis

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O2, rising partial pressure of CO2). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.

Protective effects of anti-C5a in sepsis-induced thymocyte apoptosis

Multiorgan apoptosis occurs during sepsis. Following cecal ligation and puncture (CLP) in rats, thymocytes underwent apoptosis in a time-dependent manner. C5a blockade dramatically reduced thymocyte apoptosis as measured by thymic weight, binding of annexin V to thymocytes, and laddering of thymocyte DNA. When C5a was generated in vivo by infusion of purified cobra venom factor (CVF), thymocyte apoptosis was significantly increased. Similar results were found when CVF was injected in vivo during the early stages of CLP. In animals 12 hours after induction of CLP, there was an increase in the activities of caspase-3, -6, and -9, but not caspase-1 and -8. Cytosolic cytochrome c levels increased by twofold, whereas mitochondrial levels showed a 50% decrease. Western blot analysis revealed that the content of Bcl-X(L) (but not of Bcl-2, BAX, Bad, and Bim) significantly decreased in thymocytes after CLP. C5a blockade in the sepsis model almost completely inhibited caspase-3, -6, and -9 activation, significantly preserved cytochrome c in the mitochondrial fraction, and restored Bcl-X(L) expression. These data suggest that systemic activation of complement induces C5a-dependent apoptosis of thymocytes and that the blockade of C5a during sepsis rescues thymocytes from apoptosis.

Inhibition of NF-κB activation and augmentation of IκBβ by secretory leukocyte protease inhibitor during lung inflammation

In earlier experiments, exogenous administration of secretory leukocyte protease inhibitor (SLPI) suppressed acute lung injury induced by deposition of IgG immune complexes. In the current studies we examined the mechanism of the protective effects of SLPI in this model. The presence of SLPI in the IgG immune complex-model of lung injury reduced the increase in extravascular leakage of 125I-albumin, the intensity of up-regulation of lung vascular intercellular adhesion molecule-1, and the numbers of neutrophils accumulating in the lung. The presence of SLPI caused greatly reduced activation (ie, nuclear translocation) of the transcription nuclear factor-κB (NF-κB) in lung cells but did not suppress activation of lung mitogen-activated protein kinase. SLPI did not alter NF-κB activation in alveolar macrophages harvested 30 minutes after initiation of lung inflammation. In the presence of SLPI, content of tumor necrosis factor-α, CXC chemokines, and C5a in bronchoalveolar fluids was unaffected. In the inflamed lungs, inhibition of NF-κB activation by SLPI was associated with elevated levels of lung IκBβ (but not IκBα) protein in the absence of elevated mRNA for IκBβ. When instilled into normal lung, SLPI also caused similar changes (increases) in lung IκBβ. Finally, in the lung inflammatory model used, the presence of anti-SLPI caused accentuated activation of NF-κB. These data confirm the anti-inflammatory effect of SLPI in lung and point to a mechanism of anti-inflammatory effects of SLPI. SLPI appears to function as an endogenous regulator of lung inflammation.

Role of CC Chemokines (Macrophage Inflammatory Protein-1β, Monocyte Chemoattractant Protein-1, RANTES) in Acute Lung Injury in Rats

The role of the CC chemokines, macrophage inflammatory protein-1β (MIP-1β), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1β, MCP-1, and RANTES were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1β and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response. Treatment of rats with anti-MIP-1β Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-α in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti-rat RANTES had no effect on the development of lung injury. In animals pretreated intratracheally with blocking Abs to MCP-1, RANTES, or MIP-1β, significant reductions in the bronchoalveolar lavage content of these chemokines occurred, suggesting that these Abs had reached their targets. Conversely, exogenously MIP-1β, but not RANTES or MCP-1, caused enhancement of the lung vascular leak. These data indicate that MIP-1β, but not MCP-1 or RANTES, plays an important role in intrapulmonary recruitment of neutrophils and development of lung injury in the model employed. The findings suggest that in chemokine-dependent inflammatory responses in lung CC chemokines do not necessarily demonstrate redundant function.

Regulation by C5a of Neutrophil Activation during Sepsis

In sepsis, there is evidence that excessive C5a generation leads to compromised innate immune functions, being associated with poor outcome. We now report that in vitro exposure of neutrophils to C5a causes increased levels of IkappaBalpha, decreased NF-kappaB-dependent gene transcription of TNFalpha, and decreased lipopolysaccharide (LPS)-induced TNFalpha production. Similar findings were obtained with neutrophils from cecal ligation/puncture (CLP)-induced septic rats. Such changes were reversed by antibody-induced in vivo blockade of C5a. In contrast, in vitro exposure of alveolar macrophages to C5a and LPS resulted in enhanced production of TNFalpha and no increase in IkappaBalpha. These data suggest that CLP-induced sepsis causes a C5a-dependent dysfunction of neutrophils, which is characterized by altered signaling associated with NF-kappaB activation.

Expression and Function of the C5a Receptor in Rat Alveolar Epithelial Cells

Although alveolar epithelial cells (AEC) form an important barrier for host defenses in the lung, there is limited information about ways in which AEC can directly participate in the lung inflammatory response. In the current studies, primary cultures of rat AEC (RAEC) have been shown to specifically bind recombinant rat C5a at high affinity and in a saturable manner. This binding was enhanced in a time-dependent manner by pre-exposure of RAEC to LPS, IL-6, or TNF-α, the increased binding of C5a being associated with increased levels of mRNA for the C5a receptor (C5aR). Exposure of RAEC to C5a also caused increased expression of mRNA for C5aR. As compared with exposure of RAEC to LPS or to C5a alone, exposure to the combination caused enhanced production of TNF-α, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant-1, as well as increased intracellular levels of IL-1β. These data indicate that RAEC, when activated, have enhanced binding of C5a in association with increased mRNA for C5aR. The functional outcome is enhanced release of proinflammatory mediators. These data underscore the phlogistic potential of RAEC and the ability of C5a to enhance the phlogistic responses of RAEC.

Role of IL-18 in Acute Lung Inflammation

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2–4 h in inflamed lungs and remained elevated at 24 h, and the “mature” protein form of IL-18 (18 kDa) appeared in BAL fluids 1–8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-α, IL-1β, and cytokine-induced neutrophil chemoattractant, whereas intratracheal instillation of anti-IL-18 greatly reduced these changes and prevented increases in BAL content of IFN-γ. Intratracheal administration of the natural antagonist of IL-18, IL-18 binding protein, resulted in suppressed lung vascular permeability and decreased BAL content of neutrophils, cytokines, and chemokines. These findings suggest that endogenous IL-18 functions as a proinflammatory cytokine in this model of acute lung inflammation, serving as an autocrine activator to bring about expression of other inflammatory mediators.