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Expression of Interleukin-6 in Association with Rat Lung Reimplantation and Allograft Rejection
Abstract
Organ transplantation has become a therapeutic option for the replacement of malfunctioning tissues and organs. Since the advent of the first combined heart-lung transplant in 1981, there has been a rapid growth in the popularity of lung transplantation for a number of end-stage pulmonary disorders. Interestingly, these lung transplant patients experience more complications of acute and chronic allograft rejection compared with recipients of other solid organs. These episodes of rejection are related to a complex series of events that depend on the interaction of many cells and soluble mediators leading to cellular and tissue injury. The histopathology of lung allograft rejection has been actively studied and is associated with the sequestration of activated mononuclear phagocytes, T and B lymphocytes. These cells secrete a number of soluble mediators, that is, cytokines, that participate in the evolution of the immune response via autocrine, paracrine, or endocrine mechanisms. The interaction of cytokines with their targets leads to cellular activation, proliferation, and differentiation. In this study, we postulated that interleukin-6 (IL-6) may have a central role in the pathogenesis of acute lung allograft rejection. To test this hypothesis, we employed an unmodified RT1-incompatible rat lung allograft model and assessed the time course and major tissue compartment(s) of IL-6 production during the evolution of lung allograft rejection. The expression and production of IL-6 during the pathogenesis of lung allograft rejection was measured at the whole-animal, organ, cellular, and molecular levels. The expression of IL-6 was found to be bimodal in character, initially related to the reimplantation response and finally to the maximal allograft rejection.(ABSTRACT TRUNCATED AT 250 WORDS)
... The allograft/isograft ischemic time was maintained at 120 min and recorded for each recipient animal. The thorax was closed over an 18-gauge chest tube, which was removed before the animal recovered from anesthesia (21)(22)(23)(24)(25). Postoperatively the animals were kept in an oxygenated cage for the first 24 h. ...
... We determined the full kinetics of RANTES in our rat model of acute allograft rejection. LEW rats were subjected to lung transplantation with allografts from BN rats or from syngeneic donors as previously described (21)(22)(23)(24)(25). Animals were sacrificed on days 1, 4, and 6 post-transplantation, and lungs were harvested for isolation of RANTES mRNA by RT-PCR and protein measurement by specific . ...
... To determine whether mononuclear cell infiltration was associated with cells expressing the appropriate receptors for RANTES, we assessed the expression of CCR1 and CCR5 during acute lung allograft rejection. LEW rats were subjected to lung transplantation with allografts from BN rats or from LEW syngeneic donors as previously described (21)(22)(23)(24)(25). Animals were sacrificed on days 1, 4, and 6 post-transplantation, and lungs were harvested for analysis of CCR1 and CCR5 expression by RT-PCR, FACS, and Western blot analysis. ...
Lung transplantation is a therapeutic option for patients with end-stage lung disease. Acute allograft rejection is a major complication of lung transplantation and is characterized by the infiltration of activated mononuclear cells. The specific mechanisms that recruit these leukocytes have not been fully elucidated. The CC chemokine, RANTES, is a potent mononuclear cell chemoattractant. In this study we investigated RANTES involvement during acute lung allograft rejection in humans and in a rat model system. Patients with allograft rejection had a 2.3-fold increase in RANTES in their bronchoalveolar lavages compared with healthy allograft recipients. Rat lung allografts demonstrated a marked time-dependent increase in levels of RANTES compared with syngeneic control lungs. RANTES levels correlated with the temporal recruitment of mononuclear cells and the expression of RANTES receptors CCR1 and CCR5. To determine RANTES involvement in lung allograft rejection, lung allograft recipients were passively immunized with either anti-RANTES or control Abs. In vivo neutralization of RANTES attenuated acute lung allograft rejection and reduced allospecific responsiveness by markedly decreasing mononuclear cell recruitment. These experiments support the idea that RANTES, and the expression of its receptors have an important role in the pathogenesis of acute lung allograft rejection.
... For example, frailty is characterized by increased circulating levels of IL-6 [26], which is associated with disability [27] and decreased muscle mass and strength [27,28]. IL-6 has been implicated in the major complications of lung transplantation, including primary graft dysfunction [29][30][31][32][33][34], acute rejection [32,[35][36][37], infection [37] and bronchiolitis obliterans syndrome [38][39][40]. Interestingly, cytomegalovirus seropositivity in community-dwelling elderly women has also been linked to frailty [41], and higher donor age in kidney, liver and lung transplantation has been linked to higher rates of early [42,43] and late [44][45][46] graft dysfunction. ...
... For example, frailty is characterized by increased circulating levels of IL-6 [26], which is associated with disability [27] and decreased muscle mass and strength [27,28]. IL-6 has been implicated in the major complications of lung transplantation, including primary graft dysfunction [29][30][31][32][33][34], acute rejection [32,[35][36][37], infection [37] and bronchiolitis obliterans syndrome [38][39][40]. Interestingly, cytomegalovirus seropositivity in community-dwelling elderly women has also been linked to frailty [41], and higher donor age in kidney, liver and lung transplantation has been linked to higher rates of early [42,43] and late [44][45][46] graft dysfunction. ...
In 2010, 1770 lung transplant procedures were performed in the USA, yet 2469 new candidates were added to the waiting list the same year. The shortage of suitable donor lungs requires that transplant professionals select patients for lung transplantation only if they are likely to sustain a survival benefit from the procedure. However, 20% of lung transplant recipients die within the first year of transplantation, suggesting that we are failing to identify those at high risk for severe early complications. In this perspective, we review the current guidelines for the selection of lung transplant candidates, which are based largely on expert opinion and small case series. We also propose the study of new extrapulmonary factors, such as frailty and sarcopenia, that might help improve the prediction of complications and early death after lung transplantation, leading to an improved candidate selection process.
... Other studies have shown that high in situ levels of IL-6 and IFN-␥ expression, both independently (35)(36)(37)(38) or in parallel (39,40), are associated with acute rejection of lung allografts. These results have been corroborated in an animal model of LT in rats in which IL-6 expression is associated with acute lung allograft rejection (41). Therefore, the aim of this study was to determine whether a correlation exists between genetic polymorphisms in the TNF-␣, TGF-1, IFN-␥, IL-6, or IL-10 genes and BOS development after LT. ...
BACKGROUND: A number of genetic polymorphisms have been shown to regulate the production and secretion of tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, interferon (IFN)-gamma, interleukin (IL)-6, and IL-10. Several of these genetic polymorphisms have been shown to be associated with either acute or chronic rejection of kidney, liver, and heart allografts and with development of allograft fibrosis after lung transplantation. The aim of this study was to assess the effect of these genetic polymorphisms on the development of bronchiolitis obliterans syndrome (BOS) after lung transplantation.
METHODS: Genetic polymorphisms were detected by means of polymerase chain reaction in 93 lung allograft recipients for functional polymorphisms in the TNF-alpha (-308), TGF-beta1 (+869 and +915), IL-6 (-174), IFN-gamma (+874), and IL-10 (-1082, -819, and -592) genes. Then, a correlation between BOS development and the presence of these cytokine genotypes was determined using Kaplan-Meier actuarial analysis.
RESULTS: A significant correlation was detected between the presence of high-expression polymorphisms of the IL-6 and IFN-gamma genes and BOS development after lung transplantation (P =0.045 and 0.039, respectively). Also, patients with high-expression polymorphisms in both genes developed BOS significantly earlier than patients with low-expression polymorphisms in one or both genes, suggesting a synergistic effect of the alleles during BOS pathogenesis (P =0.016). No correlation was detected between polymorphisms of the TNF-alpha, TGF-beta1, and IL-10 genes and development of BOS after lung transplantation.
CONCLUSIONS: The presence of high-expression polymorphisms at position -174 of the IL-6 gene and position +874 of the IFN-gamma gene significantly increases the risk for BOS development after lung transplantation.
... Interleukin-6 is a pro-inflammatory cytokine related to the acute phase of inflammation which can induce B and T lymphocyte differentiation and activation and generation of cytotoxic T lymphocytes. Several studies have demonstrated a relationship between elevated IL-6 levels and the magnitude of the histologic mononuclear cell infiltration of acute rejection in the transplanted lung (16)(17)(18)(19). In accordance with this hypothesis, we found in a preliminary analysis that changes in IL-6 mRNA levels followed the institution of aerosolized cyclosporine for treatment of both acute and chronic rejection (10). ...
This study evaluated the effectiveness of aerosolized cyclosporine as rescue therapy for refractory acute rejection in lung-transplant patients that is unresponsive to conventional therapy. Over 2 yr, nine allograft recipients with histologic evidence of persistent acute rejection and worsening pulmonary function were enrolled. Twenty-two patients with similar degrees of unremitting rejection served as historical controls. Aerosolization of cyclosporin A (300 mg in 4.8 ml propylene glycol) using an AeroTech II jet nebulizer was instituted daily for 12 consecutive days followed by a maintenance regimen of 3 d/wk. Cyclosporine and tacrolimus blood and plasma levels were maintained within therapeutic ranges throughout this trial. Efficacy was assessed by histologic grade of rejection, interleukin-6 (IL-6) mRNA expression by graft bronchoalveolar lavage cells, and pulmonary function testing before and during cyclosporine therapy. In seven patients, results were correlated to deposition of cyclosporine aerosol in the allograft(s) as measured by radioisotopic techniques. At a mean of 37 d after initiation of aerosolized cyclosporine, graft histology improved in eight of the nine patients. Cellular IL-6 mRNA expression decreased significantly in seven patients (mean IL-6/actin +/- SD, 40.96 +/- 118 versus 0.33 +/- 0.57 [p = 0.038]). Pulmonary function (FEV1), which had decreased posttransplant (over a mean of 347 d of observation) from a best value of 1.98 +/- 0.8 L to 1.59 +/- 0.6 L (p = 0.0077), improved over time (152 d) to a posttransplant value of 1.90 +/- 0.8 (p = 0.025). In the control subjects, FEV1 inexorably declined over a comparable period of observation (best posttransplant value 2.36 +/- 0.86 to 1.32 +/- 0.53, p < 0.0001). There was a strong correlation between cyclosporine deposition in the allograft and improvement in FEV1 (r = 0.900, p < 0.01). Fewer cycles of pulsed corticosteroids (1.4 +/- 0.9 versus 0.2 +/- 0.4, p = 0.011) and anti-thymocyte globulin 0.8 +/- 0.4 versus 0, p = 0.018) and reduced doses of oral prednisone (10.8 +/- 3.1 versus 6.1 +/- 4.2 mg/d, p = 0.026) were observed during treatment with aerosolized cyclosporine. Episodes of pneumonia also were reduced significantly during aerosol therapy (2.6 versus 0.95 episodes/100 d, p = 0.029). Nephrotoxicity and hepatotoxicity did not occur, and no patients withdrew from the study. Aerosolized cyclosporine appears to be safe and effective therapy for refractory acute rejection, but confirmation by a larger, randomized trial is necessary. The correlation observed between deposition of cyclosporine aerosol and physiologic improvement of lung function suggests that there is a dose-response relationship between the concentration of cyclosporine in the allograft and immunologic tolerance.
... Lung allograft reperfusion also results in cytokine release. First reports of elevated levels of BAL IL-2, TNF-α, interferon- gamma and IL-6 after lung allotransplantation in animal models concluded that cytokine production was compartmentalized since no change was detected in the plasma levels (9,10). Later Palace et al (11) demonstrated a transient release of TNF-α in the plasma after the initiation of reperfusion in a rat model. ...
... The aim of the present study was to evaluate the role of IL-6 and eosinophils in fetal porcine ICC xenograft rejection. Earlier studies on allogeneic transplantation have suggested that IL-6 might be of major importance to allograft rejection [12,33,35,36,47,49,52]. Hitherto, the significance of IL-6 in xenogeneic transplantation has not been fully established. ...
Earlier work on primate cardiac xenotransplantation has demonstrated a correlation between interleukin (IL)-6 levels and severity of vascular rejection. IL-6 was originally identified as a lymphokine inducing final maturation of B lymphocytes into antibody-secreting cells. The present study aimed to evaluate the role of IL-6 in fetal porcine islet-like cell cluster (ICC) xenograft rejection. Moreover, other authors have reported that eosinophils dominate the cellular response following discordant islet xenograft transplantation. Here, a technique for specific detection of eosinophils was applied. IL-6-deficient mice and wild-type controls were implanted with fetal porcine ICCs under the kidney capsule and killed 4-, 7-, and 10 days after transplantation. Xenografts were histologically evaluated, and serum samples were analyzed for IgM and IgG antibodies against ICC membrane antigens. IL-6-deficient mice and wild-type controls readily rejected the xenograft. On day 7 after transplantation, abundant numbers of F4/80+ and Mac-1+ cells were found distributed throughout the collapsing graft accompanied by small amounts of eosinophils and peripherally accumulated CD3+ T cells (predominantly CD4+). Significantly lower serum levels of IgM and IgG antibodies against ICC membrane antigens were observed in IL-6-deficient mice on day 4 or 7 after transplantation when compared to wild-type controls. No significant differences were seen on day 10 after transplantation. In both experimental groups, specific IgM and IgG antibody levels remained stable over time. In the pig-to-mouse model, IL-6 seems to be of minor importance to fetal porcine ICC xenograft rejection. Macrophages, and not eosinophils, dominate the cellular response associated with this process.
The role of differential cytology patterns in peripheral blood and bronchoalveolar lavage samples is increasingly investigated as a potential adjunct to diagnose acute and chronic allograft dysfunction after lung transplantation. While these profiles might facilitate the diagnosis of acute cellular rejection, low sensitivity and specificity of these patterns limit direct translation in a clinical setting. In this context, the identification of other biomarkers is needed. This review article gives an overview of cytokine profiles of plasma and bronchoalveolar lavage samples during acute cellular rejection. The value of these cytokines in supporting the diagnosis of acute cellular rejection is discussed. Current findings on the topic are highlighted and experimental settings for future research projects are identified.
Purpose: To study the mechanism of lung ischemia-reperfusion injury and the protective effects of ulinastatin on lung ischemia-reperfusion injury. Methods: Sixty healthy Sprague-Dawley rats were randomly divided into three groups: C group, U group and IR group. Five rats were put to death at one of four time points (45 min ischamia 30, 60 and 120 min reperfusion) in each group after blood was collected from the left carotid and them discarded the left lung. The TNF-α concentration in plasma, the dry/wet (D/W) ratio and the contents of superoxide dismutase (SOD) in lung tissue were determined and lung biopsies were also obtained. Results: (1) The TNF-α concentration in plasma: In IR group TNF-α was increased obviously at the 30 min, 60 min, and 120 min reperfusion and it was significantly higher than that in the C group and U group (P < 0.05). The increase of TNF-α was meaningless in the U group. (2) The contents of SOD in lung tissue: SOD in both the IR group and U group were significantly lower in each same time point after ischemia-reperfusion than that in the C group (P < 0.05), but the decreased level of SOD in the U group was obviously less than that in the IR group (P < 0.05). (3) The D/W ratio of lung tissue: After ischemia-reperfusion, the D/W in the IR group and U group were decreasing progressively and reached the lowest at 120 min after reperfusing (60, 120 min reperfusion vs 45 min ischemia, P < 0.01); the degree for D/W decreasing in the U group was obviously less than that in the IR group (at 60, 120 min reperfusion, the U group vs the IR group, P < 0.05). (4) Histological evaluation: In the process of ischemia-reperfusion the lung injury was aggravating progressively in the IR group; there was marked pulmonary capillary congestion, interstital edema and intraalveolar hemorrhage, infiltration; the electron microscopic section of alveolar showed the type II pneumocyte was damaged and lamellar bodies disappeared. The pulmonary pathologic alterations occurred to a lesser degree in the U group compared with the IR group. Conclusions: (1) The lung ischemia-reperfusion injury may have close relationship with cytokinin released increasingly and unbalancingly between free radicals generating and clearing; (2) Ulinastatin, a protease inhibitor, could decrease cytokinin releasing, inhibit neutrophils aggregating and activated in the lungs and increase free radicals scavenging. It could be used to protect lung ischemia-reperfusion injury.
Background. A number of genetic polymorphisms have been shown to regulate the production and secretion of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, interferon (IFN)-γ, interleukin (IL)-6, and IL-10. Several of these genetic polymorphisms have been shown to be associated with either acute or chronic rejection of kidney, liver, and heart allografts and with development of allograft fibrosis after lung transplantation. The aim of this study was to assess the effect of these genetic polymorphisms on the development of bronchiolitis obliterans syndrome (BOS) after lung transplantation. Methods. Genetic polymorphisms were detected by means of polymerase chain reaction in 93 lung allograft recipients for functional polymorphisms in the TNF-α (−308), TGF-β1 (+869 and +915), IL-6 (−174), IFN-γ (+874), and IL-10 (−1082, −819, and −592) genes. Then, a correlation between BOS development and the presence of these cytokine genotypes was determined using Kaplan-Meier actuarial analysis. Results. A significant correlation was detected between the presence of high-expression polymorphisms of the IL-6 and IFN-γ genes and BOS development after lung transplantation (P =0.045 and 0.039, respectively). Also, patients with high-expression polymorphisms in both genes developed BOS significantly earlier than patients with low-expression polymorphisms in one or both genes, suggesting a synergistic effect of the alleles during BOS pathogenesis (P =0.016). No correlation was detected between polymorphisms of the TNF-α, TGF-β1, and IL-10 genes and development of BOS after lung transplantation. Conclusions. The presence of high-expression polymorphisms at position −174 of the IL-6 gene and position +874 of the IFN-γ gene significantly increases the risk for BOS development after lung transplantation.
Complications after lung transplantation include the development of rejection and an increased incidence of infection, particularly with cytomegalovirus (CMV). Several recent-studies have suggested that interleukin (IL)-6 may be used to detect both infection and rejection after lung transplantation. In addition, IL-6 may play a role in the development of bronchiolitis obliterans after transplantation. Because CMV is also associated with the development of bronchiolitis obliterans after transplantation, we determined whether CMV induces IL-6 gene expression. We demonstrated that CMV infection increased both IL-6 protein and mRNA in peripheral blood mononuclear cells. We also demonstrated that the CMV immediate early 1 gene product increased expression of the IL-6 promoter. This effect of the CMV immediate early 1 gene product was dependent upon the presence of specific transcription factor binding sites in the IL-6 promoter. These studies demonstrate that CMV may be an important cofactor in the development of rejection and infection after transplantation through its effects on IL-6.
Abstract Earlier work on primate cardiac xenotransplantation has demonstrated a correlation between interleukin (IL)-6 levels and severity of vascular rejection. IL-6 was originally identified as a lymphokine inducing final maturation of B lymphocytes into antibody-secreting cells. The present study aimed to evaluate the role of IL-6 in fetal porcine islet-like cell cluster (ICC) xenograft rejection. Moreover, other authors have reported that eosinophils dominate the cellular response following discordant islet xenograft transplantation. Here, a technique for specific detection of eosinophils was applied. IL-6-deficient mice and wild-type controls were implanted with fetal porcine ICCs under the kidney capsule and killed 4-, 7-, and 10 days after transplantation. Xenografts were histologically evaluated, and serum samples were analyzed for IgM and IgG antibodies against ICC membrane antigens. IL-6-deficient mice and wild-type controls readily rejected the xenograft. On day 7 after transplantation, abundant numbers of F4/ 80+ and Mac-1+ cells were found distributed throughout the collapsing graft accompanied by small amounts of eosinophils and peripherally accumulated CD3+ T cells (predominantly CD4+). Significantly lower serum levels of IgM and IgG antibodies against ICC membrane antigens were observed in IL-6-de-ficient mice on day 4 or 7 after transplantation when compared to wild-type controls. No significant differences were seen on day 10 after transplantation. In both experimental groups, specific IgM and IgG antibody levels remained stable over time. In the pig-to-mouse model, IL-6 seems to be of minor importance to fetal porcine ICC xenograft rejection. Macrophages, and not eosinophils, dominate the cellular response associated with this process.
Acute allograft rejection is a major complication postlung transplantation and is the main risk factor for the development of bronchiolitis obliterans syndrome. Acute rejection is characterized by intragraft infiltration of activated mononuclear cells. The ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11) are potent chemoattractants for mononuclear cells and act through their shared receptor, CXCR3. Elevated levels of these chemokines in bronchoalveolar lavage fluid have been associated with human acute lung allograft rejection. This led to the hypothesis that the expression of these chemokines during an allogeneic response promotes the recruitment of mononuclear cells, leading to acute lung allograft rejection. We performed studies in a rat orthotopic lung transplantation model of acute rejection, and demonstrated increased expression of CXCL9 and CXCL10 paralleling the recruitment of mononuclear cells and cells expressing CXCR3 to the allograft. However, CXCL9 levels were 15-fold greater than CXCL10 during maximal rejection. Inhibition of CXCL9 decreased intragraft recruitment of mononuclear cells and cellular expression of CXCR3, resulting in lower acute lung allograft rejection scores. Furthermore, the combination of low dose cyclosporin A with anti-CXCL9 therapy had more profound effects on intragraft leukocyte infiltration and in reducing acute allograft rejection scores. This supports the notion that CXCL9 interaction with cells expressing CXCR3 has an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of acute lung allograft rejection.
Systemic hypotension may complicate the early postoperative period after lung transplantation. A release of proinflammatory cytokines secondary to lung ischemia/reperfusion injury could be involved in the pathogenesis of this early hemodynamic failure (EHF). Study objective: To assess prospectively whether the occurrence of EHF is associated with a release of cytokines in the systemic circulation.
Blood samples were taken daily during the first postoperative week in 26 patients who underwent a double or a single-lung transplantation. These patients were divided into three groups: 7 patients who experienced EHF and subsequently died (EHF group); 15 patients without EHF (control group); and 4 patients without EHF but with an identified sepsis (sepsis group). The serum levels of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 were compared among the three groups.
In the EHF group, the levels of each cytokine peaked at day 1 postoperatively. Cytokine levels at day 1 were significantly higher in the EHF group than in the control group (p<0.0006) or in the sepsis group (p<0.003 except for TNF-alpha).
We conclude that EHF is associated with a massive release of proinflammatory cytokines that could play a determinant role in the pathogenesis of this complication.
No evidence has emerged which suggests that the principles of immunity derived from studies on cells from other body sites are contradicted in the lung and its associated lymphoid tissue. What is clear, however, is that the environment dictates the types of cells, their relationship to one another, and what perturbing events will set in motion either the development of an "active" immune response or tolerance. Investigating mechanisms for the development of lung immunity has increased our understanding of how human diseases develop and is continuing to suggest new ways to manipulate pulmonary immune responses. Demonstration that lung cells regulate both nonspecific inflammation and immunity through the expression of adhesion molecules and the secretion of cytokines offers hope for ways to design more effective vaccines, enhance microbial clearance in immunosuppressed hosts, and to suppress manifestations of immunologically mediated lung disease. Important lung diseases targeted for intensive research efforts in the immediate future are tuberculosis, asthma, and fibrotic lung disease. Perhaps even the common cold might be conquered. Considering the pace of current research on lung immunity, it may not be too ambitious to predict that these diseases may be conquered in the next decade.
The adult mature fetal, but not immature fetal, lung is capable of actively transporting Na+ from the alveolar space. The reason for the impaired Na+ transport in the immature lung is not known; however, the apical membrane Na+ channel is the rate-limiting step for epithelial Na+ transport. This study determined whether transcripts coding for the adult rat colonic epithelial Na+ channel (alpha rENaC) were present in the fetal and adult lung and whether they were developmentally regulated. Similarly sized alpha rENaC transcripts were identified in RNA isolated from fetal and adult whole rat lung, primary cultures of fetal and adult alveolar epithelium, and adult rat whole kidneys, suggesting that the lung alpha rENaC is a similar transcript to that found in the salt-deprived rat colonic epithelium. There were low mRNA levels in 17- to 18-day gestational age (GA) fetal lungs and epithelium (term GA = 22 days), but these levels increased markedly during the saccular stage of lung development (20 days GA) and remained high in adult lungs. The combined administration of thyroid-releasing hormone and dexamethasone to pregnant rats between 16 and 18 days GA induced the expression of lung alpha rENaC in their fetuses. We conclude that alpha rENaC is expressed in mature fetal and adult alveolar epithelium and that it is influenced by hormones known to alter maturation of the fetal lung.
The practice of lung transplantation is constrained by a shortage of suitable donor organs. Furthermore, even "optimal" donor lung grafts are at risk of significant dysfunction perioperatively. Significant insights into the cellular and molecular mechanisms of pulmonary ischemia-reperfusion injury have occurred since the publication of previous reviews on lung preservation 3 to 4 years ago. Recent evidence indicates that the endothelium plays an essential role in regulating the dynamic interaction between pulmonary vasodilatation and vasoconstriction and is a major target during lung injury. In addition, the composition, function, and metabolism of pulmonary surfactant produced by alveolar type II cells are increasingly being recognized as important factors in pulmonary ischemia-reperfusion injury. We hypothesize that reperfusion after a period of pulmonary ischemia results in significant endothelial and alveolar type II cell dysfunction and that an important strategy in lung preservation is to preserve the integrity of these cells in the face of this injury. Given the persistent shortage of lungs available for transplantation, laboratory studies need to focus also on the "rescue" of compromised donor lungs that would have been previously regarded as unsuitable. Importantly, innovative work from the laboratory needs to be translated into clinical practice via prospective, randomized trials to ensure that the prevalence of postoperative lung graft dysfunction is reduced and the shortage of lung grafts for transplantation is alleviated.
After human lung transplantation acute rejection and cytomegalovirus (CMV) infections may occur, probably contributing to the development of chronic rejection. We established a model of subacute allograft rejection in rats to analyze leukocyte activation and effects of a CMV infection. Histoincompatible lung transplants (BN/LEW) without immunosuppression (group A) and lungs of initially immunosuppressed animals (group B) were analyzed. The production of inflammatory mediators (interleukin-6, tumor necrosis factor alpha, nitric oxides) and the expression of MHC class II antigens by alveolar and lung tissue macrophages were significantly enhanced during the alloresponse. In recipients without immunosuppression (group A) allograft necrosis was detected by day 6, whereas group B allografts were fully rejected by day 25. In allografts of immunosuppressed, CMV-infected animals (group C) the CMV infection was clearly aggravated and the number of activated lung tissue macrophages was increased when compared with noninfected allografts or isografts. The subacute model provides the advantage of allowing us to study mechanisms of acute rejection without the effects of reperfusion injury. Furthermore these findings underline the role of inflammatory mediators produced by macrophages during rejection.
The lung transplant is an immunocompetent organ, it induces anomalies of the means of defence and a powerful immunological disturbance. After transplantation a depression of the means of defence is seen, in particular non-immunological with a diminution in certain macrophage functions and the maintenance of a local alloreactive phenomenon. During an acute rejection, the graft is the centre of an accumulation of effector cells of the allograft reaction. The syndrome of bronchiolitis obliterans, associates the presence of these same cells to the increase of immunogenicity of the lung transplant. During infections the observed anomalies are similar to those seen during a rejection, further more a link exists between the pathophysiology of infection and rejection. Overall, lung transplantation induces an immunological deficit as is evident by the frequent increase of rejections and infections.
Oral cyclosporin A used in addition to high-dose intravenous pulse methylprednisolone has been shown to have an adjunctive effect in reversing the rejection of liver and renal transplants. The aim of this prospective study was to evaluate the benefits and risks of this combined drug therapy in acute corneal graft rejection.
Eleven patients with acute corneal graft rejection received the combined regimen of a single pulse of intravenous methylprednisolone (500 mg) and a low dose of oral cyclosporin A (to maintain a trough blood level of 100-200 micrograms/l).
At a mean follow-up of 16.5 months (range 8-22 months) from the presentation of the graft rejection, reversal of graft rejection was achieved in 10 of 11 cases (90.9%). No recurrence of graft rejection was encountered during the study period. One patient developed a duodenal ulcer, which healed after medical treatment. No other complications were encountered.
The high efficacy and low risk of the combined regimen demonstrated in this preliminary study call for a larger-scale prospective double-masked study to confirm the value of this treatment protocol.
Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) has become a crucial tool in the management of lung transplant recipients. Detection of pulmonary infectious pathogens by culture, cytology, and histology of BAL, protected brush specimens, and transbronchial biopsies (TBB) is highly effective. Morphologic and phenotypological analyses of BAL cells may be suggestive for certain complications after lung transplantation. For interpretation of BAL findings, the natural course of BAL cell morphology and phenotypology after lung transplantation must be considered. During the first 3 months after pulmonary transplantation, elevated total cell count in BAL and neutrophilic alveolitis are common, representing the cellular response to graft injury and interaction of immunocompetent cells of donor and recipient origin. With increasing time after transplantation the CD4/CD8 ratio decreases due to lowered percentages of CD4 cells in BAL. During bacterial pneumonias, the cellular profile of BAL is characterized by a marked granulocytic alveolitis. Lymphocytic alveolitis with a decreased CD4/CD8 ratio is suggestive of acute rejection, but is also found in viral pneumonias and obliterative bronchiolitis. In the case of a combined lymphocytosis and neutrophilia without any evidence of infection, obliterative bronchiolitis should be considered. Functional analyses of BAL cells can give additional information about the immunologic status of the graft, even before histologic changes become evident but have not been established in routine transplant monitoring. However, functional studies suggest an important role of activated, alloreactive and donor-specific T lymphocytes in the pathogenesis of acute and chronic lung rejection. Investigations of soluble components in BAL have given further insight into the immunologic processes after lung transplantation. In this overview, the characteristics of BAL after lung transplantation will be summarized, and its relevance for the detection of pulmonary complications will be discussed.
Wallerian degeneration is a post-traumatic process of the peripheral nervous system whereby damaged axons and their surrounding myelin sheaths are phagocytosed by infiltrating leukocytes. Our studies indicate that Schwann cells could initiate the process of Wallerian degeneration by releasing proinflammatory cytokines involved in leukocyte recruitment and differentiation including IL-1beta, MCP-1, IL-8 and IL-6. A comparison of the secretory pattern between nerve explants and cultured Schwann cells showed that each cytokine was differentially regulated by growth factor deprivation or axonal membrane fragments. Since Wallerian-like degeneration occurs in a wide variety of peripheral neuropathies, Schwann cell-mediated cytokine production may play an important role in many disease processes.
Major advancements in the field of lung transplantation have occurred over the past thirty-five years. Despite these advancements, limitations in our ability to obtain sufficient numbers of organs and in our comprehension of the problems associated with the procedure persist. The purpose of this article is to review the current understanding of both the surgical procedure and its most unfortunate complication, bronchiolitis obliterans. Even now, after over three decades of experience, this complication remains the most significant cause of morbidity and mortality following lung transplantation. This article is not meant to be an exhaustive review, and certainly there are important topics not covered herein. We have focused the discussion on ongoing studies, which attempt to understand bronchiolitis obliterans at both the clinical as well as the immunopathological level.
Inflammatory cytokines play an important role in the development of experimental obliterative airway disease (OAD) after transplantation. To further determine the immunologic mechanisms associated with OAD development, we used a murine tracheal transplant model in which a single mismatched HLA-A2-transgenic molecule is indirectly recognized by the recipient CD4+ T cells and then determined whether neutralization of several inflammatory cytokines affected the development of OAD.
Tracheas from HLA-A2+ C57BL/6 mice were heterotopically transplanted into C57BL/6 mice. Recipients were treated with neutralizing antibodies against tumor necrosis factor (TNF), interferon-gamma (IFN-gamma), or interleukin-1 (IL-1). Allograft histology as well as anti-HLA-A2 antibody development and T cell proliferative responses were determined at days +5, +15, +28, and +60.
Allografts in untreated and anti-IFN-gamma-treated recipients demonstrated full development of OAD by day +28. Allografts in anti-TNF-treated recipients showed no evidence of OAD, even at day +60. Allografts in anti-IL-1-treated recipients showed airway epithelium changes by day +28 but minimal evidence of OAD by day +60. Spleen cells from untreated and anti-IFN-gamma-treated recipients showed significantly higher proliferative responses to HLA-A2+ cells, compared with syngeneic recipients (negative controls). In contrast, anti-TNF and anti-IL-1-treated recipients showed significantly lower proliferative responses to HLA-A2+ cells, compared with untreated recipients. Development of anti-HLA-A2 antibodies was detected in all recipients by day +15, with the exception of those treated with anti-TNF.
Among the inflammatory cytokines, TNF seems to play a crucial role in the immunopathology of OAD developed after transplantation.
Cytokines are recognized as critical early mediators of organ injury. We attempted to determine whether or not severe hepatic ischemia/reperfusion injury results in tumor necrosis factor-alpha (TNF-alpha) release with subsequent local and systemic tissue injury. After 90 min of lobar hepatic ischemia, TNF was measurable during the reperfusion period in the plasma of all 14 experimental animals, with levels peaking between 9 and 352 pg/ml. Endotoxin was undetectable in the plasma of these animals. Pulmonary injury, as evidenced by a neutrophilic infiltrate, edema and intra-alveolar hemorrhage developed after hepatic reperfusion. The neutrophilic infiltrate was quantitated using a myeloperoxidase (MPO) assay; this demonstrated a significant increase in MPO after only 1 h of reperfusion. Anti-TNF antiserum pretreatment significantly reduced the pulmonary MPO after hepatic reperfusion. After a 12-h reperfusion period, there was histologic evidence of intra-alveolar hemorrhage and pulmonary edema. Morphometric assessment showed that pretreatment with anti-TNF antiserum was able to completely inhibit the development of pulmonary edema. Liver injury was quantitated by measuring serum glutamic pyruvic transaminase which showed peaks at 3 and 24 h. Anti-TNF antiserum pretreatment was able to significantly reduce both of these peak elevations. These data show that hepatic ischemia/reperfusion results in TNF production, and that this TNF is intimately associated with pulmonary and hepatic injury.
Tumor necrosis factor-alpha (TNF) has been implicated strongly as a principal mediator in the pathogenesis of septic shock. The authors investigated the in vivo production of TNF in CBA/J and CD-1 mice that had been primed by an intraperitoneal injection of complete Freund's adjuvant followed 2 weeks later by an intraperitoneal injection of lipopolysaccharide (LPS). TNF bioactivity peaked in both the ascites and plasma one hour after challenge, and TNF mRNA expression in the ascites cells peaked 30 minutes after LPS. After the induction of bioactivity, an interstitial pulmonary neutrophilic infiltrate occurred that was quantitated both morphometrically and by a myeloperoxidase (MPO) assay. Peripheral blood neutrophilia and lymphopenia developed after the LPS injection (PMNs: control, 46 +/- 2%; LPS, 65 +/- 3%; Lymphs control, 53 +/- 2%; LPS, 37 +/- 3%). Treatment with dexamethasone (Dex) completely inhibited the pulmonary neutrophilic infiltrate as measured by the (MPO) assay. Because Dex will inhibit the production of several cytokines, anti-TNF antiserum was given to mice at the same time as the LPS challenge to assess specifically the role of TNF in inducing these changes. This antiserum partially blocked the pulmonary neutrophil infiltrate, and completely blocked the peripheral blood changes at one hour after LPS. These data demonstrate that TNF plays an important role in the early pathophysiologic alterations that occur after systemic exposure to LPS.
Human B-cell differentiation factor (BCDF) was purified to homogeneity by sequential filtration and chromatography of culture supernatants from TCL-Na1 cells on an AcA34 gel column and then on a Mono P column with fast protein liquid chromatography and reversed-phase HPLC. A 5300-fold enrichment in specific activity of BCDF with about 25% recovery was attained. The homogeneity of purified BCDF was evidenced by the following: (i) the specific activity was 1.7 X 10(7) units/mg of protein, (ii) only two bands, Mr 19,000 and 21,000, were identified by NaDodSO4/PAGE under reduced as well as nonreduced conditions, and (iii) BCDF activity was recovered from the gel after NaDodSO4/PAGE in the fractions corresponding to protein bands of Mr 19,000 or 21,000. Purified BCDF induced Ig secretion in Epstein-Barr virus-transformed cell lines; as little as 3 pM gave 50% of the maximum reaction achieved by 30-80 pM BCDF. Purified BCDF induced Ig production in activated B cells without any effect on cell growth. Purified BCDF did not show any activity of interleukin 1 or 2, B-cell stimulatory factor (BSF)p-1, B-cell growth factor II (BCGF-II), or interferon. Since BCDF was isolated and characterized as described, we propose that the BCDF that induces the final differentiation of B cells into high-rate Ig-secreting cells be designated BSFp-2.
One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response.
Rat peritoneal exudate cells produce two interleukin 6 (IL6) messenger RNA species, a major 1200 nucleotide and a 5-fold less abundant, 2400-nucleotide species. A cDNA clone representing the major species was isolated, and sequenced. The 1055-base pair insert covered the 3′-nontranslated region, the 211 triplet coding region and most of the 5′-nontranslated region. The derived rat IL6 amino acid sequence was 93 and 58% identical, respectively, with mature murine and human IL6. Rat IL6 lacks N-glycosylation sites but contains a fifth cysteinyl residue in addition to the 4 residues shared in conserved positions with murine and human IL6. Stable murine L cell and human HeLa-derived cell lines were established by cotransfection with rat IL6 cDNA and a selectable neomycin resistance marker. These lines secrete 9-fold increased amounts of functional IL6 over their respective parental cells. A stable rat macrophage-derived cell line, RM-SV1, was established by transformation with simian virus 40. IL6 and Il1 mRNA levels are inducible 20- and 3.5-fold, respectively, in this line by treatment with lipopolysaccharide with kinetics characteristic of macrophages.
A set of three overlapping genomic DNA clones was isolated and a 10-kilobase DNA segment was sequenced containing the rat IL6 gene plus 2.9 kilobases of 5′-flanking and 1.3 kilobases of 3′-flanking sequences. The two transcription start sites used in RM-SV1 cells were mapped within 5 base pairs of each other. The exon/intron boundaries are conserved with the murine and human IL6 genes. The two IL6 mRNA species are generated by alternative polyadenylation at sites separated by a distance of 1.2 kilobases. The intervening region contains a repetitive element 72–80% identical with the rat and murine consensus L1 family sequences.
Combined heart and lung transplantation was used to treat seven patients with end stage lung disease. All were severely disabled, and their disease carried a poor prognosis. Six patients were well four to 33 months after transplantation. One patient died after 44 days from a primary cytomegalovirus pneumonia transmitted from the donor. All the survivors had normal exercise tolerance and greatly improved lung function. It is concluded that heart and lung transplantation is a suitable treatment for selected patients with end stage chronic lung disease.
Growth and differentiation of thymocytes and mature T lymphocytes is regulated by cellular interactions that are in part mediated by soluble factors. We identify IL-6, formerly called B cell stimulating factor (BSF-2). IFN-beta 2, or hybridoma-plasmacytoma growth factor (HPGF) as a novel T cell costimulant rIL-6 induced a six-to seven-fold increase in proliferation of human thymocytes stimulated with suboptimal doses of PHA. A similar effect with added IL-6 could be observed using peripheral blood T lymphocytes, but only if the cultures were first rigorously depleted of monocytes that release high levels of IL-6. Analysis of the mechanism of the IL-6 effect on thymocytes and T lymphocytes showed that IL-6 did not lead to an increase in IL-2-R expression. Concentrations of antibody to IL-2-R inhibiting IL-2 effects did not block the IL-6-induced proliferation, indicating that the IL-6 effect was relatively IL-2 independent. These results identify IL-6 as a novel costimulant of human thymocytes and mature T lymphocytes, and suggest that IL-6 is also an important regulatory of cellular immunity.
Two mRNA species that produce biologically active interferon were isolated from human fibroblasts and studied by size fractionation and cloning in Escherichia coli plasmid pBR322. The major fibroblast interferon (Hu IFN-beta 1) is coded for by the smaller of the two mRNAs, an 11S species, 900 nucleotides long, which in cell-free systems yields a 20,000 Mr protein. The second interferon mRNA species (Hu IFN-beta 2) is 14S, about 1300 nucleotides long, and codes for another protein of 23,000-26,000 Mr. The two interferon mRNAs do not cross-hybridize. Both are induced by poly(rI.rC), but IFN-beta 2 mRNA is induced to about 10% in cells by cycloheximide treatment alone whereas under these conditions IFN-beta 1 is not induced.
Transplantation for end-stage lung disease remains in its infancy, but recent successes suggest that transplantation for end-stage interstitial lung disease can achieve the same degree of success demonstrated with transplantation of other organs. Many problems remain to be solved. These include improved means of immunosuppression, the ability to diagnose rejection accurately, and the need to secure more transplantable lungs from the available donor population. This will require improved means for donor maintenance (to avoid deterioration of the lung before and after declaration of brain death) and the ability to preserve lungs for 12 hours or more to allow transportation of suitable lungs from greater distances.
At the present time, single lung transplantation is the procedure of choice for patients with pulmonary fibrosis. Double lung transplantation is indicated for patients with emphysema or cystic fibrosis when right ventricular function is preserved, and combined heart-lung transplantation is indicated for individuals who have a combination of pulmonary hypertension and irreversible right heart failure. Single or double lung transplantation may prove useful in patients with severe pulmonary hypertension prior to the occurrence of irreversible right heart failure and tricuspid regurgitation.
Success with lung transplantation has been achieved by virtue of the experimental and clinical contributions of numerous investigators over a 40-year period. The demonstration of clinical success with lung transplantation should provide additional stimulus for rapid advances in the near future.
The function of transplanted lungs may be critically impaired in the early postoperative period by the reimplantation response. Several factors of the transplantation procedure, such as disruption of hilar structures (hilar stripping), stenotic anastomoses, and graft ischemia, are considered to cause this reimplantation response. In this study the individual contributions of these factors have been analyzed in rats, after isogeneic transplantation or hilar stripping of left lungs. Marck’s technique for orthotopic transplantation of the left lung in rats was refined so that an 85% postoperative survival rate was achieved. Transplanted and hilar-stripped lungs were investigated by lung perfusion scintigraphy and chest roentgenography at regular intervals up to 168 days after operation. Macroscopic and histologic morphology was examined at corresponding intervals. Our results show that perfusion and ventilation of lung grafts are independently affected by distinct factors of the transplantation procedure. Hilar stripping did decrease graft perfusion transiently. Permanent decrease of perfusion was found to be caused by stenosis of the anastomosed pulmonary artery. Hilar stripping also unpaired ventilation, by causing interstitial and alveolar edema. After transplantation, edema and consequent impairment of ventilation were aggravated by graft ischemia, proportionally to its duration. Our improved technique for transplantation of left lungs in rats provides a new opportunity for investigating the immunologic problems of lung transplantation.
Improvements in immunosuppression and surgical techniques have made unilateral lung transplantation feasible in selected patients with end-stage interstitial lung disease. We report two cases of successful unilateral lung transplantation for end-stage respiratory failure due to pulmonary fibrosis. The patients, both oxygen-dependent, had progressive disease refractory to all treatment, with an anticipated life expectancy of less than one year on the basis of the rate of progression of the disease. Both patients were discharged six weeks after transplantation and returned to normal life. They are alive and well at 26 months and 14 months after the procedure. Pulmonary-function studies have shown substantial improvement in their lung volumes and diffusing capacities. For both patients, arterial oxygen tension is now normal and there is no arterial oxygen desaturation with exercise. This experience shows that unilateral lung transplantation, for selected patients with end-stage interstitial lung disease, provides a good functional result. Moreover, it avoids the necessity for cardiac transplantation, as required by the combined heart-lung procedure, and permits the use of the donor heart for another recipient.
To find out to what extent rejection of lungs differs from that of other organs, functional rejection of lung allografts was studied in five combinations of inbred rat strains. Rejection could be monitored accurately by perfusion scintigraphy, and equally well by chest roentgenography. The rejection of lung grafts was found to proceed remarkably fast, when compared with heart grafts, in combinations with strong RT1-incompatibilities. This accelerated rejection pattern could be converted into rejection at a normal pace by pretreatment of the donor with 10 Gy roentgen irradiation one day before transplantation. Donor pretreatment depleted the lung graft's bronchus-associated lymphoid tissue (BALT) of lymphocytes. When grafts were depleted of all other passenger cells as well-by retransplantation from a cyclosporine-treated intermediate host-they showed an even more reduced immunogenicity, probably because of the loss of donor-type dendritic cells. These results indicate that lymphocytes from the BALT of lung grafts are capable of accelerating the rejection response.
Single lung transplantation (SLT) has been considered physiologically inappropriate for patients with chronic ohstructive pulmonary disease (COPD). It has been postulated that the high static compliance and elevated pulmonary vascular resistance of the native lung functioning in parallel with the more normal allografted lung could cause unacceptable ventilation-perfusion mismatching and/or overinfiation of the native lung with encroachment on the expansion of the transplanted lung. While some degree of ventilation-perfusion imbalance may be physiologically obligatory after SLT for COPD, a significant disruption in gas exchange may not occur unless a complication, such as rejection or infection, arises in the transplanted lung. A 60year-old man with COPD who underwent successful SLT is presented and discussed. In spite of scintigraphic evidence of ventilation-perfusion mismatching between the native lung and the allograft during the first six postoperative weeks, the recipient had normal resting gas exchange on room air after the second postoperative week. Fourteen weeks after transplantation, his maximum oxygen uptake was 37.3 percent of the predicted maximal value, and no evidence of ventilatory limitation was detected. His functional status and lifestyle have been markedly improved by SLT. The role of SLT for COPD should be reconsidered. It may be a reasonable transplantation alternative for selected patients with COPD who are not candidates for double lung transplantation (DLT). (Chest 1989; 96:738-42)
Growth and differentiation of thymocytes and mature T lymphocytes is regulated by cellular interactions that are in part mediated by soluble factors. We identify IL-6, formerly called B cell stimulating factor (BSF-2). IFN-beta 2, or hybridoma-plasmacytoma growth factor (HPGF) as a novel T cell costimulant rIL-6 induced a six-to seven-fold increase in proliferation of human thymocytes stimulated with suboptimal doses of PHA. A similar effect with added IL-6 could be observed using peripheral blood T lymphocytes, but only if the cultures were first rigorously depleted of monocytes that release high levels of IL-6. Analysis of the mechanism of the IL-6 effect on thymocytes and T lymphocytes showed that IL-6 did not lead to an increase in IL-2-R expression. Concentrations of antibody to IL-2-R inhibiting IL-2 effects did not block the IL-6-induced proliferation, indicating that the IL-6 effect was relatively IL-2 independent. These results identify IL-6 as a novel costimulant of human thymocytes and mature T lymphocytes, and suggest that IL-6 is also an important regulatory of cellular immunity.
Retinal and choroidal inflammatory lesions are important causes of visual loss, but the mechanisms regulating intraocular inflammation remain poorly understood. By virtue of its position at the blood-retina barrier, the retinal pigment epithelium (RPE) cells may be critical to the initiation and propagation of ocular inflammation. Previously we showed that cytokine-stimulated RPE cells produce interleukin-8, a well-defined chemotactic factor for neutrophils and lymphocytes. In this study, we found that human RPE cells stimulated by human recombinant interleukin-1-beta (rIL-1 beta) or tumor necrosis factor-alpha (rTNF-alpha) produce interleukin-6 (IL-6). Using a plasmacytoma proliferation assay, significant levels of IL-6 were found in media of RPE cells stimulated with either rIL-1 beta or rTNF-alpha for 4 hr. Progressive accumulation of IL-6 in media overlying stimulated RPE cells occurred over the subsequent 20 hr. IL-1 beta was a significantly more potent stimulator of RPE IL-6 production than TNF-alpha, RPE IL-6 production in response to each of these cytokines was also dose-dependent over a range of 20 pg to 20 ng ml-1. Specific anti IL-6 antibody, but not control immunoglobulin, neutralized RPE-derived IL-6 activity in the plasmacytoma proliferation assays. RPE IL-6 mRNA levels were detectable 1 hr after cytokine stimulation, plateaued within 8 hr in 24-hr assays, and demonstrated dose-dependent kinetics in 6 hr assays. Lipopolysaccharide failed to induce RPE IL-6 mRNA expression or RPE IL-6 production.(ABSTRACT TRUNCATED AT 250 WORDS)
This article covers the basic biological functions of interleukin-6 (IL-6) in man. Three major topics are addressed more closely: the involvement of IL-6 in various disease states, particularly those of hematopoietic origin; the diagnostic usefulness of IL-6 measurements in biological fluids; the possible use of IL-6, IL-6 antagonists or IL-6 derivatives as therapeutic means.
The expression of the interleukin 6, tumor necrosis factor alpha, and interferon gamma (IFN-gamma) genes was studied in human renal biopsies from individuals without evidence of kidney disease and from patients undergoing acute renal allograft rejection using a method of in situ hybridization capable of detecting 1-5 copies of a specific cellular messenger RNA in individual cells. IL-6, TNF-alpha, and IFN-gamma RNA transcripts were not detected in any of the sections of normal human kidneys. Elevated levels of IL-6 mRNA but not IFN-gamma were, however, detected in the sections of the renal biopsies from six of eight patients exhibiting acute rejection. A uniform level of expression of IL-6 mRNA was observed in all the cells examined, including glomerular cells, tubular epithelia, smooth muscle cells, and vascular endothelia, as well as the interstitial mononuclear infiltrate. Juxtatabular clusters of TNF-alpha mRNA were detected in the absence of IL-6 mRNA in one patient exhibiting acute rejection. Only a small number of grains (1-5 per high-power field) was detected in the urinary space or in the tubular or vascular lumen following hybridization with the IL-6 or TNF-alpha probes. In contrast, in kidney transplant patients with stable renal function no significant labeling was observed with the IL-6, TNF-alpha, or IFN-gamma probes. A similar level of expression of actin mRNA was observed in all the sections of normal and transplanted kidneys studied, suggesting that the overall level of RNA synthesis was similar in both groups. These results suggest that cytokines such as IL-6 play a role in acute allograft rejection.
Current evidence suggests that the development of allosensitized cytotoxic T lymphocytes within sponge matrix allografts takes place primarily in situ and may be regulated by the secretory products of the cells infiltrating the graft. In vitro studies have implicated IL-2, IL-4, and IL-6 in CTL development. We have reported that TNF-alpha, macrophage colony-stimulating factor, IL-1, IFN-alpha, and IFN-beta are present in the allograft, but that IL-2 and IL-4 cannot be detected at any time using specific bioassays. In this study, we found significantly higher levels of IL-6 within the allografts compared with the syngeneic grafts. Peak IL-6 activity coincided with the appearance of allosensitized CTL in the allografts. IL-6 concentration in the serum of sponge allografted mice was less than 1% of that found in the graft. The sponge fluid exhibited both hybridoma growth factor and hepatocyte-stimulating factor activities in vitro, and both these activities were neutralized by antibody to murine IL-6 but not by antibody to murine IL-1-beta or TNF-alpha. Messenger RNA for murine IL-6 was detected in the graft-infiltrating cells. The high level of IL-6 found in the allograft coincident with the appearance of cellular immunity suggests that this cytokine might play some role in the development of allospecific CTL in vivo.
Interleukin-6 (IL-6) modulates a number of processes relevant to host immunity and inflammation. We investigated the capacity of the human alveolar macrophage to elaborate IL-6 in response to lipopolysaccharide (LPS), recombinant interleukin-1 (rIL-1), and recombinant tumor necrosis factor (rTNF), and compared macrophage IL-6 production to that of blood monocytes and lung fibroblasts. Unstimulated and TNF-stimulated alveolar macrophages and monocytes produced little or no detectable IL-6. In contrast, macrophages and monocytes produced large amounts of IL-6 in response to LPS and monocytes produced lesser but readily detectable amounts in response to rIL-1. Monocytes and alveolar macrophages differed significantly in their capacity to produce IL-6, with macrophages making more IL-6 in response to LPS and less IL-6 in response to rIL-1 than autologous blood monocytes. Monocytes aged in vitro produced little detectable IL-6 in response to LPS or rIL-1, suggesting that differences in cell maturity may account for the diminished capacity of the alveolar macrophage to produce IL-6 in response to IL-1 but not its enhanced capacity to produce IL-6 in response to LPS. Mononuclear phagocytes and lung fibroblasts also differed in their ability to produce IL-6. Lung fibroblasts produced more IL-6 in response to rIL-1 and less IL-6 in response to LPS than monocytes and macrophages. In addition, monocytes and macrophages elaborated electrophoretically identical IL-6 moieties that differed from those produced by lung fibroblasts. These differences could be at least partially attributed to differences in sialylation and/or glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Between November 1983 and August 1989, we performed single-lung transplantation for end-stage pulmonary fibrosis in 20 patients. Nine patients (45 percent) who survived for more than one year form the basis of this report. Before surgery, the nine survivors had severe restrictive lung disease, with a mean (+/- SD) vital capacity (VC) of 43 +/- 9 percent, a forced expiratory volume in one second (FEV1) of 50 +/- 9 percent, and a single-breath diffusing capacity (DLCO) of 36 +/- 9 percent of predicted values. One year after transplantation, the patients' VC had reached 69 +/- 10 percent, FEV1 79 +/- 15 percent, and DLCO 62 +/- 16 percent of predicted values. Relative perfusion to the transplanted lung rose from 63 +/- 14 percent (three days after surgery) to 77 +/- 7 percent within three months and stayed constant or increased slightly thereafter. Before surgery, despite supplemental oxygen at flow rates varying from 1 to 9 liters per minute, none of the patients could exercise beyond stage 1/2 (2.7 km [1.7 miles] per hour, 5 percent grade) on a modified Bruce treadmill-exercise protocol. All eight patients tested one year or more after transplantation achieved at least stage 1 (2.7 km [1.7 miles] per hour, 10 percent grade), and usually a higher stage, without supplemental oxygen. Arterial oxygen tension returned to normal values in most patients (87 +/- 13 mm Hg), and supplemental oxygen, which all patients required before surgery, was no longer needed by any patient after transplantation. We conclude that in carefully selected patients with end-stage pulmonary fibrosis, single-lung transplantation is an effective treatment.
Chronic rejection of the lung in patients with heart-lung transplants has most often been associated with the development of obliterative bronchiolitis. Previously only one patient receiving a single-lung transplant suffered from the development of this problem. We describe a patient whose obliterative bronchiolitis developed 9 months after single-lung transplantation. Progressive deterioration occurred until his death from obliterative bronchiolitis at 21 months after transplantation. The functional and histologic changes are described and the possible mechanisms discussed.
We characterized the effects of rIL-1 and rTNF on human lung fibroblast IL-6 production. rIL-1 was a potent stimulator, rTNF only marginally stimulated, and rIL-1 and rTNF in combination synergistically stimulated IL-6 protein production. These changes were associated with proportionate alterations in IL-6 mRNA accumulation. Nuclear run-on analysis demonstrated that the effects of rIL-1 and rTNF individually were associated with increased IL-6 gene transcription. In contrast, alterations in gene transcription could not fully explain the synergistic effects of rIL-1 and rTNF in combination. However, IL-6 mRNA was significantly more stable in cells stimulated with rIL-1 plus rTNF than in cells stimulated with rIL-1 alone. Thus the synergistic effects of rIL-1 and rTNF in combination were mediated, at least partially, by an alteration in the stability of IL-6 mRNA. Alterations in mRNA stability may be an important mechanism of cytokine-cytokine synergy.
Most cytokines involved in the regulation of the immune responses and hematopoiesis have been molecularly cloned. The studies with recombinant molecules clearly demonstrate that the function of these cytokines is not specific to a certain lineage of cells as originally expected but they show a wide variety of biological functions on various tissues and cells. One of the most typical examples of these multifunctional cytokines is IL-6. As described, it regulates immune responses, hematopoiesis, and acute phase reactions, indicating that it plays a central role in host defense mechanism. Among many cytokines, IL-6 is the first one, the abnormal expression of which is directly related to the pathogenesis of several diseases, such as myeloma/plasmacytoma, Castleman's disease, and mesangium proliferative glomerulonephritis, in which IL-6 functions as an autocrine growth factor for kidney mesangium cells. Therefore, the study on the regulatory mechanism of the IL-6 gene expression is indispensable for unraveling the molecular pathogenesis of those diseases. Neutralization of IL-6 with specific inhibitors may be applied for the treatment of such diseases. Soluble receptors are possible candidates as the specific inhibitor. The signal transduction through cytokine receptors may be unique: (a) the number of receptors is approximately 100-fold less than that of hormone or growth factor receptors and (b) any known biochemical reactions, such as phosphatidyl inositol turnover, tyrosine phosphorylation, and Ca++-ion influx, are not invoked following stimulation with cytokines. Recently, cDNAs for cytokine receptors, such as IL-6, IL-1 and γ-IFN have been cloned. The receptor molecules do not have any unique structure for the signal transduction, such as tyrosine kinase domain. Therefore, the presence of associated molecules for the signal transduction is assumed. In fact, IL-6 stimulation triggers the association of the IL-6 receptor with a nonligand binding signal transducer. The unique mechanism of signal transduction through cytokine receptors will hopefully be elucidated in the near future.
Human tumor necrosis factor-alpha (TNF), a mononuclear phagocyte (MO)-derived peptide, is increasingly being recognized for its pleomorphic immunologic effects. A number of studies have demonstrated that LPS can induce TNF synthesis, but data examining the production and regulation of TNF in human MO populations are lacking. In this study, we present data demonstrating that alveolar macrophages (AMO) and peripheral blood monocytes (PBM) obtained from 10 normal volunteers display a significant difference in both the production of TNF and their susceptibility to TNF regulation by prostaglandin E2 (PGE2) and dexamethasone (Dex). Adherent populations of PBM and AMO were incubated for 18 h in the presence of either LPS (10 micrograms/ml) alone, PGE2 for 1 h prior to LPS challenge, Dex for 1 h prior to LPS challenge, or control media alone. Cell-free supernatants were examined for TNF bioactivity and cellular TNF mRNA was assessed via in situ hybridization and Northern blot analysis. PGE2 and Dex treatment of PBM suppressed LPS-induced TNF production by 78% and 72%, respectively, while AMO-TNF production was suppressed by only 22% and 33%. The accumulation of TNF mRNA in PBM was reduced 63% by PGE2 and 45% by Dex, as assessed by laser densitometry. Similar studies demonstrated that TNF mRNA accumulation in AMO was reduced 12% and 13% by PGE2 and Dex, respectively. A 1,000-fold increase in PGE2 levels was necessary to induce 50% suppression of the maximal response to AMO as compared to PBM. These data support the notion that human MO derived from different compartments or stages of differentiation exhibit differential responsiveness to immunomodulators.
We have achieved repeated success with unilateral lung transplantation for pulmonary fibrosis and have developed an en bloc, double-lung transplant procedure for patients with advanced lung disease of an obstructive or infective nature. Six such procedures have now been performed for end-stage emphysema, and all recipients are alive and well 5 to 15 months later. A seventh transplant for primary pulmonary hypertension was unsuccessful. All recipients were judged to have a life expectancy of 12 to 18 months on the basis of the degree of disability and the documented rate of disease progression. We feel the double-lung procedure is more appropriate than the combined heart-lung transplant for patients requiring replacement of both lungs when right heart function is adequate or deemed recoverable. With this procedure, the recipient is able to retain his or her own heart, avoiding the liabilities associated with cardiac transplantation. Furthermore, the donor heart is available for a separate recipient, and this sharing of the heart and lungs greatly increases the supply of transplantable lungs for patients with end-stage lung disease. Ischemia of the donor airway has been a source of complication, including the one death to date, but this appears to be a surmountable problem.
Tumor necrosis factor-alpha (TNF) has been implicated as an important, proximal mediator of many of the pathophysiologic effects observed during septic shock. In vitro studies have demonstrated that the glucocorticoid dexamethasone (Dex) will suppress the production of TNF; yet, clinical studies have shown that glucocorticoids are not protective in septic shock. In this paper we described the in vivo effects of lipopolysaccharide (LPS) on the kinetics of local and systemic TNF production, the time dependent expression of TNF mRNA, and the suppression of both TNF mRNA and bioactive protein using a defined treatment protocol of Dex. Peritoneal macrophages were elicited by CBA/J mice in the injection of complete Freunds adjuvant and the mice challenged with an intraperitoneal injection of LPS 2 weeks later. Kinetic studies showed that the peak of TNF production occurred 1 hour post LPS injection and reached a maximum of 775 units/ml within the ascites and 26 units/ml within the plasma. Northern blot analysis of mRNA extracted from peritoneal cells showed a peak of mRNA 30 minutes post LPS challenge. Dose-response studies disclosed that 10 micrograms of LPS/mouse produced maximal TNF within the ascites fluid, and half-maximal stimulation occurred at 70 ng LPS/mouse. Mice treated with Dex in vivo before LPS challenge showed a dramatic reduction in TNF production within both the ascites and plasma, and Northern blot analysis showed a corresponding reduction in the TNF specific mRNA. Further studies revealed that mice treated with 4 mg/kg of Dex intraperitoneally 4 hours before, or at the time of LPS challenge, had dramatic reductions in TNF levels within both the ascites and plasma. However, delaying the treatment only 20 minutes after LPS injection failed to significantly reduce TNF in either compartment. These data may provide a rationale why glucocorticoids are not clinically efficacious in the treatment of septic shock, since there is rapid upregulation of LPS-induced TNF gene expression. By the time patients develop clinical signs and symptoms of septic shock there are already preformed, circulating levels of TNF.
The interaction between human endothelial cells and leukocytes during immunologic and inflammatory responses is in part mediated through the release of soluble mediators. We report that cultured human umbilical vein endothelial cells secrete IL-6 when stimulated with LPS. This effect was inhibited by polymyxin-B. The monokines IL-1 and TNF-alpha were also potent inducers of IL-6, whereas lymphotoxin was only effective at much higher concentrations. Endothelial cell supernatant IL-6 was active as hybridoma-plasmacytoma growth factor and as B-cell stimulating factor. Endothelial IL-6 activity was neutralized by a specific anti-IL-6 antibody and by immunoprecipitation it was shown to be identical in size to human fibroblast-derived IL-6. As IL-6 is possibly an important regulator of host defense responses, production of this cytokine by endothelial cells may contribute to the pathogenesis of various inflammatory and immunologic diseases.
We have investigated changes in serum interleukin 6 (IL-6) in patients undergoing elective cholecystectomy. Serum IL-6 increased in all patients within 1.5 hour of incision, reaching a maximum between 1.5-4 hours after incision (median 50 U/ml; range 22-79 U/ml). The maximum serum IL-6 correlated with the length of the operation (r = 0.95). Serum C-reactive protein was not detectable until 8-12 hours post-incision, but maximum serum C-reactive protein did not correlate with maximum serum IL-6 concentration or length of operation. There was no consistent increase in plasma interleukin 1 or tumour necrosis factor following surgery. Serum IL-6 is an early marker of tissue damage and may be of value in the study of the metabolic response to injury.
Recombinant B cell stimulatory factor 2 (rBSF-2) did not display any detectable level of antiviral activity when using human diploid fibroblasts, DIP-2, FS-4, FS-7, amnion-derived WISH and FL cells with vesicular stomatitis virus (VSV) and Sindbis virus as challenging agents (less than 2.5 X 10(2) IU/mg). Furthermore anti-IFN-beta could not neutralize the immunoglobulin-inducing activity of BSF-2. Moreover anti-BSF-2 could not neutralize the antiviral activity of IFN-beta. The data indicate that BSF-2 is functionally and immunologically not related to IFNs.
Hybridoma growth factor (HGF) is a 20-25 kD protein, supporting the growth of hybridoma cells in vitro and capable of replacing feeder cells. It was shown to be produced by human monocytes and a number of cultured cell lines. Recently, HGF was found to be identical to interferon-beta 2 or 26 kD protein and BSF-2, and was renamed interleukin 6 (IL-6). Using a sensitive bio-assay we were able to measure IL-6 activity in the serum and urine of healthy volunteers and renal transplant recipients. Low levels of IL-6 were present in the serum but not in the urine of healthy individuals. In contrast, both serum and urine of renal transplant recipients contained high levels of IL-6 directly after transplantation and during acute rejection episodes. On the basis of kinetic studies of the IL-6 response, it is concluded that serial measurement of IL-6, especially in urine, may be of value in monitoring renal transplant recipients. Moreover, the sensitivity of the bioassay will allow for detailed studies as to the biological significance of IL-6 in health and disease.
Rejection of RT1-incompatible lung grafts has been found in the study reported in our accompanying article to result in four consecutive morphological rejection phases: the latent, the vascular, the alveolar, and the destruction phase. The most prominent signs of rejection, however, occur early in the vascular phase in the bronchus-associated lymphoid tissue (BALT) of these grafts. In this study we investigated whether these four phases and the early rejection signs in BALT are universal phenomena of lung allograft rejection. Therefore, various donor-recipient combinations of inbred rat strains, incompatible for the MHC or for minor loci, were compared with respect to histological rejection phenomena--both in the lung graft and in the recipient's spleen--and alloantibody formation. The four rejection phases appeared sequentially in grafts of all combinations. Duration of the phases depended on the degree of histoincompatibility of the graft. Again, BALT was involved early in the rejection process. During the vascular phase a strong immune response developed in the spleen, and in the alveolar phase antibodies circulated in the blood. We conclude that these morphological rejection phases are universal phenomena of the rejection process against lung allografts in rats. Corresponding phenomena have been described for other species, even in immunosuppressed recipients. Based on these data, a new concept of the universal rejection process of lung allografts is postulated.
The immunological mechanism of lung allograft rejection was studied in inbred rats, in order to explain the rapid progress of the rejection response against RT1-incompatible lung grafts. Histological appearances of the graft and of the recipient's spleen were studied, migration patterns of graft and recipient lymphocytes were assessed, and titers of circulating alloantibodies were determined. Histologically, we discriminated four phases of the rejection response in lung grafts: sequentially the latent, vascular, alveolar, and destruction phases. Early in the vascular phase, recipient lymphocytes primarily infiltrated the bronchus-associated lymphoid tissue (BALT) of the graft, causing a local immune response. Concurrent with these local rejection phenomena in the graft, a strong systemic immune response developed in the recipient's spleen, presumably induced by the great number of lymphocytes that migrated from the graft's BALT into the recipient's lymphoid tissues. We conclude that BALT facilitates a fast and intensive interaction between lung graft and recipient that is likely to accelerate the induction of the rejection response both locally in the graft and systemically in the recipient's lymphoid organs.
The function of transplanted lungs may be critically impaired in the early postoperative period by the reimplantation response. Several factors of the transplantation procedure, such as disruption of hilar structures (hilar stripping), stenotic anastomoses, and graft ischemia, are considered to cause this reimplantation response. In this study the individual contributions of these factors have been analyzed in rats, after isogeneic transplantation or hilar stripping of left lungs. Marck's technique for orthotopic transplantation of the left lung in rats was refined so that an 85% postoperative survival rate was achieved. Transplanted and hilar-stripped lungs were investigated by lung perfusion scintigraphy and chest roentgenography at regular intervals up to 168 days after operation. Macroscopic and histologic morphology was examined at corresponding intervals. Our results show that perfusion and ventilation of lung grafts are independently affected by distinct factors of the transplantation procedure. Hilar stripping did decrease graft perfusion transiently. Permanent decrease of perfusion was found to be caused by stenosis of the anastomosed pulmonary artery. Hilar stripping also impaired ventilation, by causing interstitial and alveolar edema. After transplantation, edema and consequent impairment of ventilation were aggravated by graft ischemia, proportionally to its duration. Our improved technique for transplantation of left lungs in rats provides a new opportunity for investigating the immunologic problems of lung transplantation.
IgG-secretion was induced in a human B blastoid cell line, CESS, by the addition of partially purified T cell-derived helper factor(s) (TRF), which had been obtained from PHA-stimulated human T cells. The number of IgG-producing cells in CESS cells reached its maximal level (10% of total cells) within 48 hr after the addition of TRF. TRF did not affect the proliferation of CESS cells and the block of cell proliferation with hydroxyurea did not inhibit the increase of IgG-producing cells, showing that TRF induced IgG-production in CESS cells without any requirement of cell division. TRF activity was completely removed by CESS cells but TCGF-activity in the same preparation was not absorbed with CESS cells. On the other hand, TCGF-dependent human killer cells absorbed TCGF activity but not TRF activity in the same preparation. The binding of 125I-labeled factor(s) on CESS cells was also demonstrated. These results showed the presence of acceptors for TRF on the surface of CESS cells and this cell line will provide useful means for the chemical characterization of acceptors and for the study of the mechanisms of the signal transmission through acceptors.
For immunogenetic and economical reasons, an operative technique for lung transplantation in the rat was developed. With the use of an operation microscope and 8-0, 9-0, and 10-0 sutures, the left pulmonary artery, vein, and brochus were anastomosed. The mean operation time was 4 hours; the mean graft ischemia time, 87 minutes. After frequent failures initially, due to anesthetic problems and lack of experience with microsurgical operative technique, a final peroperative survival of about 80% was obtained. Postoperative mortality approximated 50%. The main cause of postoperative death was thrombus formation at the site of an anastomosis. In a final series of 28 isogeneic transplantations, proper data for a one-month follow-up was obtained from 36% of the animals that underwent operation, a percentage comparable with initial canine pulmonary and rat renal transplantation studies. Left lung transplantation in the rat proved to be technically feasible and may provide an immunogenetically well defined and economically advantageous animal model in lung transplantation research.
- Prop J