Neutralization Of Tumor Necrosis Factor Alpha Research Articles

Tumor necrosis factor alpha neutralization attenuates immune checkpoint inhibitor-induced activation of intermediate monocytes in synovial fluid mononuclear cells from patients with inflammatory arthritis

ObjectiveDuring treatment with immune checkpoint inhibitors (ICI) such as the anti-PD-1 antibody pembrolizumab, half of patients with pre-existing inflammatory arthritis experience disease flares. The underlying immunological mechanisms have not been characterized. Here, we investigate the effect of pembrolizumab on cells involved in inflammation and destruction in the synovial joint and how immunosuppressive treatments affect the pembrolizumab-induced immune reactions.MethodsWe included synovial fluid mononuclear cells (SFMCs, n = 28) and peripheral blood mononuclear cells (PBMCs, n = 6) from patients with rheumatoid arthritis and peripheral spondyloarthritis and PBMCs from healthy controls (n = 6). Fibroblast-like synovial cells (FLSs) were grown from SFMCs. The in vitro effect of pembrolizumab was tested in SFMCs cultured for 48 h, FLS-PBMC co-cultures and in SFMCs cultured for 21 days (inflammatory osteoclastogenesis). Cells and supernatants were analyzed by ELISA, flow cytometry, and pro-inflammatory multiplex assay. Finally, the effect of the disease-modifying anti-rheumatic drugs (DMARDs) adalimumab (TNFα inhibitor), tocilizumab (IL-6R inhibitor), tofacitinib (JAK1/JAK3 inhibitor), and baricitinib (JAK1/JAK2 inhibitor) on pembrolizumab-induced immune reactions was tested.ResultsPembrolizumab significantly increased monocyte chemoattractant protein-1 (MCP-1) production by arthritis SFMCs (P = 0.0031) but not by PBMCs from patients or healthy controls (P = 0.77 and P = 0.43). Pembrolizumab did not alter MMP-3 production in FLS-PBMC co-cultures (P = 0.76) or TRAP secretion in the inflammatory osteoclastogenesis model (P = 0.28). In SFMCs, pembrolizumab further increased the production of TNFα (P = 0.0110), IFNγ (P = 0.0125), IL-12p70 (P = 0.0014), IL-10 (P = 0.0100), IL-13 (P = 0.0044), IL-2 (P = 0.0066), and IL-4 (P = 0.0008) but did not change the production of IL-6 (P = 0.1938) and IL-1 (P = 0.1022). The SFMCs treated with pembrolizumab showed an increased frequency of intermediate monocytes (P = 0.044), and the MCP-1 production increased only within the intermediate monocyte subset (P = 0.028). Lastly, adalimumab, baricitinib, and tofacitinib treatment were able to attenuate the pembrolizumab-induced MCP-1 production (P = 0.0004, P = 0.033, and P = 0.025, respectively), while this was not seen with tocilizumab treatment (P = 0.75).ConclusionPembrolizumab specifically activated intermediate monocytes and induced the production of several cytokines including TNFα but not IL-6. These findings indicate that flares in patients with pre-existing inflammatory arthritis involve monocyte activation and could be managed with TNFα neutralization.

Tumor necrosis factor alpha neutralization has no direct effect on parasite burden, but causes impaired IFN-γ production by spleen cells from human visceral leishmaniasis patients

The pro-inflammatory cytokine tumor necrosis factor (TNF)-α has an important role in control of experimental Leishmania donovani infection. Less is known about the role of TNF-α in human visceral leishmaniasis (VL). Evidence for a protective role is primarily based on case reports of VL development in individuals treated with TNF-α neutralizing antibody. In this study, we have evaluated how TNF-α neutralization affects parasite replication and cytokine production in ex vivo splenic aspirates (SA) from active VL patients. The effect of TNF-α neutralization on cell mediated antigen specific responses were also evaluated using whole blood cultures. Neutralization of TNF-α did not affect parasite numbers in SA cultures. Interferon (IFN)-γ levels were significantly reduced, but interleukin (IL)-10 levels were unchanged in these cultures. Leishmania antigen stimulated SA produced significant TNF-α which suggests that TNF-α is actively produced in VL spleen. Further it stimulates IFN-γ production, but no direct effect on parasite replication.

Nociceptive neurons detect cytokines in arthritis.

Proinflammatory cytokines are major mediators in the pathogenesis of diseases of joints such as rheumatoid arthritis and osteoarthritis. This review emphasizes that proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-17 are also mediators of pain by directly acting on the nociceptive system. Proportions of nociceptive sensory neurons express receptors for these cytokines, and the application of cytokines rapidly changes the excitability, ion currents and second messenger systems of these neurons. By inducing persistent sensitization of nociceptive sensory neurons (C- and a proportion of Aδ-fibers) for mechanical stimuli in the joint (a process called peripheral sensitization), these cytokines significantly contribute to the persistent hyperalgesia typical for many disease states of the joint. In addition, the disease-associated release of cytokines in the spinal cord supports the generation of central sensitization. The therapeutic neutralization of proinflammatory cytokines thus not only reduces the process of inflammation but may directly reduce hyperalgesia and pain by reversing the neuronal effects of cytokines. It is emerging that different cytokines have different actions on neurons. The neutralization of tumor necrosis factor-alpha reduces both mechanical and thermal hyperalgesia of the joint. The neutralization of interleukin-1beta attenuates thermal hyperalgesia whereas the neutralization of interleukin-6 and interleukin-17 mainly reduces mechanical hyperalgesia. These different effects are partly explained by influencing different target molecules in sensory neurons. For example, in cultured sensory neurons tumor necrosis factor-alpha and interleukin-1beta upregulate the TRPV1 ion channel, which is involved in the transduction of heat stimuli, consistent with an effect of these cytokines in thermal hyperalgesia. By contrast, interleukin-17 upregulates the TRPV4 ion channel, which has a role in the transduction of mechanical stimuli. Thus, the analgesic potential of neutralizing cytokines seems to depend on which cytokine is mainly involved in the particular pain state.

Central nervous system effects of tumor necrosis factor alpha blockade

The therapeutic success of TNFa blockade in RA is unique and has been largely considered to result from rapid and efficient neutralization of joint inflammation based on breakdown of the inflammatory cytokine network in the affected joint, which results in an improvement of the signs and symptoms of the disease (4). It has, however, always been stunning, how fast the blockade of TNFa improves the patient condition, in particular since diseases like RA are highly chronic, building up a vast amount of inflammatory tissue and leading to irreversible damage of the cartilage and the bone. Thus, rapid resolution of this highly organized inflammatory tissue and/or tissue damage is very unlikely to explain the fast effect of TNFa blockade. There has always been a gap in understanding of how tumor necrosis factor alpha (TNFa) blockade affects the disease state of arthritis patients so rapidly, considering that diseases like rheumatoid arthritis are very chronic conditions. We thus hypothesized that blockade of TNFa acts through the central nervous system (CNS) before directly affecting joint inflammation. By use of functional magnetic resonance imaging (fMRI), we demonstrate that within 24 hours after neutralization of TNFa nociceptive CNS activity in the thalamus and somatosensoric cortex but also the activation of the limbic system is blocked. Brain areas showing blood oxygen level dependent (BOLD) signals, a validated method to assess neuronal activity elicited by pain, were significantly reduced as early as 24 hours after initiation of TNFa blockade. In contrast, clinical and laboratory markers of inflammation such as joint swelling and acute phase reactants were not affected by TNFa blockade at these early time points. Moreover, arthritic mice overexpressing human TNFa showed an altered pain behavior and a more intensive, widespread and prolonged brain activity upon nociceptive stimuli as compared to wild-type mice. Similar to humans, these changes as well as the rewiring of CNS activity resulting in tight clustering in the thalamus were rapidly reversed after neutralization of TNFa. These results suggest that neutralization of TNFa affects nociceptive brain activity in the context of arthritis, long before it achieves anti-inflammatory effects in the joints.

Tumor necrosis factor-alpha neutralization reduced cerebral edema following transient focal cerebral ischemia

Following focal cerebral ischemia, tumor necrosis factor-alpha deteriorates cerebral edema and survival rate. Therefore, tumor necrosis factor-alpha neutralization could reduce cerebral microvascular permeability in acute cerebral ischemia. Left middle cerebral artery occlusion for 120 min followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague-Dawley rats. Anti-rat tumor necrosis factor-alpha neutralizing monoclonal antibody with a rat IgG Fc portion (15 mg/kg) was infused intravenously right after reperfusion. Stroke index score, infarct volume, cerebral specific gravity, and the endogenous expression of tumor necrosis factor-a, matrix metalloproteinase-2, matrix metalloproteinase-9, and membrane type 1- matrix metalloproteinase in the brain tissue were quantified in the ischemic and matched contralateral non-ischemic hemisphere. In the anti-tumor necrosis factor-alpha neutralizing antibody treated rats, infarct volume was significantly reduced (p = 0.014, n = 7; respectively), and cerebral specific gravity was dramatically increased in cortex and caudate putamen (p < 0.001, n = 7; respectively) in association with a reduction in matrix metalloproteinase-9 and membrane type 1- matrix metalloproteinase upregulation. Tumor necrosis factor-alpha in the brain tissue was significantly elevated in the ischemic hemisphere 6 hours after reperfusion in the non-specific IgG treated rats (p = 0.021, n = 7) and was decreased in the anti-tumor necrosis factor-alpha neutralizing antibody treated rats (p = 0.001, n = 7). Post-reperfusion treatment with anti-rat tumor necrosis factor-alpha neutralizing antibody reduced brain infarct volume and cerebral edema, which is likely mediated by a reduction in matrix metalloproteinases upregulation. (See Figure 1).

Elevated monocyte chemoattractant protein-1 levels following thermal injury precede monocyte recruitment to the wound site and are controlled, in part, by tumor necrosis factor-alpha.

In previous studies, mice given a full-thickness scald injury had an influx of neutrophils into the skin that followed a local increase in a neutrophil chemoattractant. Because macrophages are known to infiltrate the wound area after neutrophils and are essential for normal wound repair, studies were designed to characterize the time course of macrophage accumulation in the wound and to identify the factor(s) responsible for this influx. A macrophage infiltrate into the wound was observed at 4 days post-injury and persisted through at least 10 days. This influx was preceded by an initial fourfold increase in dermal monocyte chemoattractant protein-1 levels at 24 hours post-injury (p < 0.05). This elevation in monocyte chemoattractant protein-1 was enhanced at 4 and 10 days postburn resulting in a sixfold increase over baseline (p < 0.01). Levels of tumor necrosis factor-alpha, a proinflammatory cytokine known to induce chemokine production, were elevated at 90 minutes after injury in burn- versus sham-injured groups (p < 0.05). Furthermore, administration of tumor necrosis factor-alpha neutralizing antibody in vivo reduced the dermal levels of monocyte chemoattractant protein-1 seen at 10 days postburn by 57% (p < 0.01); however, macrophage accumulation was not altered. Thus, elevated systemic TNF-alpha levels may influence the local chemokine milieu following burn injury.

Acute graft-versus-host disease does not require alloantigen expression on host epithelium.

Alloantigen expression on host antigen-presenting cells (APCs) is essential to initiate graft-versus-host disease (GvHD); therefore, alloantigen expression on host target epithelium is also thought to be essential for tissue damage. We tested this hypothesis in mouse models of GvHD using bone-marrow chimeras in which either major histocompatibility complex class I or class II alloantigen was expressed only on APCs. We found that acute GvHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-alpha and interleukin-1 prevents acute GvHD. These results pertain particularly to CD4-mediated GvHD but also apply, at least in part, to CD8-mediated GvHD. These results challenge current paradigms about the antigen specificity of GvHD effector mechanisms and confirm the central roles of both host APCs and inflammatory cytokines in acute GvHD.

Cellular and cytokine effectors of acute graft versus host disease.

Graft-versus-host-disease (GVHD) is the major complication of allogeneic Bone Marrow Transplant (BMT) and Older BMT recipients are at greater risk for acute graft-versus-host-disease. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older recipients. GVHD mortality and morbidity, as well as pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice. In a haploidential GVHD model, CD4+ donor T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2FI BM chimeras created with either old or young BM. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. We also evaluated whether alloantigen expression on host target epithelium is essential for tissue damage induced by GVHD in mouse models. In bone marrow chimeras recipients in which either MHC II or MHC I alloantigen was expressed only on APCs, we found that acute GVHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-alpha and interleukin-1 prevents acute GVHD. These results suggest new strategies for the prevention and treatment of this toxic complication of BMT.

Pathogenic mechanisms of Acute Graft versus Host Disease

Graft-versus-host-disease (GVHD) is the major complication of allogeneic Bone Marrow Transplant (BMT). Older BMT recipients are a greater risk for acute GVHD after allogeneic BMT, but the causes of this association are poorly understood. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older mice. GVHD mortality and morbidity, and pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice. In a haploidential GVHD model, CD4+ donor T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using bone marrow chimera recipient created with either old or young bone marrow. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. In a separate set of experiments we evaluated whether alloantigen expression on host target epithelium is essential for tissue damage induced by GVHD. Using bone marrow chimeras recipients in which either MHC II or MHC I alloantigen was expressed only on APCs, we found that acute GVHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-alpha and interleukin-1 prevents acute GVHD. These results pertain to CD4-mediated GVHD and to a lesser extent in CD8-mediated GVHD, and confirm the central role of most APCs as well as inflammatory cytokines.

Chemokine Expression Dynamics in Mycobacterial (Type-1) and Schistosomal (Type-2) Antigen-Elicited Pulmonary Granuloma Formation

Transcript expression of 24 chemokines (CKs) was examined throughout 8 days in mouse lungs with type-1 (Th1) or type-2 (Th2) cytokine-mediated granulomas induced by bead-immobilized mycobacterial purified protein derivative or Schistosoma mansoni egg antigens. Where possible, CK protein levels were also measured. In addition, we examined effects of in vivo cytokine depletions. Findings were as follows: 1) bead challenge induced increases in 18 of 24 CK transcripts with type-1 and type-2 responses displaying different patterns. CKs fell into four categories: a) type-1-dominant (gamma-interferon-inducible protein (IP-10), monokine induced by INF-gamma (MIG), macrophage inflammatory protein-2 (MIP-2), lipopolysaccharide-induced chemokine (LIX), rodent growth-related oncogene homologue (KP), macrophage inflammatory protein-1alpha (MIP-1alpha) and -1beta (MIP-1beta), lymphotactin), b) type-2-dominant (eotaxin, monocyte chemotactic protein-2 (MCP-2) and -3 (MCP-3), liver and activation-regulated chemokine (LARC), T cell activation protein-3 (TCA-3), c) type-1 and type-2 co-dominant (MCP-1, MCP-5, monocyte-derived chemokine (MDC), thymus and activation-related chemokine (TARC), C10), and d) constitutive (lungkine, secondary lymphoid-tissue chemokine (SLC), EBI1-ligand chemokine (ELC), fractalkine, macrophage inflammatory protein-1gamma (MIP1-gamma), and stromal cell derived factor-1alpha (SDF1-alpha). 2) CKs displayed characteristic temporal patterns. CXC (IP-10, MIG, MIP-2, LIX, KC) and certain CC (MCP-1, MCP-5, MIP-1alpha, MIP-1beta) CKs were produced maximally within 1 to 2 days. Others (MCP-2, MCP-3, eotaxin, lymphotactin, LARC, TCA-3) displayed peak expression later. 3) Interferon-gamma neutralization profoundly abrogated MIG, but had little effect on other CKs. Tumor necrosis factor-alpha neutralization caused up to 50% reduction in a range of CKs. These findings indicate that type-1 and type-2 granulomas display characteristic CK profiles with coordinated expression that is under cytokine-mediated regulation.

Neutralization of tumor necrosis factor-alpha or interleukin-1 prevents obliterative airway disease in HLA-A2 transgenic murine tracheal allografts.

Neutralization of tumor necrosis factor-alpha or interleukin-1 prevents obliterative airway disease in HLA-A2 transgenic murine tracheal allografts.

Tumor necrosis factor-alpha neutralization reduces lung injury after experimental allogeneic bone marrow transplantation.

Idiopathic pneumonia syndrome (IPS) is a frequent and potentially fatal complication of bone marrow transplantation (BMT). We have previously shown that experimental IPS is associated with increased levels of lipopolysaccaride (LPS) and tumor necrosis factor-alpha (TNFalpha) in the bronchoalveolar lavage (BAL) fluid, and that administration of LPS to animals with extensive graft versus host exacerbated underlying lung injury (Blood 1996; 88: 3230). Lethally irradiated CBA mice received BMT from allogeneic (B10.BR) or syngeneic (CBA) donors. The role of TNFalpha in the exacerbation of pulmonary toxicity caused by LPS injection and in the evolution of IPS after allogeneic BMT was examined by neutralizing TNFalpha after BMT using a soluble binding protein (rhTNFR:Fc). Five weeks after BMT, administration of rhTNFR:Fc dramatically reduced mortality and prevented the exacerbation of lung injury caused by LPS administration. This protective effect was associated with preservation of pulmonary function and with marked reductions of cells, neutrophils, and LPS in the BAL fluid of treated animals. TNFalpha neutralization from week 4 to 6 after allogeneic BMT effectively halted the progression of systemic GVHD and significantly reduced, but did not prevent lung injury that developed during the treatment period. We conclude that TNFalpha is central to early LPS induced toxicity in this model and is a significant, but not the exclusive contributor to the development of IPS after allogeneic BMT.

Agarose plug instillation causes goblet cell metaplasia by activating EGF receptors in rat airways.

We hypothesized that foreign bodies in airways cause inflammation leading to goblet cell metaplasia. Instilled agarose plugs lodged in the bronchi of pathogen-free rats caused a time-dependent increase in Alcian blue-periodic acid-Schiff staining that was detected within 24 h and markedly increased at 72 h. Control bronchi contained no pregoblet or goblet cells, but plugged bronchi contained many pregoblet and goblet cells and a decrease in nongranulated secretory cells. In situ hybridization showed no expression of MUC5AC in control airways, but plugged airways showed a marked expression. Control bronchi showed sparse staining for epidermal growth factor receptor (EGFR) protein, but plugged bronchi showed intense EGFR staining in the epithelium. Pretreatment with an EGFR tyrosine kinase inhibitor (BIBX1522) prevented Alcian blue-periodic acid-Schiff staining and MUC5AC gene expression in plugged bronchi. Pretreatment with tumor necrosis factor-alpha neutralizing antibody or pretreatment with cyclophosphamide abolished plug-induced EGFR protein expression and goblet cell metaplasia. Thus instillation of agarose plugs induces profound goblet cell metaplasia by causing EGFR expression and activation.

Anti-TNF alpha: new therapy for Crohn's disease.

Immunological technology has paved the way for a promising new therapy for patients suffering from chronic active Crohn's disease. Neutralization of tumor necrosis factor-alpha (TNF alpha), a cytokine secreted by cells in the immune system, may prove useful in decreasing bowel inflammation and aid in the closure of fistulae. Two multicenter, randomized, double-blinded, placebo-controlled trials were conducted to evaluate the safety and efficacy of inflixmab (Remicade [Centocor]), a chimeric monoclonal antibody form of anti-TNF alpha, hypothesized to provide a prolonged therapeutic effect in chronic inflammation. The 5 mg/kg dose regimen of infliximab was the lowest effective dose and produced a high degree of benefit lasting 8 weeks to 3 months. Infusion preparation, cost, and patient indications bear special considerations. Further investigation is needed in long-term safety and efficacy for chronic administration of infliximab.

Cellular Fibronectin is Induced in Ultraviolet-Exposed Human Skin and Induces IL-10 Production by Monocytes/Macrophages

CD11b+ monocytic/macrophagic cells that infiltrate human skin after in vivo ultraviolet exposure potently produce interleukin-10. We hypothesized that binding of monocyte beta1 integrins to ultraviolet-induced extracellular matrix ligands, such as fibronectin, after entry of blood monocytes into the dermis, is involved in the modulation of immunoregulatory monocytic cytokines. Immunostaining of human skin and reverse transcriptase-polymerase chain reaction studies revealed that the embryonic isoform of cellular fibronectin, in which the extra domain A (EDA) segment is spliced in (EDA+ cellular fibronectin), and confers enhanced binding to beta1 integrins, is newly induced and is associated with infiltrating CD11b+ cells post in vivo ultraviolet exposure. We then tested the effect of fibronectin on resting purified peripheral monocytes in vitro. We found that monocyte interleukin-10, but not interleukin-12, was significantly induced in a concentration-dependent manner by in vitro binding to cellular fibronectin (n = 6), but not plasma fibronectin. Tumor necrosis factor-alpha was also induced in a concentration-dependent manner, but to a lesser extent. Monoclonal antibodies to beta1 integrins beta-subunit (CD29) also strongly induced tumor necrosis factor-alpha and interleukin-10 production, but not interleukin-12. Neutralization of tumor necrosis factor-alpha reduced by 54% the interleukin-10 production that was induced by monocytes binding to cellular fibronectin, indicating that interleukin-10 induction is at least in part dependent upon concomitant autocrine tumor necrosis factor-alpha release. In conclusion, ultraviolet skin injury results in increased production and deposition of EDA+ cellular fibronectin in the papillary dermis, which may be one of the key signals capable of inducing interleukin-10 but not interleukin-12 in monocytes that infiltrate micromilieu of human skin after ultraviolet exposure.

In vivo neutralization of tumor necrosis factor-alpha and interferon-gamma abrogates resistance to Yersinia enterocolitica infection in mice.

Cytokines are important mediators of the inflammatory host response against infectious agents. In this study, the role of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the elimination of a primary infection with highly virulent Yersinia enterocolitica serotype 0:8 strain WA-P has been investigated in C57BL/6 mice. The injection of anti-TNF-alpha or anti-IFN-gamma antibodies ("serotherapy") prior to the intravenous challenge of a sublethal dose of Y. enterocolitica caused an increased bacterial net-growth in the spleens, although this effect was more pronounced for anti-TNF-alpha treatment. The later treatment with anti-TNF-alpha or anti-IFN-gamma antibodies on day 3 post infection likewise abrogated resistance to Y. enterocolitica and, subsequently, led to death from progressive infection. Our data demonstrate for the first time that the endogenous production of both the cytokines TNF-alpha and IFN-gamma is required for the restriction of a primary Y. enterocolitica infection in mice.

Protection by pentoxifylline against graft failure from storage injury after orthotopic rat liver transplantation with arterialization

Destruction of the endothelial cell lining and activation of Kupffer cells after reperfusion limits the safe storage of livers for transplantation surgery. Tumor necrosis factor-alpha (TNF) release by activated Kupffer cells may contribute to graft failure from storage injury. Accordingly, we evaluated whether pentoxifylline, which suppresses macrophage TNF release, would improve graft survival after orthotopic rat liver transplantation with arterialization. Livers from syngeneic Lewis rats were stored for 12–24 h in cold UW solution. Prior to implantation, the livers were flushed with cold Ringer's solution or warm Carolina rinse solution B. With either rinse, pentoxifylline treatment of graft recipients significantly improved graft survival. Combined use of pentoxifylline (50 mg/kg for 5 days) and Carolina rinse solution doubled the safe storage time to 24 h. Acidotic pH and antioxidants were essential components of Carolina rinse solution that acted synergistically with pentoxifylline. Pentoxifylline was also shown to suppress TNF release by lipopolysaccharide (LPS)-stimulated cultured rat Kupffer cells. Thus, pentoxifylline may protect against primary non-function and failure of grafts from storage injury by suppressing excessive TNF release by activated Kupffer cells. However, neutralization of TNF with excess anti-TNF antibody did not improve survival. This may mean that depletion of TNF is as deleterious as excess TNF production. Alternatively, other Kupffer cell secretions [e.g., interleukin-1 (IL-1), interleukin-6 (IL-6) and other cytokines] may be involved in the pathogenesis of graft failure. In conclusion, pentoxifylline could protect against graft failure from storage injury.

Protection by pentoxifylline against graft failure from storage injury after orthotopic rat liver transplantation with arterialization.

Destruction of the endothelial cell lining and activation of Kupffer cells after reperfusion limits the safe storage of livers for transplantation surgery. Tumor necrosis factor-alpha (TNF) release by activated Kupffer cells may contribute to graft failure from storage injury. Accordingly, we evaluated whether pentoxifylline, which suppresses macrophage TNF release, would improve graft survival after orthotopic rat liver transplantation with arterialization. Livers from syngeneic Lewis rats were stored for 12-24 h in cold UW solution. Prior to implantation, the livers were flushed with cold Ringer's solution or warm Carolina rinse solution B. With either rinse, pentoxifylline treatment of graft recipients significantly improved graft survival. Combined use of pentoxifylline (50 mg/kg for 5 days) and Carolina rinse solution doubled the safe storage time to 24 h. Acidotic pH and antioxidants were essential components of Carolina rinse solution that acted synergistically with pentoxifylline. Pentoxifylline was also shown to suppress TNF release by lipopolysaccharide (LPS)-stimulated cultured rat Kupffer cells. Thus, pentoxifylline may protect against primary non-function and failure of grafts from storage injury by suppressing excessive TNF release by activated Kupffer cells. However, neutralization of TNF with excess anti-TNF antibody did not improve survival. This may mean that depletion of TNF is as deleterious as excess TNF production. Alternatively, other Kupffer cell secretions [e.g., interleukin-1 (IL-1), interleukin-6 (IL-6) and other cytokines] may be involved in the pathogenesis of graft failure. In conclusion, pentoxifylline could protect against graft failure from storage injury.

Anti-TNF-alpha and Pentoxifylline for Prophylaxis of aGvHD in Murine Allogeneic Bone Marrow Transplantation

Clinical studies analyzing sequential serum levels suggest pathophysiological involvement of Tumor-Necrosis-Factor-alpha (TNFa) in acute graft-versus-host disease (aGvHD) and endothelial complications following allogeneic bone marrow transplantation (BMT). For further investigation of the role of TNFa in aGvHD a neutralizing rb-anti-ms-TNFalpha antibody or pentoxifylline (PTX) as an inhibitor of TNFa-transcription were injected in lethally irradiated mice before or following transplantation of semi- or fully allogeneic bone marrow and various T-cell doses. Prophylactic application of anti-TNFa either prior to irradiation and BMT (p < .05) or at weekly intervals from d7 until d35 (p < .Ol) significantly reduced aGvHD-related mortality and cachexia, when low doses of semiallogeneic T cells were used for induction of aGvHD. Under these conditions, only a marginal protective effect could be obtained by long term application of PTX. In addition, aGvHD induced by high doses of T cells or by transfer of fully allogeneic T lymphocytes could not be prevented by anti-TNFa. These results further indicate that neutralization of TNFa might be helpful as an additive strategy for prevention of aGvHD especially in HLA-identical BMT.

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha).

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.