Severe intrahepatic cholestasis in patients treated with recombinant interleukin-2 and lymphokine-activated killer cells

Summary Immunotherapy with recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells (LAK) has become a new form of therapy that has been shown to induce remissions in patients with renal cell carcinoma and melanoma. Despite encouraging results, this form of therapy has been associated with increasing toxicity, often requiring therapy in an intensive-care unit. In this report severe intrahepatic cholestasis occurred in two patients receiving rIL-2 and LAK cells. This form of cholestasis appeared to be directly related to rIL-2 administration at a doses of 2×10⁶ U/m² and 3x10⁶ U/m² t.i.d. A liver biopsy showed moderate hepatocellular bile stasis, with lobular and portal inflamation. All other studies for potential cause of this cholestasis were negative, including studies for metastatic disease. When therapy was discontinued, evidence for cholestasis and bile stasis resolved. We conclude that rIL-2 is a drug with a potential to induce severe hepatic injury that is reversible upon cessation of therapy with rIL-2. Further care should be exercised when rIL-2 is administered to patients with abnormal liver function.Supported in part by Dr. I. Fund Foundation, Hoffmann-La Roche Inc., Nutley, New Jersey, and the Sarah C. Upham Trust     

Perforin-2 Restricts Growth of Chlamydia trachomatis in Macrophages

Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that preferentially infects epithelial cells. Professional phagocytes provide C. trachomatis only a limited ability to survive and are proficient killers of chlamydiae. We present evidence herein that identifies a novel host defense protein, perforin-2, that plays a significant role in the eradication of C. trachomatis during the infection of macrophages. Knockdown of perforin-2 in macrophages did not alter the invasion of host cells but did result in chlamydial growth that closely mirrored that detected in HeLa cells. C trachomatis L2, serovar B, and serovar D and C. muridarum were all equally susceptible to perforin-2-mediated killing. Interestingly, induction of perforin-2 expression in epithelial cells is blocked during productive chlamydial growth, thereby protecting chlamydiae from bactericidal attack. Ectopic expression of perforin-2 in HeLa cells, however, does result in killing. Overall, our data implicate a new innate resistance protein in the control of chlamydial infection and may help explain why the macrophage environment is hostile to chlamydial growth.     

Molecular reorganization of lipid bilayers by complement: a possible mechanism for membranolysis.

The interaction between the membrane attack complex (MAC) of complement and flat lipid bilayers was investigated. Using spin-labeled derivatives of phospholipids and cholesterol and electron paramagnetic resonance spectroscopy, we measured the penetration of the MAC into bilayers and its influence on the order of bilayers. The MAC precursor components C5b--6, C7, C8, and C9 did not exert any measurable influence on lipid membranes. Functional C5b--7 was shown to interact strongly with the bilayer surface without deep penetration into the bilayer. Formation of C5b--8 and especially C5b--9 caused a marked change in the anisotropy of spectra from probes located within the hydrocarbon phase. The spectral changes are not caused by changes in probe rotation and, in the case of the cholesterol probes, are not due to direct probe--protein interactions. For these reasons we interpret the spectral changes to be the result of reorientation of ordered bilayer lipids effected by strong binding of phospholipids to MAC proteins.     

Cytolysis by H-2-specific T killer cells. Assembly of tubular complexes on target membranes

Cloned T killer cells derived from one-way mixed lymphocyte reactions were characterized with regard to their Lyt phenotype and specificity. Two clones of Lyt-1-2+ phenotype that recognized H-2Dd were selected and examined for their cytolytic function by negative staining and thin section electron microscopy. When incubated with the H-2d target S194, they assemble two types of tubular complexes, polyperforin 1 and 2. Both structures appear to arise by polymerization of precursors that may originate in dense granules and/or Golgi of T killer cells. Polyperforins appear to be associated with vesicles that are released during the lytic reaction and transferred to target membranes as shown by immunoelectron microscopy. Since there is a close correlation between target lysis and the appearance of polyperforins on target membranes, it is suggested that polyperforins take part in the cell-mediated killing reaction. Although polyperforins are different in size and several molecular properties from complement, there are striking similarities between these circular complexes and polyperforin (C9). It is therefore possible that they belong to a closely related family of cytolytic effector molecules.     

Effector cells derived from host CD8 memory T cells mediate rapid resistance against minor histocompatibility antigen-mismatched allogeneic marrow grafts without participation of perforin, Fas ligand, and the simultaneous inhibition of 3 tumor necrosis factor family effector pathways.

Reduced-intensity conditioning regimens for transplant recipients have heightened awareness of immunologic resistance to allogeneic bone marrow transplants (BMT). Although T cell-mediated cytotoxicity has been assumed to play a role in the resistance against donor allogeneic hematopoietic stem and progenitor cell grafts, several studies have reported relatively unimpaired resistance by recipients who lack perforin, Fas ligand (FasL), and other cytotoxic mediators. This study compared the early kinetics of T cell-mediated resistance in B6 (H2b) cytotoxically normal versus deficient recipients after transplantation with major histocompatibility complex-matched, minor histocompatibility antigen (MiHA)-mismatched allogeneic marrow grafts. Wild-type B6 or cytotoxic double-deficient perforin-/-/gld+/+ (B6-cdd) mice were sensitized against major histocompatibility complex-matched BALB.B or C3H.SW (H2b) MiHA and transplanted with a high dose (1 x 10(7)) of T cell-depleted bone marrow. CD8 T memory cells were shown to be present in recipients before BMT, and anti-CD8 monoclonal antibody infusion abolished resistance, thus demonstrating that CD8 T cells are the host effector population. Donor-committed and high proliferative potential progenitor numbers were markedly diminished by 48 hours after transplantation in both wild-type B6 and B6-cdd anti-donor MiHA-sensitized recipients. These observations indicate that the resistance pathway used in the cytotoxic deficient mice was both potent and rapidly induced--consistent with a CD8 memory T-cell response. To examine the role of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)- and TL1A-mediated cytotoxicity in this strong resistance, newly generated monoclonal antibodies specific for these ligands were administered to B6-cdd recipients sensitized to donor antigens. Recipients of syngeneic B6-gfp bone marrow exhibited significant donor colony-forming unit numbers after BMT. In contrast, low or absent colony-forming unit levels were detected in allogeneic recipients, including those that lacked perforin and FasL and that received anti-TWEAK, anti-tumor necrosis factor-related apoptosis-inducing ligand, and anti-TL1A monoclonal antibodies. These findings extend previous observations by demonstrating the existence of a rapidly effected resistance pathway mediated by memory CD8 effector T cells independent of the 2 major pathways of cytotoxicity. Together with previous findings, these results support the notion that effector cells derived from memory CD8 T-cell populations can mediate strong resistance against donor allogeneic MiHA-disparate hematopoietic engraftment by using a mechanism that is independent of the contribution of perforin, FasL, and the known death ligand receptor pathways.     

CD30 supports lung inflammation

The physiological functions of CD30 have not been fully elucidated. Here we show that in CD30-deficient mice (CD30(-/-)), lung inflammation is significantly diminished in the ovalbumin (OVA) model of airway hyperreactivity. In CD30(-/-) mice, the recruitment of eosinophils into the airways after OVA-aerosol challenge of OVA-primed mice was significantly diminished when compared with wild-type (w.t.) mice. IL-13 levels were also significantly reduced in CD30(-/-) mice while levels of IFN-gamma, IL-4, IL-5 and IgE in bronchoalveolar lavage fluid, lung tissue and serum were comparable to w.t. mice. Peribronchial lymph node cells from CD30(-/-) mice, re-stimulated in vitro with OVA, secreted significantly lower levels of IL-13 than those from w.t. mice, but showed normal proliferative response and other cytokine production. Exogenous IL-13 reconstituted airway recruitment of leukocytes in OVA-challenged CD3O(-/-) mice. Adoptive transfer to naive w.t. mice of in vitro OVA-re-stimulated spleen cells from CD30(-/-) mice failed to induce eosinophilic pulmonary inflammation in contrast to transfer of primed cells from w.t. mice. These results indicate that CD30 is a regulator of T(h)2 responses in the effector-memory phase and a regulator of IL-13 production in memory cells in the lung.     

Secreted heat shock protein gp96-Ig: an innovative vaccine approach

Heat shock proteins (HSPs) are a large family of proteins with different molecular weights and different intracellular localizations. These proteins undertake crucial functions in maintaining cell homeostasis, and therefore they have been conserved during evolution. HSP gp96 also known as glucose-regulated protein grp94, is the primary chaperone of the endoplasmatic reticulum. Gp96/grp94, because of its peptide chaperone capacity and its ability to interact actively with professional antigen-presenting cells (APCs), is also endowed with crucial immunological functions such as natural adjuvant for priming innate and adaptive immunity. To make gp96 accessible to the immune system without biochemical purification and without cell lysis, we generated a secreted form of gp96. The immunological properties of secreted gp96 and its implications for vaccine in human cancer and infectious diseases will be discussed.