Essential Immune Functions Research Articles

On regulation of phagosome maturation and antigen presentation

Phagocytosis has essential functions in immunity. Here we highlight the presence of a subcellular level of self-non-self discrimination in dendritic cells that operates at the level of individual phagosomes. We discuss how engagement of Toll-like receptor signaling controls distinct programs of phagosome maturation. An inducible mode of phagosome maturation triggered by these receptors ensures the selection of microbial antigens for presentation by major histocompatibility class II molecules during the simultaneous phagocytosis of self and non-self.

Gene therapy in the treatment of autoimmune diseases.

Complex pathogenic processes lead to heterogeneous manifestations in autoimmune diseases. Except for rare patients in whom a single genetic defect — in genes for the Fas ligand or for the complement factors C4, C2, or C1q — is associated with autoimmune manifestations, these diseases are multifactorial. Complex genetic, environmental, and immunoregulatory factors appear to play out in a process that we understand in only a fragmentary sense and that we have little ability to influence (1). Therefore, gene therapy involving the reconstitution of a single missing gene should be expected to be of minimal help in treating these diseases. Our current practice of helping patients symptomatically, or at best by indiscriminate suppression of the immune system, is obviously inadequate. Correction of the identified immunoregulatory aberrations has become the mainstay of the efforts to treat autoimmune disorders in a rational manner. Therapeutic intervention in autoimmune disease faces formidable challenges, since it requires a balance between the control of ongoing pathogenic immune responses and the maintenance of essential immune surveillance functions. Therapies directed at general pathways of immune activation or amplification are antigen-nonspecific and may allow for widespread application across multiple diseases, but they also carry the risk of global immunosuppression. Antigen-specific therapeutics, on the other hand, are potentially much more selective and less deleterious, but they require a priori knowledge of precise immunologic targets relevant in each autoimmune setting. Cytokines, cytokine antagonists, anti–T cell monoclonal antibodies, inhibitors of signal transduction, and conventional pharmacologic agents fall into the former group, whereas specific peptide antigens, antagonists, and MHC-antigen complexes fall into the latter group of treatments. Table ​Table11 summarizes many of the approaches that have been tested in mouse models of various autoimmune disorders. In this issue of the JCI, articles by Agarwal et al. (2) and Lawson et al. (3) apply novel forms of gene therapy to introduce directed immune therapeutics in autoimmune animals, illustrating both antigen-nonspecific and antigen-specific strategies. Table 1 Gene therapy in the treatment of autoimmune diseases

Inhibition of the Defense System Stimulating Interleukin-12 Interferon-γ Pathway During Critical Illness

AbstractInterleukin-12 (IL-12) and interferon-γ (IFN-γ) exert protective effects during experimental endotoxemia through upregulation of cellular immunity and phagocytic functions. They are part of a positive regulatory feedback loop that enhances the production of the other. Because critically ill patients show a marked suppression of T-cell and macrophage functions with a high susceptibility to infection, potential defects in the immunity/inflammation upregulating IL-12 IFN-γ pathway were studied. As an ex vivo model of endotoxemia, lipopolysaccharide (LPS) stimulated whole blood from 25 critically ill patients and 12 healthy individuals was incubated with either recombinant human (rh) IL-12 or rhIFN-γ, respectively. IFN-γ dose-dependently (P < .05) increased the release of IL-12 p40 and p70 into LPS-stimulated whole blood from healthy humans without effect in whole blood from critically ill patients. RhIL-12 p70 enhanced (P < .05) the secretion of IFN-γ in controls, while it was ineffective in LPS-stimulated whole blood from critically ill patients. The observed inhibition of the IL-12 IFN-γ pathway is not specific to LPS, since Staphylococcus aureus Cowan strain I (SAC)-stimulated whole blood from critically ill patients showed similar suppression. The secretion of IL-12 and IFN-γ was less reduced in critically ill patients when using isolated cultures of adherent cells or lymphocytes. Although preculture of whole blood from healthy humans with IL-10, but not with IL-4, mimicked suppression of the IL-12 IFN-γ pathway similar to that observed during critical illness, the release of antiinflammatory reacting cytokines (IL-4, IL-10, transforming growth factor [TGF]-β1 ) was decreased into LPS-stimulated whole blood from critically ill patients. These results indicate at least two mechanisms responsible for dramatic disturbances of the IL-12 IFN-γ pathway during critical illness: (1) deactivation of IL-12 and IFN-γ producing leukocytes in vivo early after the primary insult, and (2) presence of serum suppressive factors different from IL-4, IL-10, or TGF-β1 . Because IL-12 and IFN-γ upregulate essential immune functions, the marked inhibition of IL-12 and IFN-γ release may be pivotal for high susceptibility of critically ill patients to infection.

Inhibition of the Defense System Stimulating Interleukin-12 Interferon-γ Pathway During Critical Illness

Interleukin-12 (IL-12) and interferon-γ (IFN-γ) exert protective effects during experimental endotoxemia through upregulation of cellular immunity and phagocytic functions. They are part of a positive regulatory feedback loop that enhances the production of the other. Because critically ill patients show a marked suppression of T-cell and macrophage functions with a high susceptibility to infection, potential defects in the immunity/inflammation upregulating IL-12 IFN-γ pathway were studied. As an ex vivo model of endotoxemia, lipopolysaccharide (LPS) stimulated whole blood from 25 critically ill patients and 12 healthy individuals was incubated with either recombinant human (rh) IL-12 or rhIFN-γ, respectively. IFN-γ dose-dependently (P &lt; .05) increased the release of IL-12 p40 and p70 into LPS-stimulated whole blood from healthy humans without effect in whole blood from critically ill patients. RhIL-12 p70 enhanced (P &lt; .05) the secretion of IFN-γ in controls, while it was ineffective in LPS-stimulated whole blood from critically ill patients. The observed inhibition of the IL-12 IFN-γ pathway is not specific to LPS, since Staphylococcus aureus Cowan strain I (SAC)-stimulated whole blood from critically ill patients showed similar suppression. The secretion of IL-12 and IFN-γ was less reduced in critically ill patients when using isolated cultures of adherent cells or lymphocytes. Although preculture of whole blood from healthy humans with IL-10, but not with IL-4, mimicked suppression of the IL-12 IFN-γ pathway similar to that observed during critical illness, the release of antiinflammatory reacting cytokines (IL-4, IL-10, transforming growth factor [TGF]-β1 ) was decreased into LPS-stimulated whole blood from critically ill patients. These results indicate at least two mechanisms responsible for dramatic disturbances of the IL-12 IFN-γ pathway during critical illness: (1) deactivation of IL-12 and IFN-γ producing leukocytes in vivo early after the primary insult, and (2) presence of serum suppressive factors different from IL-4, IL-10, or TGF-β1 . Because IL-12 and IFN-γ upregulate essential immune functions, the marked inhibition of IL-12 and IFN-γ release may be pivotal for high susceptibility of critically ill patients to infection.

Liposomal Formulation of a Cytokine Restraining Agent, (CRA)-HP 228: Encapsulation, Stability, and Release Characteristics

Cytokine restraining agents (CRAs) are a family of compounds having profound anti-inflammatory properties. Among them, HP 228 is a heptapeptide, containing some unnatural amino acids, with the unique ability to restrain the pathological effects of cytokines without interfering with their essential immune functions. A liposomal formulation of HP 228 was developed to protect the drug from proteolytic enzymes and to provide controlled release of the drug. The liposomes were prepared using either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine and cholesterol by the thin-film hydration method. Dicetyl phosphate was added to the lipids to provide negative charge to the liposomes. Homogeneous size distribution of multilamellar vesicles (MLVs) was achieved by extruding them through the 800-nm polycarbonate membranes. The size distribution was characterized using a submicrometer particle size analyzer. Encapsulation efficiency of HP 228 was determined by adding protamine to MLVs and separating the supernatant from the liposome protamine aggregate. Enzymatic stability of the drug was evaluated in phosphate-buffered saline, pH 7.0 and 37°C. The studies were conducted in the presence of trypsin and chymotrypsin. The encapsulation of HP 228 increased with the increase in fatty acid chain length. Encapsulation was further increased in negatively charged liposomes compared with neutral liposomes. The encapsulation efficiency was over 50%. Also, the negative charge helped in preventing the aggregation of the liposomes and promoted the electrostatic interaction between the lipid and the drug. Encapsulating the drug in the aqueous compartment of the liposomes completely protected HP 228 from trypsin and chymotrypsin. The rate of release of HP 228 was measured using a side-by-side diffusion chamber and was found to be drastically lower from the liposomes, as compared with the drug solution, making the formulation suitable for controlled release of HP 228.

Effect of anesthesia and operation on essential immune functions

Study of the references listed leaves no doubt that essential specific and unspecific immune functions are more or less strongly influenced by a wide variety of anaesthetics. In clinically employed concentrations these negative effects are rapidly and completely reversible. No clinically relevant adverse actions on the immune system have ever been identified in short-term anaesthesia with any substance, not even with the thiobarbiturates although these are known to be highly inhibitory. However, matters are entirely different in respect of highly dosed long-term sedation. In such cases, it may occur with e.g. thiobarbiturates or diazepam that in the course of days or weeks concentrations are attained in the tissue which may lead us to expect in vivo quite considerable suppression of both unspecific and antigen-specific cellular immune mechanisms. This expectation, which is first of all based on in-vitro results, has been indirectly supported by a clinical study in patients suffering from craniocerebral trauma who received artificial respiration and in whom an association was seen between thiopental administration and the incidence of bacterial pneumonias. Over and above this, however, the preoperative condition of the patient and of course mainly the operation as such with its associated stress reaction will cause direct and indirect changes in the immune system (Fig. 4). Trauma and anaesthesia exert not only a direct action, but act as inhibitors or stimulators of important immune functions chiefly by modulating the stress response. We are still unaware as to whether and to what extent the prognosis of the individual patient is actually adversely affected by the combined action of these factors. Many findings, however, seem to point to an essential role played by both the surgical trauma and the stress response it induces, as well as by the pre-operative immune status of the patient, in the occurrence of subsequent infectious complications (Fig. 4).

The activation of resting lymphocytes is accompanied by the biogenesis of lysosomal organelles

The degradative activity of lymphocytes plays by important role in a number of essential immune functions. In the present study we have examined how the activation of resting lymphocytes, by the mitogen concanavalin A (Con A), affects three major components of the lysosomal compartment: the lysosomal enzyme beta-glucuronidase (Gus); an integral lysosomal membrane protein (LAMP-1); and the mannose 6-phosphate receptor (MPR) which directs lymphocyte enzyme transport. Resting T cells were found to contain only very low levels of these proteins, but they were actively synthesized by, and far more abundant in, stimulated lymphoblasts. Although the lysosomal antigens did not have a distinct cytoplasmic localization in the resting lymphocytes, in the activated T lymphoblasts they were present in several highly developed intracellular structures, including the rough endoplasmic reticulum and the Golgi complex. Furthermore, in these latter cells Gus was also found to be accumulated within the lumen of large vesicles which we characterized as lysosomes by the presence of LAMP-1 at the periphery and by the absence of MPR. Subcellular fractionation confirmed that these organelles were present in the activated lymphocytes only, and not in the resting T cells. Our results demonstrate that lymphocyte activation is accompanied by the synthesis of the enzymic and structural components of the lysosomal compartment which are sorted and assembled into distinct organelles in the activated cell.