J. Edwin Blalock

Human leukocyte interferon (HuIFN-α): Potent endorphin-like opioid activity

Human leukocyte interferon, but not fibroblast or immune interferons, binds to opiate receptor in, vitro. When injected intracerebrally into mice, human leukocyte, but not fibroblast or immune interferon, caused potent endorphin-like opioid effects. These effects include analgesia, lack of spontaneous locomotion and catalepsy. All of these actions of human leukocyte interferon were preventable and reversible by the opiate antagonist naloxone. The findings suggest that some of the side effects of leukocyte interferon therapy may be mediated by opiate receptor binding. They also provide evidence for a regulatory circuit between the immune and neuroendocrine system. This putative circuit could be an etiologic site for certain psychopathological states.

Neuroendocrine peptide hormones and their receptors in the immune system: production, processing and action

Recent findings indicate that the immune and neuroendocrine systems interact and modulate one another functionally. The mechanism for this seems to be that the 2 systems share a set of receptors and ligands (hormones). Cells of the immune system are able to synthesize neuroendocrine peptide hormones which are biologically active and produced in physiologically significant quantities. Furthermore, leukocytes possess functional receptors for these same neuroendocrine hormones which will specifically modulate immune responses. The structural and functional evidence for these interactions is reviewed and discussed in the context of a bidirectional regulatory circuit between the immune and neuroendocrine systems.

Molecular characterization of a corticotropin (ACTH) receptor

We have used a new methodology to generate a monospecific antiserum to the corticotropin (ACTH) receptor on mouse Y-1 adrenal cells. Using immunoaffinity chromatography the ACTH receptor was purified, and the molecular structure and 125I-ACTH binding characteristics were determined. A molecular weight (Mr) of 225 000 was determined for the complete ACTH receptor as analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The receptor was composed of 4 subunits with Mr 83 000, 64 000, 52 000 and 22 000. The 83 and 52 kDa subunits were disulfide linked and non-covalently associated with the 64 and 22 kDa subunits. The ability to specifically bind 125I-ACTH was localized to the 83 kDa subunit. The purified receptor possessed binding affinities of 3.4 X 10(10) M-1 and 1.0 X 10(9) M-1 as determined by Scatchard analysis.

Regions of complementarity between the messenger RNAs for epidermal growth factor, transferrin, interleukin-2 and their respective receptors

Messenger (m)RNA sequences complementary to the mRNA sequences for the receptors to epidermal growth factor (EGF), interleukin-2 (IL-2) and transferrin (TF) were written out and compared for homologies with their ligands (EGF, IL-2 and TF, respectively). Highly significant amino acid and nucleotide homologies between the ligands and their appropriate receptor complements were detected in each case. For example, EGF and its receptor complement contained two homologous segments, each being six amino acids in length. When these segments were screened for matches against a protein sequence bank (3060 proteins and 616,748 test segments), only EGF contained either sequence. Similar results were obtained with IL-2 and TF. In each case, the homologous segments corresponded to complementary regions in the ligand binding portion of the receptor.

Enhancement of the in vitro antibody response by thyrotropin.

The pituitary hormone thyrotropin (TSH) has been shown to enhance in a dose dependent manner the in vitro antibody response. Highly purified preparations of bovine and human TSH enhanced up to 375% the number of cells producing antibody to sheep erythrocytes. TSH had to be present prior to 24-48h of the initiation of culture for enhancement of the antibody response. An analogy is discussed between TSH and B lymphocyte growth and differentiation factors.

The Pituitary-Adrenocortical Axis and the Immune System

Recent findings indicate that interactions between the pituitary-adrenocortical axis and the immune system involve more than simply the effects of glucocorticoid hormones. This altered view has resulted from the observations that: 1) cells of the immune system have receptors for and are directly acted upon by ACTH and endorphins, and 2) the immune system is an important non-pituitary source of these peptide hormones. In this chapter, while we review in a cursory way the older findings with glucocorticoid hormones, we concentrate on the newer developments which suggest that leukocyte- and pituitary-derived ACTH and endorphins perform regulatory functions within and between the immune system and the pituitary-adrenocortical axis.

Separation of mitogen-induced suppressor and helper cell activities during inhibition of interferon production by cyclic amp.

Abstract The effect of exogenous cyclic AMP on mitogen-induced suppression and enhancement of the in vitro plaque-forming cell (PFC) response and on mitogen induction of immune interferon (also called type II) in cultures was examined. Mitogen induction of immune interferon was quantitatively associated with mitogen-induced suppressor activity, and cyclic AMP blocked both the suppressor activity and the production of immune interferon in mouse (C57B1/6) spleen cell cultures. The evidence is as follows: (a) The concentrations of dibutyryl cyclic AMP that blocked T-cell mitogen (staphylococcal enterotoxin A) suppressor activity were the same as those that blocked mitogen induction of immune interferon. (b) The blocking action of dibutyryl cAMP on both the suppressor and interferon effects of mitogen was a function of the time of dibutyryl cAMP addition to cultures relative to mitogen addition. (c) A dramatic immunoenhancing effect of mitogen occurred in the presence of dibutyryl cAMP under conditions that blocked production of immune interferon. Specifically, mitogen-induced helper cell function is dramatically enhanced in the presence of dibutyryl cyclic AMP, if the mitogen is added to cultures 24 to 48 hr after SRBC and dibutyryl cyclic AMP. Dibutyryl cyclic GMP did not affect the mitogen- or cyclic AMP-induced effects under the conditions of our test system. Under the conditions described here, then, cyclic AMP appears to selectively block suppressor cell activity while allowing or aiding mitogen-induced helper cell activity. It is possible that the immune response is a reflection of the ratio of helper to suppressor activities in the system.

A Complete Regulatory Loop between the Immune and Neuroendocrine Systems Operates Through Common Signal Molecules (Hormones) and Receptors

Infection of lymphocytes with Newcastle disease virus induces the cells to synthesize immunoreactive (ir) adrenocorticotropin (ACTH) and endorphins. The irACTH is synthesized de novo, and common properties of lymphocyte and pituitary ACTH include: antigenicity, bioactivity, molecular weight, and retention time on reverse phase high-pressure liquid chromatography. The irACTH appears to be active in vivo because a rise in serum corticosterone levels in hypophysectomized mice corresponds with spleen cell production of irACTH. Furthermore, preliminary experiments showed that B cell depletion blocked the normal rise in serum corticosterone levels after herpes simplex virus infection of intact mice. It seems that a similar system operates in vivo in humans. Typhoid vaccine, which induces lymphocyte-derived irACTH production in vitro, caused a time-dependent increase in the number of irACTH-positive lymphocytes in both hypopituitarism and normal short children. A rise in serum cortisol levels was seen in one patient with hypopituitarism and all normal patients. The above regulatory circuit also seems able to act in the reverse direction. Pituitary ACTH and alpha-endorphin can behave like lymphokines by being immunosuppressive at 0.5 microM in an in vitro antibody synthesis system. Further, lymphocytes were shown to have high-affinity receptors for both of these hormones. Thus, it appears that the immune and neuroendocrine systems have the ability to signal each other through common or related peptide hormones and receptors.

Generation of a soluble IFN-γ inducer by oxidation of galactose residues on macrophages

Abstract Depletion of macrophages from human peripheral blood mononuclear cells (PBMC) caused a marked decrease in galactose oxidase and sodium periodate, but not a calcium ionophore, stimulated Interferon-γ (IFN-γ) production. Reconstitution of such depleted cultures with galactose oxidase treated macrophages, but not lymphocytes, restored IFN-γ levels to those of control nonfractionated PBMC. Thus, galactose oxidase seemed to act on macrophages which in turn stimulated lymphocyte production of IFN-γ. Unlike human cells which have terminal galactose residues on glycoproteins, murine cell glycoproteins terminate their oligosaccharide component in the order N -acetyl-neuraminic acid followed by d -galactose, N -acetyl-glucosamine, and glycoprotein. Galactose oxidase or sodium periodate only activated murine macrophages to stimulate lymphocyte IFN-γ production after exposing d -galactose residues by the removal of the terminal N -acetyl-neuraminic acid residues with neuraminidase. Removal of such exposed terminal galactose residues with β-galactosidase inhibited the effect of galactose oxidase on murine macrophages. Taken together, these results strongly suggest that oxidation of terminal galactose residues on macrophages is the initial site of action of galactose oxidase and sodium periodate. Studies with Boyden chambers have shown that galactose oxidase-treated macrophages released a soluble factor which stimulates lymphocyte production of IFN-γ. Based on these findings, it appears that the oxidation of terminal galactose residues on the surface of macrophages leads to the induction and transmission of a soluble signal for lymphocyte production Of IFN-γ.

Vitamin A inhibition of polyoma virus replication.

Vitamin A (retinoic acid) inhibited polyoma virus replication in confluent mouse embryo cells. A significant, dose dependent inhibition was observed when cell monolayers were pretreated with concentrations of vitamin A (10(-8) to 10(-6) M) thought to approximate those found in vivo. This inhibitory effect could be reduced by increasing the input multiplicity of infection. Growth curves of polyoma virus in the presence and absence of vitamin A suggested that vitamin A actually inhibited, and did not simply delay, virus replication. The cell density dependence of this inhibitory effect suggested its association with the prevailing level of cellular DNA synthesis. Vitamin A caused a significant decrease in overall (viral plus cellular) DNA synthesis. Other viruses which do not require induction of host cell DNA synthesis for their replication in confluent, non-dividing cells were not inhibited by vitamin A. These results are consistent with the known inhibitory effects of vitamin A on papovavirus infection in vivo and suggest a mechanism of vitamin A action at the level of the infected cell.