Charles J. Grossman

Possible underlying mechanisms of sexual dimorphism in the immune response, fact and hypothesis

It is a confirmed fact that in females both the humoral and cell mediated immune response is more active than in males. A large amount of information supports the view that hormones of the endocrine system are intimately involved in this immunological dimorphism. Such hormones include the gonadal steroids, the adrenal glucocorticoids, growth hormone (GH) and prolactin (Prl) from the pituitary, thymic hormones, and substances generated by activated lymphocytes. It is suggested that a complex medley of these hormonal interactions effect both developing lymphocytes within the microenvironment and regulate adult effector cells. The most important of these hormonal interactions leading to immunological dimorphism are the effects elicited by estrogen (E) elaborated at elevated levels from the female ovary after puberty. Elevated E leads to basal GH secretion, increased Prl, and increased thymosin release, all of which are hypothesized to effect lymphocyte development and stimulate adult T- and B-cell function in females. Interactions of hormonal regulatory axes involving the hypothalamus, pituitary, gonads, adrenals, and thymus are also thought to be involved. Factors elaborated by activated immune cells including IL-1 and IL-2 may also play a role in down regulation of these responses. Finally, genetic components are also considered pertinent especially under conditions of pathological disequilibrium leading to autoimmune disease. While the benefits provided by immunological dimorphism are still not entirely clarified, since sex hormones are intimately involved in immunological regulation it is quite possible that the increased immune response in females allows them to compensate for the increased physiological stress which accompanies reproduction. The final outcome would thus be the assurance of reproductive success of the species.

Sex steroid regulation of autoimmunity

The immune response of males and females is not identical but instead has been shown to be dimorphic in its nature, with females generally demonstrating a greater overall response than males. This dimorphism extends to both the humoral and cell mediated systems and appears to be mechanistically based on the differences in type and concentration of sex steroids in males vs females. Furthermore, growth hormone and prolactin secretions which are different in males and females may also be partly responsible for the observed dimorphism. Because autoimmune disease results from a pathological perturbation of normal immune function, it follows that expression of these diseases will also demonstrate a dimorphic pattern. Examples of this autoimmune dimorphism include (but are not limited to) lupus, rheumatoid arthritis and multiple sclerosis with the two former more prevalent in females than males and the latter more severe during pregnancy. To explain autoimmune dimorphism it therefore becomes necessary firstly to describe the cellular and hormonal interactions found in normal immune regulation and thereafter extrapolate these to autoimmune phenomena.

Rat thymic estrogen receptor—I. Preparation, location and physiochemical properties

Specific, high affinity estrogen receptor has been shown to be present in the thymus tissue from both rat and bovine sources. The receptor is located in the reticuloepithelial portion of the organ in the rat and is not present in the T-lymphocyte fraction. The equilibrium association constant and concentration of specific estrogen receptor was determined in thymic cytosol by Scatchard plot analysis. The equilibrium association constant (KA) for bovine was 1.72 ± 0.12 × 109M−1 and for rat was 7.14 ± 0.52 × 109M−1. The receptor concentration for bovine thymus was 0.298 ± 0.03 pmol/g tissue and rat was 0.362 ± 0.017 pmol/g tissue or for bovine 6.57 ± 0.13 fmol/mg protein and for rat was 7.18 ± 0.61 fmol/mg protein. From competition assays, the receptor possessed specificity for estradiol and the estrogen-like compound diethylstilbestrol, but not for progesterone, testosterone, triamcinolone or cortisol, Dihydrotestosterone was shown to bind slightly (0.04% with respect to estradiol) and only at high concentrations (4 × 10−6M). By sucrose gradient centrifugation studies the thymic estrogen receptor was shown to have a sedimentation value of 7–8s in low salt or 3–4s in high salt buffer. A possible mechanism for estradiol suppression of the cell mediated immune system is discussed.

Rat thymic dihydrotestosterone receptor: Preparation, location and physiochemical properties

Castration in the male rat has been shown to produce enlargement of the thymus gland while treatment with dihydrotestosterone (DHT) results in a decrease in thymic size in these animals. To determine if these changes might be receptor mediated, thymus tissue from castrate male rats was removed and homogenized in buffer and centrifuged to produce cytosol. By Scatchard plot analysis, it was shown that a specific DHT receptor was present at a concentration of 0.24 +/- 0.02 pmoles/g tissue and it possessed a KA of 2.51 +/- 0.45 x 10(9)M-1. This thymic DHT receptor sedimented on 5--20% sucrose gradients in the 8s region. By competition analysis it was found that testosterone only partially competed (25%) for this receptor, with virtually no binding noted for estradiol, progesterone, and cortisol. The receptor was found to be localized in the reticuloepithelial matrix of the thymus and was not present in the thymic lymphocyte fraction.

Estradiol regulation of thymic lymphocyte function in the rat: Mediation by serum thymic factors

Estradiol (E2) can depress the function of the thymic lymphocytes. To determine if this response to a gonadal steroid is regulated directly or indirectly, thymic lymphocytes were incubated in vitro for 3 days in the presence of the mitogens concanavalin A (Con A) or phytohaemagglutinin (PHA) + tissue culture media + 20% specific rat serum fractions and pulse labelled with tritiated thymidine. Rat serum fractions were prepared from control, castrate, thymectomized (Tx) and castrate-Tx animals as well as from similar groups of animals treated in vivo for three days with physiological doses of E2. It was found that there was a significant enhancement of thymocyte blastogenesis in cultures incubated with castrate rat serum + Con A or PHA vs. control serum cultures (P less than 0.001). Direct replacement of E2 to castrate sera in vitro at physiological concentrations failed to depress thymocyte blastogenesis to noncastrate levels. Sera prepared from castrate animals treated with E2 at physiological concentrations was successful in depressing the blastogenic response to noncastrate levels. Sera from Tx animals did not enhance Con A induced blastogenesis, but PHA response was significantly increased (P less than 0.01) This effect was lost utilizing sera from Tx animals treated with E2. It is concluded that thymocyte function is regulated by serum factors which have their origin in the thymus, and that these factors are modulated by a gonadal steroid estradiol.

Hepatitis B vaccination

Alcoholics are at risk to develop hepatitis B infections, chronic active hepatitis, and even hepatoma. Hence, immunization with hepatitis B vaccine is recommended. However, immune abnormalities may coexist which alter their responsiveness to vaccination. This study compares the immune response to this vaccine in controls (group I), alcoholics without overt liver disease (group II), and alcoholics with clinical liver disease (group III). By the seventh month after the initial vaccination, 89% in group I, 70% in group II, and 18% in group III had a response >36 RIA units. The magnitude of the response was significantly different in groups I, II, and III (19,456 vs 8,326 vs 153 RIA units, respectively; P <0.05, group I vs III). In those who did not respond, a significant (P < 0.02) lower helper/inducer (T4)class of lymphocytes was observed as compared to patients who exhibited an adequate response. These observations suggest: (1) that the response to hepatitis B vaccine is a T-cell-dependent event and (2) that in this population, using the existing vaccine, postvaccination evaluations of antibody concentrations are needed before protection against hepatitis B infection can be assumed.

Rat thymic estrogen receptor--II. Physiological properties.

Specific, high affinity estrogen receptor has been shown to be present in the rat thymus. From in vivo and in vitro studies as well as competition assays, the receptor possesses specificity for estradiol and the estrogen-like compound diethylstilbestrol, but not for progesterone, testosterone, triamcinolone or cortisol. By sucrose gradient centrifugation studies the thymic estrogen receptor was shown to have a sedimentation value of 7–8s in low salt buffer. Castration resulted in a decrease in thymic estrogen receptor in terms of pmol/g tissue or fmol/mg soluble protein but no change in terms of pmol/mg DNA. Estradiol injection in vivo resulted in a rapid decrease of measurable cytoplasmic receptor by 2.5 min, which became undetectable by 20 min and returned to control values by 24 h. Autoradiographic studies show that the radioactive label is concentrated in single cells which may be of the reticulo-epithelial variety.

Biphasic in vivo immune function after low- versus high-dose alcohol consumption

A series of experiments was performed to assess the alterations in immune status in vivo that are associated with differences in the amount and duration of ethanol intake. Using a nonspecific delayed cutaneous hypersensitivity-like response to the intradermal injection of phytohemagglutinin, the area of induration (skin test response) was significantly enhanced (p = 0.008) after low-dose ethanol (0.5 g/kg) administered daily by gastric gavage for 5 days. High-dose ethanol (6.0 g/kg) significantly diminished this response (p = 0.03). Using an experimental model of Mycobacterium bovis hepatitis, the host immune response was also altered in a biphasic manner after chronic, 28-day ethanol consumption. With this model 0.43 +/- 0.03 g/kg/day (mean +/- SEM) of ethanol (low dose) was associated with a 40% improvement in the removal of the organisms from liver tissue (p = 0.002). High dose (12.1 +/- 0.5 g/kg/day) impaired removal, resulting in a 55% increase in the number of viable organisms (p = 0.001). The levels of three cytokines, MIF, TNF-alpha, and IL-2, known to be involved in the modulation of the host response to mycobacterial infections, were measured in sera after the infection. The serum levels of these cytokines in response to infection did not correlate with this biphasic response to different alcohol dose levels.

The interrelationship of the HPG-thymic axis and immune system regulation

It is well established that gonadal steroids play an important role in the regulation of immune response. These substances exert their effect through estradiol and possibly dihydrotestosterone receptors located in the thymic reticuloepithelial cell matrix. In vivo treatment of male rats with estradiol has been shown to depress the cell mediated immune response as mediated through thymic serum factors. Castration stimulates the cell mediated immune response while thymectomy inhibits this effect. It also appears that growth hormone at physiological concentrations can directly inhibit thymocyte blast transformation in vitro while leutinizing hormone, follicle stimulating hormone, and prolactin do not directly affect thymocyte function. A tentative scheme for immune system regulation by the HPG-thymic axis is presented.

Alcohol and immune regulation I. in vivo effects of ethanol on concanavalin a sensitive thymic lymphocyte function

Alcohol is known to suppress the immune response, but the underlying mechanism to account for this immune suppression is still not clearly elucidated. In an attempt to clarify such mechanisms, experimental rats were fed for 50 days on a 36% ethanol, Lieber diet (LED) while control (LCD) rats were fed a similar diet supplying the same amount of calories but lacking ethanol. It was found that both LCD and LED animals grew at a linear rate (LCD: r = 0.981, LED: r = 0.961) but that LCD animals grew more rapidly. While thymic weights in the LED group were significantly smaller (P less than 0.05) than in the LCD group, the ratios of thymic weight/body weight between these groups were not significantly different. To identify the effects of ethanol on immune response, thymic (Th) or splenic (S) cells were prepared and incubated in culture with the mitogen, Con A and rat serum prepared from LCD or LED groups. It was found that lymphocytes prepared from thymus of LED animals appeared to be depressed in mitogen-driven blastogenic transformation when incubated in LCD serum but not LED serum. Furthermore, lymphocytes prepared from the spleen of LED animals appeared to be depressed in mitogen driven blastogenic transformation when incubated in LED serum but not LCD serum. Since lymphocytes of the thymus and spleen are undergoing maturation and replication this implies that ethanol may alter these processes.