Alloreactivity. I. Effects of age and thymic hormone treatment on cell-mediated immunity in C57B1/6NNia mice

C57B1/6NNia mice 1, 12, and 24 months old showed loss of cellular-mediated cytotoxicity with aging. Treatment of the three age groups with different thymic hormone preparations effected their cellular mediated cytotoxicity differently. When cytotoxicity of the thymic hormone treated groups was compared to that of the physiological saline treated group, 1-month-old mice treated with serum thymic factor (FTS) at 1 microgram/mouse and 10 ng/mouse had significantly higher activity, and lower to similar activities at 12 and 24 months; TP5 (active fragment of thymopoietin) at 1 microgram and 10 ng caused significantly higher activity in 1-month-old mice, and lower to higher and significantly lower to similar activity at 12 and 24 months, respectively; TM4 (an analogue of TP5) at 1 ng showed significantly depressed activity in 1-month-old mice, and significantly enhanced activity in 12- and 24-month-old mice; thymosin at 10 micrograms and 1 microgram had slightly lower, but not significant, depression at 1 month, similar activities at 12 months and significantly depressed to higher activity at 24 months. Unimmunized control mice showed significant protection in the 12-month-old mice in comparison to 1- and 24-month-old mice. Different hormone preparations showed age- and dose-dependent effects on the ability of spleen cells to kill P815 mastocytoma. Partial restoration of cytotoxicity was observed in 24-month-old mice treated with FTS, TP5 and thymosin fraction V.     

Heterogeneity of mitogen-responsive lymphocytes in carp (Cyprinus carpio)

Peripheral blood leukocytes (PBL) of carp respond in vitro to a variety of phytomitogens, shown to be T-cell specific or B-cell specific in mammalian systems. Some basic differences have been observed in the proliferative response of carp PBL to PHA (phytohemagglutinin), ConA ( concanvalin A) and LPS (lipopolysaccharide): (1) The response to PHA and ConA was found to be highly dependent upon the continuous presence of mitogen in the medium, in contrast to LPS, where after the initial stimulation, cells could continue to proliferate for several days without mitogen. (2) Lymphoblasts grown in long term culture with either PHA or Con A could be transferred into medium containing the other mitogen without impairing cell proliferation, but cell growth was reduced to background level following transfer into LPS-containing medium. LPS grown cells continue to proliferate independently of the mitogen content of the medium. (3) Co-stimulation with LPS+PHA or LPS+ConA results in a synergistic response, while co-stimulation with PHA+ConA results in inhibition of DNA synthesis. (4) Several morphological differences have been observed between cells proliferating in the presence of PHA and those proliferating in the presence of LPS. It is suggested that while the PHA and ConA responsive cells may belong to the same lymphocyte subpopulation, they are distinct from the LPS-responsive subpopulation.     

T cell hyperproliferation in autoimmunity prone obese strain (OS) chickens is independent of abnormal mitogen binding in vitro and can be demonstrated in vivo

In contrast to systemic autoimmunity, spontaneous autoimmune thyroiditis of Obese strain (OS) chickens is associated with a marked T cell hyperreactivity in vitro, i.e. an increased proliferation and interleukin 2 (IL 2) secretion in response to Concanavalin A (ConA). In the present study we report an enhanced capacity of OS peripheral lymphoid cells (splenocytes and peripheral blood lymphocytes, PBL) to adsorb fluorescein isothiocyante (FITC) labelled ConA, but not phytohemagglutinin (PHA). However, the elevated ConA binding cannot be a prerequisite for in vitro ConA hyperreactivity as OS thymocytes are normal with respect to ConA binding but nonetheless exhibit elevated responses to this mitogen. Moreover, ConA binding does not correlate with the frequency of cells able to express IL 2 receptors upon short term ConA stimulation. The percentage of ConA activatable cells was found to be increased in OS- PBL as compared to normal control PBL, but was unaltered in OS splenocytes. This finding points to a further mechanism of T cell hyperreactivity in OS chicks in addition to the previously reported defects in nonspecific immunosuppression. Finally, enumeration of cells in the S phase revealed that enhanced proliferation of OS T lymphocytes was not restricted to the in vitro response to ConA and phytohemagglutinin (PHA) but also occurs in vivo.     

Lymphocyte cultures with small numbers of cells.

Prior experience with cultures of cerebrospinal fluid lymphocytes indicated a need to develop methods for culturing small numbers of cells. Peripheral blood lymphocytes (PBL) were obtained from 20 normal volunteers. Standard microcultures using 100 X 10(3) PBL/0.2 ml and cultures with 50, 25 and 12.5 X 10(3) in 0.1 ml or 0.2 ml were established in RPMI 1640 with autologous plasma. These cultures were incubated with PHA (1--30 microgram) for 3, 4 and 5 days, pulsed with [3H]thymidine and harvested. In unstimulated cultures, cpm declined linearly with decreasing cell numbers. Standard cultures (100 X 10(3) PBL/0.2 ml) had maximal PHA stimulation (80,916 +/- 6394) at day 3 with 30 microgram PHA. Other 0.2 ml cultures had lower cpm. By culturing 25 X 10(3) PBL in 0.1 ml for 3 days cpm were 82,874 +/- 6875 with 30 microgram PHA and 77,153 +/- 6022 with 15 microgram PHA and were similar to standard cultures. Similar cpm were seen with 12.5 X 10(3) PBL in 0.1 ml after 4 days with 30 micrograms of PHA (80,838 +/- 6674) and with 15 micrograms of PHA (72,860 +/- 6243), and also after 5 days with 30 micrograms of PHA (86,703 +/- 6732) and with 15 micrograms of PHA (74,066 +/- 6388). The maximal response (126,578 +/- 6580) was seen with 25 X 10(3) PBL/0.1 ml at day 4 with 30 micrograms of PHA. By decreasing culture volume to 0.1 ml and increasing time, the number of cells necessary to give PHA responses similar to standard cultures can be reduced by 75--88%.     

Origin and immunological hyporeactivity of canine alveolar lymphocytes.

Autochthonous canine thoracic duct lymphocytes were isolated, labelled with 111indium and injected intravenously. Direct sampling showed that the label passed from blood to lymph within 25 hr. By gamma camera imaging, the initial accumulation of 111In the liver and spleen was followed by a striking increase in lymph node associated activity between 24-36 hr. Although initial lung-associated counts were low, a bicarbonate-induced non-specific inflammation of the right lung induced a rapid and selective accumulation of labelled lymphocytes (ratio right to left lung 8:1). Cell suspensions lavaged from the alveolar surface showed only a 9% response to phytohaemagglutinin (PHA) compared to peripheral blood lymphocytes (PBL). The addition of unfractionated lavage cells to PBL caused a macrophage-dependent suppression of mitogen responsiveness. However, macrophage depletion of alveolar lavage cells did not restore the lymphocyte response to PHA. Similarly, the frequency of alloreactive precursors of cytolytic lymphocytes (CTL) was almost 10-fold less in alveolar lymphocytes compared to PBL. Thus, bronchoalveolar lavage provides a technique by which viable cells may be recovered in significant numbers from the alveoli and should be invaluable for the investigation of lung allograft rejection.     

Noncytolytic human lymphocytes injure dermal microvessels in the huPBL-SCID skin graft model

Recent transplantation experiments using perforin-deficient mice as allograft recipients have challenged the concept that allograft rejection is mediated exclusively by CTL. We sought to determine if human noncytolytic lymphocytes could mediate rejection of allogeneic human skin grafts in the huPBL-SCID mouse model of rejection. We generated short term lines of human lymphocytes from peripheral blood mononuclear cells using PHA as a mitogen. The first group was stimulated with PHA alone, the second with PHA plus IL-4 and neutralizing antibody to IL-12, and in the third group PBL were depleted of B cells and monocytes before stimulation as in group 2. After two passages, lines were tested for cytolytic ability and IFN-gamma production. Each line was injected i.p. to mice bearing allogeneic skin grafts. The grafts were harvested between day 16 and 21 after PBL injection, then the histology was scored by a blinded observer for degree of infiltration, microvessel injury, induction of epidermal MHC class II, and perforin expression. In vitro we found that PBL in groups 2 and 3 were unable to lyse cultured endothelial cells in a lectin-directed 111In release assay. In vivo 80% of the IL-4/anti-IL-12 groups maintained the IFN-gamma-low phenotype, and no perforin was detected in these grafts. Nevertheless, human microvessel injury was similar between the two groups. This was not antibody-dependent since the B-cell-depleted group showed similar injury. Moreover adjacent murine vessels were intact. We interpret these observations to show (1) these human PBL lines maintained their phenotype following in vivo restimulation, and (2) noncytolytic graft-infiltrating lymphocytes specifically promote injury of allogeneic human microvessels.     

Selective inhibition of the in vitro murine B lymphocyte response by pentamidine

A study was undertaken on the immunomodulating properties of pentamidine, a diamidine used in the treatment of African trypanosomiasis, pneumocystosis and leishmaniasis. Pentamidine inhibited the ability of mouse splenic lymphocytes to respond to mitogens. In particular inhibition of the B lymphocyte response was observed. At concentrations of 1 microgram/ml (1.7 x 10(-6) M), pentamidine markedly inhibited the response to the B cell mitogen, lipopolysaccharide (LPS), while concentrations of 10 micrograms/ml had to be attained to produce a similar effect on the response to the T cell mitogens phytohaemagglutinin (PHA) and concanavalin A (ConA). Further studies showed that pentamidine was not toxic to either resting or proliferating cells and probably acts by interacting directly with B cells rather than modifying the regulatory cell populations.     

Mitogenic responses of splenic B and T lymphocytes in neonatally capsaicin-treated mice

The proliferative response of spleen cells from neonatally capsaicin-treated mice to 5 mitogens (LPS, PWM, dextran sulfate, ConA and PHA) were tested. Both B- and T-lymphocyte responses were essentially unaffected by the capsaicin treatment, which is known to destroy certain small-sized neuropeptide containing primary sensory neurons. However, the capsaicin-treated mice displayed a shift in the dose response to PHA so that the maximal response was obtained with a lower dose of the mitogen. The results exclude major effects of the affected sensory neurons on the development or expression of immune competence, but point to a possible modulatory effect on some step in the response to PHA.     

The immunomodulatory activity of the plant proteins Momordica charantia inhibitor and pokeweed antiviral protein

The immunological activity of Momordica Charantia inhibitor (MCI) and of Pokeweed antiviral protein (PAP-S), 30,000 daltons plant proteins possessing close similarity to Ricin A chain as inhibitor of protein synthesis, was investigated in mice. In vivo, single nontoxic injections of microgram amount of these substances delayed H2-incompatible skin allograft rejection, splenocyte responsiveness to ConA and PHA, but not to LPS, and abrogated the PFC response to a T-dependent (SRBC) antigen while totally sparing that to a T-independent (S III) stimulus. Injection of these substances could also reduce NK cell activity while increasing macrophage-mediated spontaneous cytotoxicity. In vitro, MCI and PAP-S at non-cytotoxic concentrations inhibited lymphoid cell responsiveness to PHA and ConA, but not to LPS, and markedly enhanced macrophage-dependent cytotoxicity.     

Induction of lymph follicle formation with endotoxin lipopolysaccharide in the draining lymph node of athymic nude mice

Formation of lymph follicles in draining popliteal lymph nodes in athymic nude mice and their phenotypically normal littermates (PN mice) was investigated after footpad injection with either endotoxin lipopolysaccharide (LPS) or phytohemagglutinin in soluble and precipitated forms (PHA). The dose of LPS injected ranged from 10 to 100 micrograms, and that of PHA was 50 micrograms. After any dose of LPS, draining nodes of nude mice produced new lymph follicles in the peripheral cortex outside pre-existing follicles, though the number of follicles induced was somewhat less than in the case of PN mice treated with LPS. After injection of PHA in soluble or precipitated form, PN mice developed a significant number of new follicles in the draining nodes, but nude mice failed to do so. The present results are consistent with the view that nude mice have the ability to develop lymph follicles by way of a thymus-independent mechanism in response to exogenous stimuli.     

Influences of sodium diethyldithiocarbamate, DTC (imuthiolR) on T cell defective responses of aged BALB/c mice

The effect of chronic imuthiol treatment, 25 mg/kg weekly for 4 months initiated at the age of 12 months, on T-cell functions of aged female BALB/c mice was investigated. Imuthiol restored to normal value the impaired response to Concanavalin A (Con A) and enhanced the proliferative response to phytohemagglutinin (PHA). Impaired cytotoxic T-cell activity (CTL), was restored near to the value of young controls by imuthiol. Serum thymic factor (FTS) levels in serum of treated aged animals outpassed those of untreated young mice. Delayed-type hypersensitivity (DTH) reaction to oxazolone was increased. In contrast, the graft-vs-host (GVH) mortality induced by injecting H-2 histoincompatible cells to irradiated recipients, which GVH was impaired by aging, was not significantly modified by imuthiol. The excessive cytotoxicity for chicken red cells of macrophages (ADCC) from aged mice was reduced, as well as macrophage cytotoxicity for tumor cells. Natural killer cell activity remained unchanged. This finding confirms that imuthiol enhanced effectively T cell-dependent responses but the data on GVH reaction suggest that its effects are under a complex mode of action. Restoration of a normal production of FTS may be one mechanism by which imuthiol acts on the reinduction of the T-cell differentiating pathway in aged mice.     

Decline in mitogen induced proliferation of lymphocytes with increasing age.

We previously reported that mitogen-induced proliferative responses of lymphocytes in the elderly were significantly lower than in young individuals. To determine when this decline occurs, we evaluated the responses of 26-30 subjects of each decile from the third to the tenth decile to the T cell mitogens, phytohaemagglutinin (PHA) and concanavalin A (ConA), and the T dependent B cell mitogen, pokeweed mitogen (PWM). There was a significant decrease in the responses of the 70-, 80- and 90-year-olds to PHA and ConA (less than 40% of the 20-year-olds; P less than 0.01). The 80- and 90-year-olds also showed a decreased response to PWM (approximately 50%; P less than 0.01). The 60-year-olds showed a decreased response to all three mitogens but only the PHA and ConA responses were significantly decreased (P less than 0.01). The 50-year-olds showed a decreased response to ConA, while the 40-year-olds showed decreased responses to both PHA and ConA; both significant at P less than 0.01. The decreased response of the 40-year-olds, however, was only seen in the females. This may be due to the hormonal changes associated with menopause. The general trend of the data suggests that there is a gradual decrease in mitogen-induced proliferative responses with increasing age, large differences become apparent at the age of 60, with a further decrease in the 70s, and most importantly, they remain fairly constant thereafter. Of interest is that only three of the 111 subjects less than 60 years old failed to mount a proliferative response and in each case this was to only one mitogen, while 42 of 118 subjects greater than 60 years old did not respond to at least one mitogen. Ten of these older subjects (2/28 of the 60-year-olds) did not respond to any of the three mitogens (P less than 0.01). This lack of response may be important since we have found a significant association between the lack of response to all three mitogens and increased mortality.     

Analysis of human lymphocyte transformation responses to graded doses of T cell mitogens by curve fitting

The between-group comparison of complete lymphocyte transformation dose-response curves is complex. We have therefore derived a mathematical model of the dose-response characteristics of human mononuclear cells to stimulation by concanavalin A (ConA) and purified phytohaemagglutinin (PHA), in order to simplify such analyses. This model describes dose-response curves in terms of the magnitude of the peak response, the dose of mitogen that elicits the peak and an estimate of the range of mitogen doses which induce a response. Responses to ConA were described by the model more precisely than those to PHA. Furthermore, use of the model revealed differences between anorexia nervosa patients and healthy subjects in terms of the dose of mitogen necessary to elicit a peak response and the range of mitogen concentrations producing a response. It is proposed that this form of mathematical treatment may be of use for the comparison of lymphocyte transformation dose-response curves and for the valid rejection of suspect results.     

In vitro effects of two immunopotentiators, Isoprinosine and NPT 15392, on murine T-cell differentiation and function

A new immunopotentiator, NPT 15392, was analyzed in vitro for its capacity to induce a T-cell marker (Thy-1 antigen) and to enhance the mitogen-induced lymphocyte proliferation. The activity was compared with that of Isoprinosine and a putative thymic hormone (thymosin Fr. V). Thymic hormone preparation (thymosin Fr. V) and the non-thymic agents (Isoprinosine and NPT 15392) induced significant percentages (20-30%) of Thy-1 positive cells in nylon wool-nonadherent nu/nu spleen cells. Maximum induction was observed the concentrations of 100 microgram/ml thymosin Fr. V, 1.0 microgram/ml Isoprinosine, and 0.1 microgram/ml NPT 15392. In addition, NPT 15392 or Isoprinosine significantly augmented proliferation of C57BL/6J spleen cells stimulated by the T-cell mitogens (Con A and PHA) and B-cell mitogen (LPS). The effect of both agents was greater on spleen cells from aged mice (18 mth) than on young mice (3 mth). The minimum effective concentration of NPT was lower than that of Isoprinosine. The data indicate that Isoprinosine and NPT 15392 have actions on precursor T cells, mature T cells, and probably B cells.     

The immunological activity of plant toxins used in the preparation of immunotoxins--II. The immunodepressive activity of gelonin

The immunological activity of Gelonin, a 30,000 dalton plant protein possessing close similarity to Ricin chain A as a protein synthesis inhibitor which may be of interest for the preparation of antibody-toxin conjugates, was studied in mice. At in vitro concentrations not affecting baseline radioactivity uptake, this substance reduced mitogen responses with the following order of sensitivity PHA less than ConA less than LPS. In microgram/ml concentrations it also markedly reduced macrophage-dependent cytotoxicity while not affecting NK activity. Macrophagic (but not NK) cytotoxicity and mitogen responses were similarly depressed after in vivo treatment. When given before (but not after) stimulus, Gelonin also reduced the primary responses to a T-dependent and, although to a lower degree, to a T-independent antigen, and decreased resistance to allogeneic tumor grafts and L. monocytogenes challenges. The immunopharmacological activity of this and similar substances should be considered in the design of antibody-toxin conjugates and in the evaluation of their therapeutic activity.     

Effects of long-term, low-dose growth hormone therapy on immune function and life expectancy of mice

We have studied effects of long-term, low-dose growth hormone therapy on the immune function and life expectancy of Balb/c mice. Sixty male Balb/c mice were aged up to the time when they started showing signs of senescence and causal death (deaths started when they became 17 months old). The aged mice were divided into two groups of 26 mice each. One group received growth hormone (30 micrograms/mouse) subcutaneously twice a week for 13 weeks. The control group received an equal volume of saline for the same period. During this treatment period, 16 control mice died (61%) whereas only 2 of the hormone-treated mice died (7%). Four mice from each group were killed and immunological functions of splenocytes were evaluated. Hormone-treated mice had higher stimulation indices for pokeweed mitogen but not for Concanavalin-A. Total IgG production was decreased but IL-1, IL-2 and TNF production was increased. After a lag period of 4 weeks, growth hormone therapy was continued for another 6 weeks. One of the growth hormone treated mice died while the control group no longer existed. Splenocyte functions of the growth hormone treated mice were compared to those of young mice. The results showed no significant difference between cytokine production (IL-1, IL-2, TNF and IgG) in the young and the hormone treated groups. Stimulation induced by concanavalin-A and pokeweed mitogen however, was higher in the young group than the old group. The mortality curve obtained suggests that long-term low-dose growth hormone treatment prolongs life expectancy.     

Carrageenan-induced suppression of chicken lymphocyte proliferation

Mitogenic responsiveness of peripheral blood lymphocytes (PBL) of chickens was suppressed by either pretreatment with or addition to the culture medium of various concentrations of carrageenan (CGN). Pretreatment for 1 hr significantly suppressed response to Concanavalin A (Con A) and Pokeweed mitogen (PWM) but did not affect Phytohemagglutinin (PHA) induced stimulation. Extension of the pretreatment period to 4 hrs suppressed response induced by all three mitogens. On the other hand, addition of carrageenan to the culture medium caused a dose-dependent suppression of PHA and Con A mediated response, but the effect on stimulation due to PWM was equivocal. In addition, low concentrations of CGN were weakly mitogenic to PBL and splenic lymphocytes.     

Lymphocyte proliferative responses in haemophiliac patients: relations to clinical and immunological findings

Blood lymphocyte proliferative responses to mitogens were studied in 65 patients with haemophilia (haemophilia A: 54 patients, haemophilia B: 11 patients) in parallel with 39 male control subjects. As a group, patients with haemophilia did not demonstrate abnormal proliferative responses to phytohaemagglutinin (PHA), Concanavalin A (ConA) and pokeweed mitogen (PWM) when compared with healthy controls. When the patients were analysed according to their seropositivity for antibody to human immunodeficiency virus (HIV), those who were positive had significantly decreased PHA, ConA and PWM responses. Haemophiliac patients with T4+/T8+ ratios less than 1 had reduced proliferative responses to PHA, ConA and PWM when compared to patients with ratios greater than 1. No significant difference in mitogen responses were found when the patients were analysed according to the presence or absence of palpable lymphadenopathy. Those patients with haemophilia A who had received more than 5 x 10(4) units of factor VIII during the two years preceding the study showed no significant difference in PHA, ConA and PWM responses when compared to patients receiving less.     

鸭外周血淋巴细胞转化试验的研究

用血清斑点杂交试验证实 DHBV DNA 阴性的芙蓉鸭,采其外周血分离淋巴细胞,用 PHA刺激,建立了鸭淋巴细胞转化试验。发现 PHA 浓度20μg/ml、细胞浓度3×10~6/ml、孵育温度以 41℃最适宜。观察到加 10％正常鸭血清刺激指数明显高于 10％小牛血清。同时研究了不同鸭龄(1周龄～12周龄)淋巴细胞转化能力,发现 1周龄鸭淋巴细胞在 PHA刺激下无转化能力,4周龄鸭已有部分鸭的淋巴细胞能转化,8周龄的鸭淋巴细胞均能转化,说明8周龄鸭细胞免疫已成熟。     

Lymphocyte response to T-cell mitogen during experimental gingivitis in humans.

This study was conducted to evaluate the dose response relationships of peripheral blood lymphocytes (PBL) by stimulation of phytohemagglutinin (PHA) during the onset of oral inflammation. Eleven dental students underwent a 3-week experimental gingivitis program (Löe et al., 1965). At time zero, weeks 1, 2, and 3, and after 1 week of reinstituted oral hygiene (week 4), the plaque accumulations were evaluated, the degree of gingival inflammation was assessed, and a blood sample was taken. Quadruplicate microcultures each containing 2 x 10(5) PBL in 0.2 ml of tissue culture medium 199 and 10% fetal calf serum were stimulated with five concentrations of PHA (10 to 0.5 mug/ml) and incubated for 78 h at 37 C in 5% CO2. [3H]thymidine was added to each culture for the final 8 h. The cultures were then harvested and counted by liquid scintillation, and stimulation indexes (SI) were determined. At time zero the maximum PBL response occurred at a PHA concentration of 5 mug/ml (SI = 100). During weeks 1, 2, and 3 the location of the maximum PBL response shifted to a lower PHA concentration (1.0 mug/ml) and increased to over SI =400. The phenomenon of shifting peak PHA responses to lower PHA concentrations could be observed after only 1 week of developing gingival inflammation. The PBL response returned to pre-experimental values after 1 week of reinstituted oral hygiene, which resolved the oral inflammation. The findings show that a dose response relationship exists between PHA concentrations and the PBL response. If these dose response changes seen during developing gingival inflammation are ignored, either a decrease, increase, or no change in PBL response can be shown depending upon the PHA concentration evaluated. Owing to the dose-dependent nature of this PBL response, it is advisable to routinely use dose response curves in order to properly evaluate the full responsiveness of PBL to mitogenic substances.