B-Lymphocyte Mediated Cytolysis: A Complement Independent Phenomenon

Spleen cell suspensions from C₅₇BI mice immunized intraperitoneally with sheep erythrocytes were highly cytotoxic towards the erythrocytes in vitro. The cytolysis was specific: human type 'O' erythrocytes were not lysed. Cytolysis was inhibited when the interacting cells were incubated in the presence of anti-K or anti-γ1 plus anti-γ2 antisera. Pre-treatment of the immune spleen cells with anti-immunoglobulin antisera in hhe presence or absence of complement completely inhibited their subsequent cytolytic activity, but had no effect on plaque-forming cell numbers. The cytolytic activity was unaffected by the removal of θ-antigen bearing cells or of glass-adherent cells (presumably macrophages).

In contrast, the lysis of DBA/2 mastocytoma cells by immune C₅₇B1 lymphocytes was unaffected by treatment with any of the anti-immunoglobulin reagents. The lytic activity of the spleen cell suspensions towards the mastocytoma cells was completely abolished by removal of θ-antigen bearing cells.

Both the B-cell mediated cytolysis of erythrocytes and the T-cell lysis of mastocytoma cells appeared to proceed independently of the complement system. Lysis in both cases was unaffected by the presence of an excess of antisera monospecific for complement components C₂, C₃ and C₅.     

Thymostimulin Administration Modulates Polymorph Metabolic Pathway in Patients with Chronic Obstructive Pulmonary Disease

Several studies outline the imbalance of phagocyte functions in chronic obstructive pulmonary disease (COPD). In this regard, here, we have assessed either monocyte- and polymorphonuclear cell (PMN)-mediated chemotactic, phagocytic and killing capacities or PMN-triggered metabolic pathway in a group of COPD patients before and at different times after thymostimulin administration. Before therapy, an increase of O₂-generation and a decrease of myeloperoxidase release were found in these individuals when compared to controls. Moreover, a reduction of either PMN-mediated chemotaxis and killing or monocyte chemotactic capacities was observed. By contrast, no differences were seen in terms of p-glucuronidase release, monocyte-mediated killing and PMN or monocyte phagocytic function. During a one-year monitoring following immunotherapy, O₂- production and myeloperoxidase activity fell within normal values, while phagocyte functional capacities were unaffected by such a treatment. Furthermore, COPD subjects exhibited a significant improvement of their clinical status as assessed during a one-year follow-up.

All together, these findings suggest a potential role for thymostimulin in the treatment of COPD patients.     

Effect of Macrophage Colony-Stimulating Factor (M-CSF) on Mouse Immune Responses in Vivo

We examined the effects of recombinant human M-CSF (rhM-CSF) on mouse macrophages and immune responses in vivo. Intraperitoneal administration of rhM-CSF (20-500 μg/ml) increased Mac-l⁺ cell numbers in the peritoneal cavity. The tumoricidal activities of the macrophages from vehicle-administered (V-MΦ) and from rhM-CSF-administered (M-MΦ) mice were the same as those observed in vitro. However, when activated by lipopolysaccharide (LPS), the tumoricidal activity of M-MΦ was stronger than that of V-MΦ. Intravenous administration of rhM-CSF (500 μ g/gk) increased the number of spleen cells. Flow cytometric analysis showed that administration of rhM-CSF increased Mac-1⁺, B220+ and NK 1.1⁺ cell counts in the spleen. However, CD4⁺ and CD8⁺ cell numbers did not change. Concomitant increases were observed in levels of IL-4 and IL-10 in mouse serum following rhM-CSF administration, but no significant changes were observed in the serum level of IFN-γ.

In experiments involving mouse immune responses, the administration of rhM-CSF reduced the contact sensitivity (CS) reaction against picryl chloride (PC) and augmented IgE production in response to 2,4-dinitrophenyl (DNP), but did not affect the production of either IgM or IgG 1.

These results suggest that administration of rhM-CSF not only activates murine macrophages, but modulates antigen-specific immune responses in vivo.     

In Vivo Capsaicin Treatment Inhibits Rat NK Cell Cytotoxicfunctions

The direct and indirect interactions between the nervous system and its transmitters with NK cell cytotoxic functions has been evaluated in the rat by using the neurotoxin capsaicin (8-methyl-N-vanillyl-6-nonenamide). When administered to neonatal rats, capsaicin (50 mg/Kg in 10% ethanol and 10% tween 80 at 2 days of age) interferes with the synthesis and intraneuronal transport of peptides by causing irreversible degeneration of c fiber afferent nerves.

Capsaicin treatment resulted in a marked inhibition of NK and ADCC activities both in the spleen and peripheral blood. Inhibition was already evident on day 15 after treatment and persisted until day 90 in the spleen; at this time NK cytotoxicity in the peripheral blood returned to control levels.

The inhibitory effect of capsaicin treatment on peripheral blood NK and ADCC activities was associated with changes in NK cell number evaluated as percentage of cells with an LGL morphology and expressing the NK-RP1 cell surface receptor. LGL numbers did not always correlate with the percentage of NK-RP1⁺ cells suggesting that capsaicin may interfere with maturation of lytic effector cells. Overall these results indicate a direct influence of the nervous system on natural immune cytotoxic functions.     

The Lack of Antigenicity of Tuftsin: A Naturally Occurring Phagocytosis Stimulating Tetrapsptide

Tuftsin (Thr-Lys-Pro-Arg) is a naturally occurring tetrapeptide that stimulates all known functions of the polymorphomaclear. leukocyte and macrophage cell lines. Tuftsin is located in the F_c region of IgG between the 289 and 292 amino acid sequence of the CH₂ domain. We describe unsuccessful attempts to generate antituftsin antibodies. In separate experiments tuftsin was chemically conjugated to methylated bovine serum albumin (CH₃ BSA), BSA, keyhole limpet hemocyanin (KLH) and purified protein derivative (PPD). Tuftsin was also polymerized with glutaraldehyde. Animals used for immunization were rabbits, roosters, and dogs. All experiments failed to produce antituftsin antibody. Probable reasons for the lack of antigenicity include:

I) Lack of “foreignness” of tuftsin in mammalian species.

II) The small size of the tetrapeptide.

III) Tuftsin may be exerting an adjuvant effect when coupled to foreign antigens and is therefore not recognized by the host immune system.     

In Vitro Effects of Tp5 Analogs on E-Rosette Formation and Cell Division

The effects of seventeen synthetic analogs of thymopentin (TP-5) have been studied in the active and azathioprine-inhibited E-rosette tests.

Thymopentin was gradually shortened from the C terminus to peptides and single amino acids. Thymopoietin 32-34 (Arg-Lys-Asp-RGH-0205-TP-3) (II) and thymopoietin 32-35 (Arg-Lys-Asp-Val-RGH-0206-TP-4) (I) were the most active peptides.

Dipeptide Arg-Lys produced significant stimulatory effect on azathioprine (ED₇₅) inhibited E-receptor. Treatment of azathioprine (ED₇₅-inhibited E-rosette forming cells (ERFC) with arginine or especially lysine increased the number of ERFC.

Some of TP-4 analogs decreased further the number of ERFC decreased by azathioprine ED₃₀. These “suppressive” peptides as well as TP-3 caused a partial arrest of K 562 cell proliferation up to 96 hours.

Results suggest that TP-5 is not the smallest active fragment of thymopoietins, since peptides (TP-3 and TP-4) exhibit similar or higher T-cell membrane activation on E-receptor. Arginine, lysine, and acidic aspartyl residue seem to be a necessary basic structure to produce a cumulative chemical signal on the activity of T-lymphocytes.     

Effects of Anti-T Cell (Theta) and Anti-B Cell (Beta) Serum on the Immune Response in Mice

Heterologous anti-B cell (anti-beta) serum was prepared in rabbits against the spleen from neonatally thymectomized mice. The anti-beta serum, after absorption with thymus, is cytotoxic for bone marrow, bone marrow-derived cells, fetal liver and peritoneal lymphocytes. The cytotoxicity to the B cell can be absorbed out with bone marrow.

The cytotoxic effects of anti-beta serum on spleen and lymph node cells is compared to that of anti-theta serum. The data suggest that spleen has relatively more B than T cells, while lymph node has relatively more theta-positive cells.

To test the effect of anti-beta and anti-theta serum on the functional activity of lymphoid cells, C57 spleen or thymus was pre-incubated with the antiserum, in the presence of complement, and tested in vivo for graft-vs-host activity or transfer of an adoptive immune response to SRBC.

Treatment with anti-beta serum does not decrease the graft-vs-host activity of thymus or spleen cells. Anti-theta serum does decrease the graft-vs-host activity of both thymus and spleen cells.

Neither anti-beta serum nor anti-theta serum affect the phagocytic activity of peritoneal macrophages.

Both anti-beta serum and anti-theta serum decrease the transfer of an adoptive primary and secondary immune response to SRBC. A combination of anti-theta and anti-beta treated spleen can transfer adoptive immunity. Thymus and bone marrow can reconstitute the immunocompetence of anti-theta or anti-beta treated spleen respectively.

The results suggest that T cells alone can mount a graft-vs-host reaction and that this activity is not affected by anti-beta serum. The transfer of a humoral antibody response, on the other hand, requires functionally active T- and B-cells. This holds true for a primary as well as secondary immune response. Our anti-beta serum does not appear to have any anti-macrophage activity.     

Effect of Lansoprazole on Human Leukocyte Function

Recent findings on the capacity of omeprazole to mfluence various leukocyte functions, in vitro, raises the question on the potential use of protonic pump inhibitors, commonly used in the treatment of acid-secretion- related disorders, as immunomodulators. The aim of this study was to evaluate the in vitro effect of lansoprazole on human natural killer (NK) cell cytotoxix activity, chemotaxis and superoxide anion (02*^-) generation exerted by polymorphonucleated cells (PMNs). NK cytotoxicity activity was assessed by a ⁵¹Cr release assay, PMN chemotaxis was determined by an under agarose method and 02*^- generation was analyzed on the basis of reduced cytochrome C. Incubation times with lansoprazole was 30 min for PMNs and 1-4.5 hours for NK cells, respectively. Lansoprazole induced significant dose dependent bitiion of NK cell activity and PMN functions at concentrations ranging fiom 100 to 1,000μM.

Ths study demonstrate that lansoprazole, like omeprazole, inhibits several leukocyte functions, in vitro, then suggesting that protonic pump inhibitors are able to provoke these effects, at least at certain doses.     

“Nk-Like” T Cytotoxicity Against B Lymphocytes in a Hypogammaglobulinemic Patient

Physiologically, cells with NK activity appear to exert a negative control on immunoglobulin production. The clinical association of large granular lymphocyte (LGL) proliferation with hypogammaglobulinemia suggests that these functional NK cells could also be involved in pathological situations.

We studied in vitro lymphocyte functions in a patient presenting LGL proliferation associated with hypogammaglobulinemia. The CD3⁺ CD8⁺ CD57⁺ CD16^- phenotype lymphocytes expressed a high NK type cytotoxicity towards K562 targets, suggesting that they may be considered as “NK-like” T cells. We cultured the patient peripheral blood mononuclear cells (PBMC) with control subject PBMC and with PBMC from two other subjects with B chronic lymphocytic leukemia (B- CLL) of the CD20⁺ CD21^- CD10^- phenotype. Patient PBMC exhibited a lytic activity on control PBMC and on the B lymphocytes of one of the two B- CLL but only in the presence of PWM. This activity was not exerted by the culture supernatant and required a cell - to - cell contact. We suggest that the hypogammaglobulinemia observed in this patient may be related to a cytotoxic effect exerted on B lymphocytes by a CD3⁺ CD8⁺ CD57⁺ CD16^- LGL proliferation.     

Monocyte Locomotion in Anergic Chronic Brucellosis Patients: The In Vivo Effect of Ascorbic Acid

In 14 patients suffering from relapsing chronic brucellosis who were anergic to brucella antigens, we have studied peripheral blood monocyte random migration and chemotaxis against non-specific and specific leukoattractants, as well as plasma and monocyte ascorbic acid levels.

We found that all parameters studied, were significantly beneath normal, when compared to normal subjects.

After the oral administration of ascorbic acid at a daily dose of 1gr for 15 conseguetive days, random and directed migration against a non-specific stimulus (casein)returned to normal. Directed migration against disease associated leukoattractants (brucella melitensis and brucella abortus) antigens improved significantly, without reaching normal values.

We concluded that ascorbic acid supplementation might partially restore peripheral, monocyte function and help the monocyte-macrophage system to mount an effective immune response against chronicity of brucella infection.     

Methadone vs morphine: comparison of their effect on phagocytic functions

A comparison of the effects of methadone and morphine on phagocytic physiology was carried out in mice, using a number of tests, to estimate the risk of using methadone in maintenance protocols for opiates addicts. Results indicate that methadone, like morphine, reduces (a) R.E.S. activity and (b) PMN superoxide anion production, while unlike morphine it (a) does not produce haematologic changes, (b) does not exacerbate C. albicans infections, (c) does not inhibit phagocytosis and killing by murine polymorphonuclear leukocytes and macrophages, or by rabbit alveolar macrophages, and (d) does not reduce spleen and liver weight. These results are in strict agreement with those previously found in human subjects receiving controlled administration of morphine or methadone. Compared to morphine methadone therefore appears to have a lower toxic potentiality.     

Liposomes in Immunology: Further Evidence for the Adjuvant Activity of Liposomes

The immune response to HSA-phosphatidylcholine complexes administered to rabbits was not markedly enhanced when compared with the response to unmodified ESA.

It was found in earlier work that HSA entrapped in liposomes (mainly composed of phosphatidylcholine) evoked a strong immune response under conditions where no response was detected to free HSA.

The present results exclude the possibility that HSA- phosphatidylcholine complexes which may arise from liposome- encapsulated HSA may be responsible for the adjuvant activity of the liposomes.

The adjuvant activity of liposomes could also be established after administration of a liposome-associated strong antigen (BGG).     

Inhibition of Graft-Vs-Host Induced Immunodeficiency with Immunosuppressive Therapy

The pathologic features of the acute graft-vs-host disease occurring in unirradiated (C57B1/6 X A/J)F₁ mice injected intravenously with lymphocytes from the C57B1/6 parent are similar to those reported for other parental → F₁ hybrid combinations.

When stimulated in culture with concanavalin A, lipopolysaccharide or alloantigen, spleen cells from B6AF₁ mice that had been injected 11 days previously with B6 lymphocytes exhibited proliferative responses that were drastically reduced in comparison to the responses of spleen cells from F₁ hosts injected with syngeneic lymphocytes. IL2 production in h7H spleen cell cultures was also diminished. Proliferative responses and IL2 production were partially restored in mice given immunosuppressive therapy with azathioprine, cyclosporin A or Sch 24937 a drug whose inhibitory effects on cellular and humoral immune responses in mice have recently been described.

Phenotypic analyses by flow cytometry of the GVH splenocyte population indicated that the most consistent chanqe in the GVH spleen was the appearance of an Lyt2⁺ L3T4⁺ T cell subset which in the majority of experiments was accompanied by an increase in cells expressing only the Lyt2 antigen. Both subpopulations were reduced in mice that had recovered immunological responsiveness following immunosuppressive therapy. The results suggest that in this GVH model the development of an immunodeficient state is directly related to the induction of an active T suppressor cell population and that such cells are effectively eliminated from the splenocyte population following treatment with some immunosuppressive drugs.     

Effects of Lentinan on Cytotoxic Functions of Human Lymphocytes

The in vitro effects of lentinan on natural killer (NK), antibody-dependent cell-mediated cytotoxicity (ADCC), lectin-dependent cell-mediated cytotoxicity (LDCC) and mitogen-induced blast transformation were studied in patients with solid tumors and chroyic lymphocytic leukemia (CLL). NK activity was measured against Cr-labelled K-562 targets, ADCC against antibody-coated chicken red cells. LDCC and natural cell-mediated cytotoxicity (NCMC) was assessed using ³H-thymidine prelabelled HEp-2 targets. Mitogen (PHA-and Con A-) induced blast transformation was measured by thymidine incorporation.

Blastogenesis and LDCC was not influenced by lentinan. 1 μg/ml lentinan increased NCMC of tumor-bearing subjects. The most prominent enhancement of NK and ADCC activity was seen in CLL patients, where a dose-related increase was seen (from 0.01 to 1 μg/ml).     

Lymphocyte Activation by the Fc Region of Immunoglobulins

The Fc region of Ig is required for numerous biological effector functions which include: 1) opsonization, 2) anaphylaxis, 3) C fixation, 4) catabolism of the Ig molecule, 5) FcR binding, and 6) immune regulation. To this latter point, the cellular and subcellular events involved in immune regulation by IC and Fc fragments of Ig have been the focus of numerous investigations.

Characterization of cyanogen bromide cleavage fragments from a human IgG₁ myeloma protein indicates that one biologically-active site is found in residues 335-357 of the CH₃ domain of the molecule. Synthesis of the biologically-active region resulted in a peptide, termed p23, which stimulates mouse and human B cells to secrete polyclonal Ig and activates AA metabolic pathways. In contrast to these findings, p23 is unable to induce B cell proliferation or IL-1 secretion from macrophages. Analysis of data obtained with overlapping peptides, based on p23, suggests that the minimal active sequence needed for B cell differentiation is leu-pro-pro-ser-arg (residues 351-355). In contrast, only p23 or p23 minus the carboxyterminal glu₃₅₆ and glu₃₅₇ were able to induce PGE release.

Release of biologically-active peptides derived from the Fc region of Ig into the cellular microenvironment may form the nucleus of a nonspecific in vivo Immunoregulatory network. The specificity of peptide regulatory activities could reside in their effectiveness at high concentrations in the cellular microenvironment. The interaction of Fc region peptides with receptors on B cells, T cells, and macrophages/monocytes could result in a dynamic control of immune reactivity.     

Regulation of Murine T-Lymphocyte Function by Spleen Cell-Derived and Exogenous Serotonin

The modulatory effects of serotonin on T-cell activity were investigated. T-cell blastogenesis of normal spleen cells was slightly stimulated by the addition of low doses (1 and 10 ng/ml) of the inducer of serotonin release, fenfluramine. In contrast to the stimulatory effects of low doses of fenfluramine, high doses of fenfluramine (1 and 10 ug/ml) or of exogenously added serotonin (≥0.1 ug/ml) inhibited T-cell activation. Both the stimulation by low dose fenfluramine and the inhibition by high dose fenfluramine were accentuated by pretreating mice with tryptophan to heighten intracellular stores of serotonin and then inducing serotonin release. Pretreatment of mice with the serotonin inhibitor p-cholorphenylalanine (PCPA) abolished the fenfluramine inhibition of T-cell activation indicating that the fenfluramine inhibitory effect was mediated via endogenous spleen cell-derived serotonin. However, the PCPA treatment diminished T-cell activation. These results suggest that endogenous serotonin causes a biphasic dose-response effect on T-cell activity with serotonin being required for optimal T-cell function, low doses being immune stimulatory and higher doses being suppressive.

Con-A, concanavain A

5-HT, 5-hydroxytryptamine

LAK, lymphokine-activated killer

NK, natural killer

PCPA, p-cholorphenylalanine

PHA, phytohemagglutinin     

Functional Modifications of Tee Cd4+and the Cd8+-H Subset Due to Maternal Serum

Numerous experiments have been performed to try to explain the successful gestation of the semiallogeneic mammalian fetus in the immuno-competent mother (1, 3, 4, 5). A popular hypothesis is that localized intra uterine suppression mediates the immune response and contributes directly to the survival of the fetus (6). Suppressive factors synthesized at the fetomaternal interface may be transported by the bloodstream and be found in the retroplacental and peripheral blood circulation (7). In this report we aim to study these modulating factors by exposing a proteinaceous antigen (Candidine or human mono nuclear cells) to unrelated human lymphocytes in the presence of a pool of maternal serum, retroplacental (MAT SR) or peripheral (MAT SP), or to a pool of male or calf serum (MALS or CS).

A significant downregulation of the candidine mediated lymphocytic stimulation was observed in the case of the maternal serum. In order to further characterize this inhibition, a quantitative evaluation of the expression of the CD₄ and CD₈ positive subpopulations was performed. A selective inhibition of the CD₄ positive subset was observed.

In the PHA stimulation assay in the presence of maternal serum there was an inhibition of the thymidine uptake of unrelated lymphocytes. When studying the different subsets which were stimulated in the presence of maternal serum and control media it was shown that the CD₄ positive subpopulation remained unchanged while there was a slight inhibition of the CD₈ positive subset in the first case (maternal serum treated).

The same CD₄ positive inhibitive property of maternal serum was observed when a neoplastic cell line (HUT cells) was used as target for candidine in a stimulation test. This CD₄ inhibited expression remained constant as long as the maternal serum was renewed.

Maternal lymphocytes however remained resistant to the inhibitive action of maternal serum and did not show any change in their CD₄ and CD₈ positive subpopulation. By investigating the different blood components, it was shown that the suppressive factor was included in the IgG fraction and synthesized at the placental level. Appearing quite early during the gestation (at the 5-6th week), this factor was vanishing 2 weeks after the delivery.     

Liposomes in Immunology: The Immune Response Against Antigen-Containing Liposomes

Three ways of antigen administration to rabbits were compared with respect to the resulting immune responses.

Antigen was administered free in solution, entrapped in liposomes or free in solution but together with empty liposomes.

Liposomal material showed adjuvant activity when injected together with free antigen, but a much stronger adjuvant effect was found when antigen was injected entrapped in liposomes.     

Role of prolactin in the modulation of NK and lak cell activity after short- or long-term morphine administration in neoplastic patients

In a previous work we demonstrated that chronic in vivo antalgic therapy of cancer patients with morphine reduced the endogenous cytotoxic activity of natural killer (NK) cells, while increasing the development of lymphokine activated killer (LAK) cell cytotoxicity. In order to investigate the mechanisms by which morphine affects NK and LAK cell function further, we evaluated the modulation exerted by short- or long-term morphine administration on either NK/LAK cell cytotoxicities or plasma levels of prolactin (PRL) and other immunomodulating neurohormones. An intravenous morphine injection (10mg) significantly increased the plasma levels of PRL, reduced the cytotoxic activity of NK cells, and increased the development of LAK cell activity 30 min after drug injection in neoplastic patients. The administration of bromocriptine before the injection of morphine prevented both PRL augmentation and the increase in LAK cell activation, although it did not prevent the inhibition of NK cytotoxicity. The chronic oral administration of morphine (90±30mg/day for 1 month) also resulted in higher PRL levels; the NK and LAK cell activities were, respectively, lower than or higher than those found in neoplastic patients untreated with morphine. The plasma levels of thyrotropin (TSH), adrenocorticotropic hormone (ACTH) and cortisol were not significantly modified in either short- or long-term experiments. The absolute number and the percentages of lymphocyte populations, as well as the percentage of IL-2 receptors, were not modified after short-term morphine administration whereas little changes of T lymphocyte populations and NK cell number were observed after oral treatment with morphine. In vitro morphine did not affect the development of LAK cell activity. In conclusion, our findings indicate that morphine reduces NK cytotoxicity and increases the development of LAK cell cytotoxicity after short- and long-term administration. The effect of morphine on LAK cell activation but not on NK cell reduction is related to the modulation of PRL levels determined by the opioid drug.     

Hypothyroidism and Antibody Prodction in Immature Male Chickens

This study was conducted to determine if hypothyroidism has an effect on humoral immunity in immature male chickens. Two week old Single Comb White Leghorn male chicks were used as experimental animals. Two experiments were conducted using different methods to induce hypothyroidism. In Experiment 1, birds were surgically thyroid-ectomized (Tx group) and in Experiment 2, hypothyroidism was induced by supplementing the feed throughout the experiment with 0.1% propylthiouracil (PTU group). Antibody production against sheep red blood cells (SRBC) (thymus-dependent antigen) and Brucella abortus (BA) (thymus-independent antigen) was tested at 4 weeks of age. Serum concentrations of T₄ and T₃ were measured in birds from each treatment group at 5 and 9 weeks of age. Body weights were recorded and birds were then autopsied and thyroid gland weights were measured.

Hypothyroidism was successfully induced in both Tx and PTU birds, as reflected by significant reduction in body weights in both groups, enlargement of thyroid glands in PTU birds and absence of thyroid glands in Tx birds. Though T₄ and T₃ were reduced in sera of treated birds, considerable amounts of these hormones were detected. Hypothyroidism did not seem to have profound or consistent effects on antibody production against SRBC or BA.

The possibility that thyroid hormones play a role in antibody production was not ruled out. However, it was suggested that within the physiological range of thyroid gland activity, thyroid hormones may not significantly regulate antibody production.