Circulating human antisperm antibodies recognize prostasomes

PROBLEM: The presence of naturally occurring antisperm antibodies (ASA) is a well-known cause of infertility in men and women, but the antigens for these antibodies are usually poorly characterized. Prostasomes, organelles secreted by human prostatic acinar cells and expelled into the seminal plasma at ejaculation, can adhere to sperm cells. Thus, we have examined whether prostasomes could be an antigen for ASA. METHOD OF STUDY: We have studied the reactivity of chicken antiprostasome antibodies with sperm cells in an agglutination test and conversely the reactivity of serum positive for ASA from 20 infertile patients, with spermatozoa using flow cytometry and with purified prostasomes using enzyme-linked immunosorbent assay. RESULTS: The chicken antiprostasome antibody caused agglutination of sperm cells similarly to the agglutination observed with patients' sera. All of these patients' sera contained IgG antibodies against prostasomes. CONCLUSIONS: The high percentage of patients with antiprostasome antibodies in this study shows that prostasomes could be one of the major targets for ASA.     

Helper activity by human large granular lymphocytes inin vitro immunoglobulin synthesis

In the present study we have examined the effect of human large granular lymphocytes (LGL) from healthy donors on Ig synthesis by autologous B lymphocytes. The results showed that this cell population has a consistent helper activity in pokeweed mitogen-activated cultures even when added at very low numbers. LGL can mediate their effect by secreting soluble helper factors capable of modulating B-cell responses as evidenced by the enhancement of IgG and IgM production by supernatants obtained from LGL cultures. Preincubation with interferon gamma further potentiated the helper activity by LGL.     

Blastogenesis of large granular lymphocytes in nonlymphoid organs

High numbers of large granular lymphocytes (LGL) accumulate in the livers and peritoneal cavities of mice during the course of viral infection. Accumulation of natural killer (NK) cells at day 3 postinfection (p.i.) was shown to be radiation-sensitive, implying that proliferation was required for this response. Accumulation occurred in splenectomized mice, indicating that the spleen, known to be an organ for mature NK cell proliferation, was not the major source for liver and peritoneal NK/LGL. Significant percentages (greater than 25%) of the LGL found in the liver and peritoneal cavity following viral infection or interferon induction with poly-inosinic:poly-cytidylic acid were defined morphologically as blasts (large cells with prominent nucleoli and intensely basophilic cytoplasms containing azurophilic granules). Most blast LGL at day 3 p.i. were sensitive to administration of anti-asialo GM1 serum in vivo, were Lyt-2-, and were enriched in populations that lysed NK cell-sensitive targets in vitro, indicating that these were NK/LGL. At day 3 p.i., leukocytes from the liver and peritoneal cavity incorporated 3H-thymidine and bound to and killed NK cell-sensitive targets in single-cell cytotoxicity assays. These data suggest that NK/LGL undergo at least one round of division in the liver and peritoneal cavity during viral infection. In contrast, blast LGL at day 7 p.i. were resistant to in vivo treatments with anti-asialo GM1 serum, were Lyt-2+, and were enriched in populations of cells that killed virus-infected histocompatible targets, indicating that they were cytotoxic T lymphocytes (CTL). These results suggest that both NK/LGL and CTL/LGL are capable of blastogenesis and presumed proliferation at sites of virus infection, providing a means for the in situ augmentation of a host's cell-mediated antiviral defenses.     

Immunoperoxidase-immunogold double labelling in immunoelectronmicroscopy of large granular lymphocytes

A method for better characterization of mononuclear cell subpopulations using detection of 2 surface antigens simultaneously in electron microscopy was developed with immunoperoxidase-immunogold double labelling. Monoclonal antibodies of IgG and IgM classes were used in the first step, and colloidal gold-labelled anti-mouse IgG antibody and peroxidase-labelled anti-mouse IgM antibody (mu chain-specific) in the second step. Immunoelectronmicroscopy with such double labelling improves ultrastructural analysis of mononuclear cell subpopulations.     

A particular case of large granular lymphocytes lymphoma

A patient with non-cutaneous lymphocytic lymphoma with leukaemic spread is reported. The large majority of the peripheral blood mononuclear cells formed rosettes with sheep erythrocytes (E), had receptors for the Fc portion of IgG (Fc gamma R) and displayed an unusual enzymatic profile. Part of these cells had also labile bound surface membrane IgG. The leukaemic cells were morphologically characterized as large granular lymphocytes (LGL) and consisted of two different types of cells: lymphoid cells and monocytoid cells. The histopathological diagnosis was T-cell lymphoma, pleomorphic type, diffuse. Despite their morphological heterogeneity, all the proliferative cells had the same immunological phenotype, showed normal ADCC activity but no NK activity. After three months, without specific treatment, the surface pattern changed: the cells resembled mature LGL and the NK activity increased. The cytochemical study of the leukaemic cells revealed an enzymatical peculiarity: besides an enzymatic profile characteristic for relatively mature LGL, the cells also displayed peroxidase activity. This unusual aspect--the expression by an individual malignant cell of markers believed to be restricted to a single cellular lineage--might be interpreted as a lineage infidelity or lineage promiscuity. The observation that this lymphoma developed in a patient with a long history of hypogammaglobulinaemia is of particular interest.     

Characterization of sperm antigens reacting with human antisperm antibodies

Antibodies reacting with human spermatozoa have been detected by various immunological techniques in the sera of subfertile men. Different patterns of sperm agglutination are observed with different sera, either head-to-head, tail-to-tail, or tail-tip-to-tail-tip. Differences have been detected between the clinically relevant antibodies in spontaneously infertile males and the less important antibodies in males who have undergone reversal of vasectomy. It has been suggested that the variations in agglutination patterns are due either to different classes of antibody or to binding of antibody to different antigens. In the present study immunoblotting techniques were used to characterize the reactivity of solubilized sperm proteins with serum samples exhibiting different modes of sperm agglutination. This involved the electrophoretic transfer of proteins from SDS gels to nitrocellulose sheets followed by overlay with serum antibody. Using these techniques we have attempted to characterize the antigens of spermatozoa which react with sera from both spontaneously infertile and vasovasostomized men. The results showed that although antisperm antibodies bind to discrete and sperm-associated antigens, there is no substantial difference between the antigenic patterns observed with antibodies producing different types of sperm agglutination. Neither the antigens detected, nor the intensity of reaction showed significant differences although there was a tendency for head-to-head agglutinating antibodies to react more strongly with the higher molecular weight antigens. Moreover, although with sequential serum samples the patterns of agglutination may change, the antigenic pattern remains unchanged.     

The role of human spleen interferon on natural killer activity of peripheral blood lymphocytes enriched in large granular lymphocytes

The elimination of monocytes as well as B- and T-lymphocytes by forming rosettes with high affinity for sheep red blood cells yielded an enriched population of both natural killer (NK) activity (cytotoxicity: 65.4 +/- 9.9% with an E/T ratio of 12:1, P less than 0.005) and large granular lymphocytes (LGL: 76 +/- 13%) compared to the untreated lymphocyte population where NK activity is 35.7 +/- 17.3% (E/T 12:1) and the percentage of LGL of 26 +/- 6%. We studied the action of type I interferon (IFN) obtained from human spleens, on NK activity of 9 peripheral blood lymphocyte populations and 9 enriched in LGL. NK activity of the total lymphocyte population is significantly increased (P less than or equal to 0.05) in 6 out of 9 cases after treatment by interferon. Cell populations enriched in LGL showed increased NK activity in only one case after treatment by interferon, but no increased activity was found in the other cases. These results are compatible with the notion of cellular cooperation in increased NK activity by interferon.     

Decrease of peripheral large granular lymphocytes in Graves' disease.

Peripheral large granular lymphocytes (LGL) were enumerated in normal subjects and patients with autoimmune thyroid diseases. In normal subjects, the percentage of LGL was significantly lower in women (mean +/- s.d., 17.0 +/- 3.6%; n = 35; P less than 0.05) than in men (19.5 +/- 5.3%; n = 20). In untreated patients with thyrotoxic Graves' disease (GD), both the percentage (11.5 +/- 2.7%; n = 12; P less than 0.001) and the absolute count (244 +/- 102/mm3; P less than 0.01) of LGL were significantly lower than those in normal women (17.0 +/- 3.6% and 334 +/- 122/mm3; n = 35). No significant differences from normal controls were observed in the percentages or absolute counts of LGL in patients with euthyroid GD under treatment or in euthyroid or hypothyroid patients with Hashimoto's disease (HD). The percentage of LGL was inversely correlated with the serum levels of T4 and T3 and the free T4 index, but not with the titre of anti-thyroid antibodies, the size of goitre or the degree of proptosis in the group of untreated patients with GD and HD. The decreased levels of LGL in thyrotoxic patients with GD may be related to the self-perpetuation of thyrotoxicosis in patients with this disease.     

Liver-associated large granular lymphocytes: morphological and functional aspects

Large granular lymphocytes (LGLs) differ from other lymphocytes in their recirculation pattern and are distributed preferentially in nonlymphoid organs such as the liver and lung. The liver-associated LGLs adhere strongly to the sinusoidal endothelium and show a natural killer (NK) cytotoxicity against incoming metastatic tumor cells; this reaction occurs very rapidly because, in contrast to the immune response, it does not require complex processes in the lymphoid tissue. They have been extensively studied morphologically in terms of pit cells. LGLs have two characteristic cell organelles which participate in the NK cytolysis, i. e., dense granules and rod-cored vesicles. The former are lysosomes derived from multivesicular bodies and contain pore-forming proteins. The latter are the secretory vesicles exclusively seen in LGLs and are markedly increased in number when the NK function is augmented by biological response modifiers. These two structures are believed to be exocytosed in the space between LGL and the conjugated tumor cell. The microenvironment of the liver sinusoids, which includes Kupffer cells, endothelial cells and other lymphocytes, is considered to regulate the function of the liver-associated LGLs. Liver-associated LGLs, as well as Kupffer cells, are intrinsically involved in the defense system of the liver under various physiological and pathological conditions.     

Human sperm antigens and antisperm antibodies. II. Age-related incidence of antisperm antibodies.

Naturally occurring human antisperm antibodies (ASA) detectable by immunofluorescence, were found to have a peak indicence of 90% in both sexes before puberty. Thereafter, the incidence declined to about 60% and persisted through life. This age-related incidence resembled that for foreign and not self antigens. The natural incidence of the six antibodies under investigation varied: the most frequent were antibodies to acrosomal antigen Acl and equatorial antigen; the least frequent were antibodies to acrosomal antigen Ac2 and sperm nuclear protamine; and between these were antibodies to the mainpiece of tail and post-acrosomal region. Irrespective of their natural incidence, these antibodies increased at a comparable rate in men following vasectomy indicating these six sperm antigens have comparable immunogenicity. These results provide evidence for the lack of immunologic tolerance in man toward many sperm antigens. The variation in the natural incidence of individual ASA is explanable by differences in prevalence of crossreaction between each sperm antigen with exogenous antigens. Thus, antibodies to acrosomal antigen Acl and equatorial antigen, which occurred most frequently, appeared to crossreact with ubiquitous microorganisms.     

Occult (non-surface expression) of T, B and monocyte markers in human large granular lymphocytes

Human large granular lymphocytes were examined for non-surface expression with a panel of monoclonal antibodies to T cell, B-cell and monocyte markers. T101, antibody to the T65 antigen, showed binding to crude fractions containing intracellular membranes but not to immobilized whole cells. Non-surface expression of T65 was also demonstrated by flow cytometry using lysolecithin to transiently permeabilize cells. With the latter technique nonsurface expression was also demonstrated with monoclonal antibodies B2 and MO-2. T65 was shown to be synthesized by large granular lymphocytes by metabolic labeling, indirect immunoprecipitation and SDS-PAGE. T65 from large granular lymphocytes was the same mol. wt as antigen for T cells derived from the same donor. These results indicate that human large granular lymphocytes synthesize, but do not express on the surface, certain monoclonal antibody-derived markers heretofore considered specific for other cell lineages.     

Suppression of human immunoglobulin synthesis by interleukin-4 in tandem with interleukin-2 through large granular lymphocytes

Recent studies have shown that interleukin (IL)-4 can affect secretion of immunoglobulins (Igs) or activation of cytotoxic cells by IL-2, while other studies have shown that natural killer (NK) cells/large granular lymphocytes (LGLs) can also affect Ig synthesis. Therefore, we examined the effect of IL-4 with and without IL-2 or human NK/LGLs on pokeweed mitogen (PWM)-stimulated production of IgM and IgG. We found that when IL-4 and/or IL-2 were incubated with peripheral blood lymphocytes and PWM for 7 days and an enzyme-linked immunosorbent assay was run to measure Ig synthesis, IL-4 with IL-2 caused a greater suppression of Ig synthesis than either cytokine alone. A further experiment was done to determine the effect IL-4 and IL-2 would have on LGL suppression of Ig synthesis. IL-4 and IL-2 alone and in combination, when added to LGL, caused the LGL to suppress Ig synthesis to a greater extent than alone. We conclude that IL-4 acts on NK/LGLs separately and jointly with IL-2, to suppress Ig synthesis (IgM and IgG).     

The predominance of IgG1 and IgG3 subclass antisperm antibodies in infertile patients with serum antisperm antibodies.

Mouse monoclonal antibodies (MAb) specific for each of the four human IgG subclasses and immunofluorescence flow cytometry were used to evaluate the subclass of the IgG antibody response to sperm in serum samples from 13 men and 6 women with a high titer (greater than 1:15,625) of IgG antisperm antibodies (ASA] determined by an indirect immunobead test. Five sera without ASA were also studied as a control. All 19 (100%) of the ASA-positive sera contained immunoglobulin (Ig)G ASA of the IgG1 and IgG3 subclasses. A 1:1 correlation was observed between the presence of IgG1 and IgG3 ASA. IgG2 was essentially undetectable, while IgG4 reactivity, although less intense than IgG1 and IgG3, was more prominent in the sera from the five vasectomized men. The ability of the IgG1 and IgG3 ASA-positive sera to deposit complement (C) on sperm was demonstrated by the concomitant binding to antibody-laden sperm of polyclonal antibodies to the membrane attack complex (C5b-9) of C. Both C-fixing and non-C-fixing ASA-positive sera were found to possess IgG1 and IgG3 antisperm antibodies. The predominance of IgG1 and IgG3 subclasses suggested a T-cell dependent immune response to sperm antigens.     

Choriocarcinoma cells modulate the cytokine production of decidual large granular lymphocytes in coculture

PROBLEM: How do major histocompatibility complex (MHC) I positive/negative choriocarcinoma (CC) cells affect the production of tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), interleukin‐10 (IL‐10), interleukin‐4 (IL‐4), and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) by decidual CD56⁺⁺ cells (large granular lymphocytes [LGL]), LGL‐depleted decidual cells (WASH) and unseparated decidual cells (DEC) in cocultures?
METHOD OF STUDY: Decidual tissue was obtained by legal abortions. CD 56⁺⁺ LGL were isolated in a magnetic cell separator. Cytokines were measured by ELISA. Differences were analyzed for significance by Wilcoxons test.
RESULTS: We found a significant increase of IL‐10 in LGL/JEG‐3 and of TNF‐α in LGL/JAR cocultures compared to noncocultured decidual cells. There was a significant increase of IL‐10 and TNF‐α and a significant decrease of GM‐CSF in WASH and DEC cocultures with both JEG‐3 and JAR. IFN‐γ was only found in 3/11 cases of LGL/JAR cocultures. No IL‐4 was found in any experiment.
CONCLUSION: MHC class I positive/negative CC modulate the cytokine production of decidual LGL in different ways.     

Human large granular lymphocytes induce immunoglobulin synthesis after bone marrow transplantation

Following T cell-depleted bone marrow transplantation, helper T cell numbers remain depressed for some months. Nonetheless, functional B cells can be adoptively transferred to the recipients of such grafts, where they continue to secrete antibody. We now show that immunoglobulin production by these transferred B cells is induced by activated large granular lymphocytes (LGL) which circulate in the recipients in substantial numbers during the immediate post-transplant period. The LGL are CD3 negative and therefore provide help in an antigen-unlinked manner. Helper effects for autologous (donor) B cells are augmented by the addition of anti-LFA-2 (anti-CD2) which appears to act by blocking recruitment of LGL inhibitory to developing B cells. In contrast antibody to the beta chain of LFA-1, which effectively reduces natural killer activity of LGL, does not influence their helper function. The peripheral blood LGL fraction thus contains both helper and cytotoxic activity, which can be distinguished by appropriate monoclonal antibodies.