Lacrimal gland-directed B cell responses

Although it is accepted that IgA plasma cells predominate in the lacrimal gland, the factors leading to this prevalence are not known. A series of 4-day LPS-driven co-culture experiments performed with dissociated lacrimal gland and lymphoid cell populations was employed to study the direct effect of lacrimal gland cells on B cell differentiation. Lacrimal gland cells, when co-cultured with spleen or mesenteric lymph node cells, were found to suppress differentiation of cells to IgA, IgG, and IgM production. Furthermore, suppression of IgG and IgM responses occurred after co-culture of lacrimal gland cells with Peyer's patch cells. However, these Peyer's patch co-cultures led to a stimulation of the IgA response, a condition that was abrogated by removal of Peyer's patch T cells before co-culturing. Pretreatment of lacrimal gland cells with mitomycin C eliminated the suppression and stimulation previously observed. These results demonstrate the effects of lacrimal gland, both directly and indirectly through T cells, on B cell differentiation. These findings explain in part the preferential accumulation of IgA-plasma cells within the gland.     

Characteristics of the lymphoid tissue reaction in compensatory reparative growth of the salivary glands in mice]

The capacity of the spleen cells of CBA mice to antibody formation as well as to the "graft-versus-host" (GVH) reaction and the change in the stem cell number determined by the spleen colony method were studied after unilateral removal or burn of the submandibular salivary gland and amputation of lower incisors. It is shown that any of these experimental changes in the salivary gland state causes an increase in the stem cell migration and makes lymphocytes more active in inducing the GVH reaction. The ability of spleen lymphocytes to react on additional antigen stimuli increases after amputation of lower incisors, accompanied by an enlargement of salivary glands, and sharply decreases after the removal or burn of the submandibular salivary gland, not causing hypertrophy of salivary glands.     

Hormonal influence on the secretory immune system of the eye: endocrine impact on the lacrimal gland accumulation and secretion of IgA and IgG

The objective of the current investigation was to explore the processes underlying the androgen control of tear IgA and to determine whether hormone exposure also modifies tear IgG content. In addition, studies evaluated the impact of diabetes on the androgen regulation of secretory immunity in the eye. Tears and lacrimal glands were collected from age-matched, adult male rats, which had undergone hypophysectomy, selective ablation of the anterior pituitary, streptozotocin-induced diabetes, sham-surgery and/or orchiectomy and had been exposed to vehicle or physiological amounts of testosterone for varying periods of time. Our findings demonstrated that testosterone administration selectively increased the accumulation of IgA, but not IgG, in tears and lacrimal glands of orchiectomized rats. This hormone effect was associated with a 2-fold enhancement of the IgA transfer from lacrimal tissue to tears; IgA movement was against a gradient. In contrast, androgen exposure had no significant influence on the lacrimal gland/tear transfer of IgG, which was down a 90-fold gradient. Testosterone action on the lacrimal gland appeared to involve an increase in IgA production, but not a consistent alteration in the total number of IgA-containing cells. Similarly, androgen exposure had no impact on the population of IgG-containing lymphocytes in lacrimal tissue. Of interest, ablation of the anterior or entire pituitary in orchiectomized rats, which procedure inhibits testosterone-induced stimulation of tear IgA levels, significantly reduced the total number of IgA-containing cells in the lacrimal gland. Induction of diabetes by streptozotocin injection to orchiectomized rats resulted in diminished tear IgA content and decreased numbers of lacrimal IgA-positive lymphocytes, but did not prevent the testosterone-associated rise in IgA antibody content. In summary, our findings demonstrate that androgens increase the lacrimal gland production and secretion of IgA, but not IgG.     

THE KINETICS OF LYMPHOCYTE RECIRCULATION WITHIN THE RAT SPLEEN

The migration of lymphocytes from the blood into the splenic pulp and the release of lymphocytes from the spleen into the blood was studied by isolating the rat spleen and perfusing it with 15 ml of recirculating, oxygenated blood. When thoracic duct lymphocytes labelled with tritiated uridine were added to the initial perfusate the concentration of these cells fell exponentially for 2–3 hr and then rose to a flat secondary peak. From this pattern it was inferred that small lymphocytes entered the spleen at a rate proportional to their instantaneous concentration in the perfusate, traversed the splenic pulp and re-entered the perfusate with a minimum transit time of 2–3 hr. The rate of release of small lymphocytes from the spleen was not influenced by the prevailing concentration of small lymphocytes in the perfusate but probably reflected the rate of migration into the spleen over a period earlier than 2 hr before. The rate of exchange of small lymphocytes between the blood and the intact spleen in vivo was estimated to be about 84 × 10⁶ cells/hr. The size of the intrasplenic pool of recirculating small lymphocytes was probably 400–500 × 10⁶ cells. The rate of migration of small lymphocytes into the spleen was not affected by prior irradiation of the spleen donor. When either of two antigenic materials were added to the perfusate no inhibition of lymphocyte migration into the spleen was noted although the release of lymphocytes from the spleen was diminished by the addition of a large dose of sheep erythrocytes.     

In vitro migration of peritoneal and spleen cells and its inhibition in some avion species

Cell migration and its inhibition was tested by the capillary tube technique with peritoneal exudate cells and spleen cells of chicken, turkey, goose, guinea fowl, and Japanese quail. Peritoneal cells were produced by ip administration of proteose peptone and harvested 24 hr later. Liquid paraffin proved to be unsatisfactory for preparation of peritoneal cells in some avian species. Mononuclear cells represented no more than 50–60% of the peritoneal cell populations, the other 50% being polymorphonuclear cells in all five avian species studied. Cell migration was demonstrated with chicken, turkey, and goose peritoneal and spleen cells, but not with those of guinea fowl and Japanese quail. The composition of the cell populations in the migration areas was nearly the same as in the initial preparations of peritoneal and spleen cells. Spleen cell migration was inhibited to a greater extent than that of peritoneal cells. Migration inhibitory factor (MIF) produced by chicken and turkey lymphocytes exhibited some species specificity.     

Regulation of lymphocyte motility by macrophages: characterization of a lymphocyte migration inhibitory factor derived from a macrophage-like cell line

An inhibitory factor on lymphocyte migration was detected using a capillary random migration assay in the culture supernatant of peritoneal exudate macrophages cultured at concentrations greater than 8 x 10(6) cells/ml. After examining different macrophage-like cell lines, J774A.1 cells were found to produce this inhibitory factor, which was termed lymphocyte migration inhibitory factor (LMIF). The inhibitory effect of LMIF on the migration of spleen lymphocytes, thymocytes, and bone marrow cells was determined. The migration of thymocytes was more sensitive to LMIF than was the migration of spleen lymphocytes and bone marrow cells. Interestingly, when the effect of LMIF was tested on the migration of spleen T cells and B cells, T cells were more sensitive than B cells. When the thymocytes were separated by peanut agglutinin into mature and immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes to the effect of LMIF, suggesting that the greatest effect of LMIF was on the migration of mature T cells. Partial purification of LMIF by ion-exchange and gel-filtration chromatography revealed that it is approximately 14,000 in molecular weight and could exist in either monomeric or dimeric forms. The possible role of this factor in an immune response is discussed.     

T and B lymphocyte migration into syngeneic tumors.

The migration of splenic T and B lymphocytes into syngeneic tumors undergoing immunologic rejection was investigates. Spleen cells were obtained from normal BALC/c mice or BALB/c mice bearing tumors induced by murine sarcoma virus (MSV). Either whole spleen cells or immunoabsorbent purified T and B cells were radiolabeled with sodium chromate-51 and injected i.v. into normal or MSV inducted-tumor bearing syngeneic recipients. Twenty-four hours later the recipient mice were sacrificed and radioactivity was assessed for tumor, contralateral normal muscle, the lymph nodes draining the tumor and contralateral draining lymph nodes, peripheral lymph nodes, spleen, and liver. Both T and B lymphocytes from either normal or MSV tumor-bearing animals show greatly increased migration into the tumor when compared with normal muscle. Migration of T cells from both normal and MSV tumor bearers was 30 times that of migration to normal muscle. B cells from tumor-bearing mice, on the other hand, localized in the tumor itself only 50% as frequently as did B cells from normal animals. In addition, T cells from MSV tumor bearers were found in the highest proportion in the lymph node draining the tumor site. We conclude that T and B lymphocytes from either normal or tumor-bearing mice migrate to a syngeneic tumor undergoing immunologic rejection. In contrast, the migration of both T and B cells from tumor-bearing animals was decreased to the peripheral lymph nodes at the time of maximum tumor growth.     

The separation of different cell classes from lymphoid organs. X. Preparative electrophoretic separation of lymphocyte sub-populations from mouse spleen and thoracic duct lymph

Conditions have been established for the separation of viable mouse lymphoid cells by continuous free-buffer film preparative electrophoresis. The detailed electrophoretic distribution profiles of T and B lymphocytes from mouse spleen and thoracic duct have been determined. Cell surface θ-antigen was used as a marker for T cells, and high surface-density of immunoglobulin as a marker for B cells. Spleen cells from athymic “nude” mice were also studied. In the unselected normal spleen cell populations B lymphocytes are heterogeneous, about 60% being of low mobility with the remainder distributing broadly, and extending into the highest mobility fractions. T lymphocytes are predominantly of high mobility. Lymphoid cells lacking markers of either the B or T lineage are of intermediate mobility. There is only partial separation of T and B cells because of the extensive overlap between the populations. The high mobility B cells, which separate along with T cells, include a substantial proportion of large cells, and include cells with high surface density of immunoglobulin. The majority of these large B cells can be selectively eliminated by their adherence on passage through a glass-bead column. By pre-selecting the 50% non-adherent lymphocytes from spleen as the starting material, a very sharp and more extensive separation of B and T cells can be achieved, with 100% pure B cells and 90% pure T cells in many fractions. However these samples are not representative of the total T and B cell populations of spleen. In thoracic duct lymph high mobility B-cells are absent, there is little overlap between T and B cell mobility. 100% pure T and B cells can be isolated.     

Cell infiltration in various organ and dilatation of the urinary tubule in NON mice

The non-obese non-diabetic (NON) mouse, which shows no glycosuria, is a subline of the non-obese diabetic (NOD) mouse. Cellular infiltrations in various organs were observed by light and electron microscopy in both sexes from 30 to 300 days after birth. These infiltrations were found in the kidney, pancreas, mandibular gland, parotid gland, exorbital lacrimal gland, and thyroid gland, but not in the adrenal gland, sublingual gland, testis and ovary. The infiltrating cells were mononuclear cells, mostly small lymphocytes. The population and frequency of these cellular infiltrations were weak generally; especially the infiltration into the pancreatic islet, which was very weak compared with that in NOD mice. Dilation of the proximal tubule occurred only in the females at 60 days or more after birth and it gradually increased with age. Numerous acidophil bodies appeared in the epithelial cells and the lumen of these dilated urinary tubules. These bodies were PAS-positive and stained with MT, and They had electron-dense complex structures.     

B cells in the appendix and other lymphoid organs of the rabbit: Stimulation of DNA synthesis by anti-immunoglobulin

Lymphocytes from rabbit lymphoid organs were cultured in the presence of class specific anti-immunoglobulin sera and of anti-allotype sera. Stimulation of tritiated thymidine uptake into DNA was taken to indicate the presence of the corresponding immunoglobulins on the cell surfaces. Thymus and bone marrow lymphocytes were unresponsive to all reagents tested. Popliteal lymph node contained cells responsive to anti-μ, anti-γ, and anti-α and therefore presumably bearing IgM, IgG, and IgA. Spleen had only IgM- and IgG-bearing-cells, and the appendix contained cells with IgM and IgA receptors only. The lymph node, spleen, and appendix cells of rabbits depleted of B lymphocytes by irradiation (900 R) and injection of thymocytes were unresponsive to anti-immunoglobulin but were stimulated at almost normal levels by PHA and Con A. T cell-depleted animals (thymectomy, irradiation with three divided doses of 450 R and bone marrow shielding) had immunoglobulin-bearing lymphocytes but were unresponsive to the mitogens. However a moderate level of mitogen-responsiveness reappeared by 3–4 wk after irradiation. Cells of morphologically distinct regions of the appendix, separated manually, showed different responses corresponding to the inferred origins of these anatomic areas. The “dome” and “corona” contained functional IgM- and IgA-bearing cells. The “TDA” reacted well to PHA, Con A, and PWM, but was depleted of immunoglobulin-bearing cells. The “follicle” cells, which are almost all in active DNA synthesis or mitosis, were relatively unresponsive to either T or B cell stimuli. Anti-allotype serum stimulated the same populations which responded to class-specific heteroantisera but at a slightly lower level. It was inferred that gut-associated lymphoid tissues like the appendix may play a special role as an amplification site for B-cells destined to produce IgM and IgA elsewhere in the organism.     

Chicken fetal antigen: association with lymphoid development and differentiation

Rabbit antisera capable of detecting chicken fetal antigen (CFA) was prepared against 1-day chick red blood cells (RBCs) and made specific by exhaustive adsorption with adult chicken peripheral RBCs (PRBCs) from the same strain. Microcytotoxicity was used to study the incidence of CFA on lymphocytes obtained from several organs at different developmental stages in the chicken. Lymphocyte-associated CFA (LA-CFA) determinants and erythrocyte-specific CFA (ES-CFA) determinants were distinguished through the use of adsorption analysis. The high incidence of CFA-positive lymphocytes found in the fetal bursa and thymus was not equaled in the peripheral organs of the spleen, cecal tonsils, and gland of Harder. CFA expression on adult lymphocytes was restricted to the thymus and peripheral blood. It is suggested that these cells may represent a subpopulation of T lymphocytes. Adult spleen, cecal tonsils, and gland of Harder were virtually devoid of CFA-bearing lymphocytes. At fetal developmental stages when greater than 94% of the bursal B cells were CFA-positive, few, if any, of the highly differentiated Harderian B cells possessed CFA. It is suggested that LA-CFA expression is dependent upon B cell differentiation and/or the bursa (central) vs gland of Harder (peripheral) microenvironment. The pattern of CFA expression on bursacytes is discussed in light of the properties of age resistance and bursal-dependent target cells associated with virally induced lymphoid leukosis.     

Immunological state of adult germfree miniature minnesota pigs

Fundamental hematological and immunological data were obtained on sexually mature germfree miniature pigs fed, after the milk diet period, with cereal-type diet sterilized by γ-radiation, and were compared with data of control conventional animals. Germfree adult pigs had a lower count of peripheral blood leukocytes with a lower percentage of neutrophil granulocytes and without any younger forms, a lower total serum protein level with a negligible amount of γ and α₂ globulin fractions and a higher serum albumin and β globulin level. In the mesenteric lymph nodes and in spleen, surface IgA-bearing cells predominated over surface IgG-bearing cells. Also a large amount of IgA-containing cells was found in the intestinal lamina propria, where the IgG cells were present in a negligible amount. IgM cells were the most frequent surface isotype in peripheral blood. The count of blood T lymphocytes was more than doubled.     

Calcein: a novel marker for lymphocytes which enter lymph nodes.

Previous studies have identified unique cell surface antigens which are associated with the specific binding of lymphocytes to high endothelial venules (HEV). Evidence is presented in this paper which demonstrates that uptake of the fluorescent dye calcein by lymphocytes represents an additional marker for the lymph node homing subpopulation of lymphocytes. Calcein exhibits a characteristic ability to label lymphocytes differentially into two distinct populations, based on fluorescence intensity, that does not occur with three other structurally related, fluorescein-based dyes. In vivo lymphocyte migration studies revealed that cells displaying the "dull" fluorescence phenotype, although entering all lymphoid organs examined, preferentially homed to the lymph nodes, particularly the popliteal lymph node (PLN). By contrast, lymphocytes displaying the "bright" phenotype were essentially excluded from entering lymphoid organs, where entry is HEV dependent, but were observed entering spleen, where entry is HEV independent. Furthermore, a high proportion (76.5%) of lymphocytes displaying the dull fluorescence phenotype expressed the PLN homing receptor MEL-14. Based on these observations it is suggested that calcein uptake may be a marker for general membrane properties, such as fluidity and plasticity, essential for the passage of lymphocytes through HEV.     

Long-lived lymphocytes include lipopolysaccharide-reactive B cells

We have used a new protocol of prolonged in vivo hydroxyurea (HU) administration which eliminates all cycling and short-lived cells. This treatment kills 99% of non-B non-T bone marrow cells, and it leaves in spleen and bone marrow "long-lived" B- and T-cell populations which represent 33 and 59%, respectively, of the total numbers of lymphocytes found in untreated controls. The relative proportions of B and T cells in spleen or blood of HU-treated mice were practically unaffected, while an increased blood-to-marrow permeability results in markedly abnormal proportions of B and T lymphocytes in bone marrow. Mitogen reactivities of these long-lived lymphocytes recovered either in spleen or bone marrow of HU-treated animals were studied. The results show that such B cells respond perfectly well to the B-cell mitogen lipopolysaccharide, by proliferation and differentiation into Ig-secreting cells, and that T cells proliferate at nearly control levels in response to concanavalin A. This protocol of long-term HU treatment offers the possibility of studying selected long-lived lymphocyte populations, the clonal repertoires and functional properties of which can now be readily approached.     

The presence of carbonic anhydrase in orbital glands. An enzyme-histochemical study in cynomolgus monkeys and rabbits

The distribution of carbonic anhydrase (CA) was studied in the lacrimal gland of the cynomolgus monkey as well as in the lacrimal, infra-orbital and harderian glands of the rabbit. In the lacrimal gland of the cynomolgus monkey, a number of acini with positive staining were found; however, another group of acini did not stain. In the positively stained acinar cells, large amounts of reaction product were located in the cytoplasm, but only weak staining was observed in the membranes. In the endothelial cells of capillaries a strong staining reaction was only seen in those vessels which were adjacent to the acinar cells containing CA. In the lacrimal and infra-orbital glands of the rabbit, there was intense staining of the cell membranes in all acinar cells and weak staining of the cytoplasm in a few acinar cells. Stained capillaries were also found here, but these were not as numerous as in the lacrimal gland of the cynomolgus monkey. In the harderian gland of the rabbit, there was no staining in the white lobe. In the red lobe the acinar cells displayed distinct staining exclusively in the basolateral membranes. There was no staining of capillaries in the harderian gland. In none of the glands studied was there staining of the epithelial cells of the excretory ducts. The functional significance of these findings is discussed.     

Characterization of lacrimal gland carbonic anhydrase VI.

We have previously demonstrated by immunohistochemistry the presence of secreted carbonic anhydrase (CA VI) in the acinar cells of the rat lacrimal glands. In this study we purified the sheep lacrimal gland CA VI to homogeneity and demonstrated by Western analysis that it has the same apparent subunit molecular weight (45 kD) as the enzyme isolated from saliva. RT-PCR analysis showed that CA VI mRNA from the lacrimal gland was identical to that of the parotid gland CA VI mRNA. An RIA specific for sheep CA VI showed the lacrimal gland tissue concentration of the enzyme to be 4.20 +/- 2.60 ng/mg protein, or about 1/7000 of the level found in the parotid gland. Immunohistochemistry (IHC) and in situ hybridization (ISH) showed that lacrimal acinar cells expressed both immunoreactivity and mRNA for CA VI. Moreover, CA VI immunoreactivity was occasionally observed in the lumen of the ducts. Unlike the parotid gland, in which all acinar cells expressed CA VI immunoreactivity and mRNA, only some of the acinar cells of the lacrimal gland showed expression. These results indicate that the lacrimal gland synthesizes and secretes a very small amount of salivary CA VI. In tear fluid, CA VI is presumed to have a role in the maintenance of acid/base balance on the surface of the eye, akin to its role in the oral cavity.     

Changes in proliferation activity and relative distributions of lymphoid cell subpopulations in congenitally athymic nu/nu mice and lurcher mice with spontaneous olivopontocerebellar degeneration

Changes observed in mice with congenital damage of some part of the CNS-neuroendocrine-immune regulatory system are described. nu/nu mice with congenital absence of thymus and Lurcher mice with spontaneous olivopontocerebellar degeneration displayed changes in the histoarchitecture of adrenal gland, immune organs (thymus, spleen, axillar lymph nodes) and intestine. Changes were also observed in IgM+, IgG+, CD4+ and CD8+ lymphoid cell subpopulations in the main lymphoid organs--the spleen and axillar lymph nodes and in the proliferative ability of whole lymphoid cell populations. The extreme decrease of lymphoid T-cell subpopulations in athymic nu/nu mice is the consequence of the absence of thymus, the organ of their maturation. On the other hand, a relative increase of B-cell subpopulations was found in this mouse strain. A relative decrease of CD4+ lymphocytes and a different influence of immunization on B-cell subpopulations were found in the spleen in neurodeficient Lurcher mice. The high percentage of apoptotic cells, cells in the S-phase of cell cycle and increased proliferation index in nu/nu mice suggest that the turnover and renewal of lymphoid cells in the spleen in nu/nu mice is more rapid than in control immunocompetent BALB/c mice.     

Anti-immunoglobulin M induces both B-lymphocyte proliferation and differentiation in Xenopus laevis

Anti-IgM induced the proliferation of spleen lymphocytes of the amphibian Xenopus laevis as determined by 3H-thymidine uptake. The responding cells were B lymphocytes, since lymphocyte populations enriched in surface-Ig-positive cells exhibited an increased proliferative response, and spleen cells from larvally thymectomized animals still responded to anti-IgM. Immunofluorescence analysis and gel electrophoresis of biosynthetically labeled Ig polypeptides revealed that lymphoblasts induced by anti-IgM differentiated into plasmablasts that synthesized and secreted mainly IgM and small amounts of IgY. The in vitro differentiation of B lymphocytes also occurred in spleen cells obtained from thymectomized animals. These findings are in contrast with those obtained in mammals and suggest that the differentiation of B lymphocytes in X. laevis is subject to different regulatory mechanisms.     

Detection of mitogen induced stimulation of leukocytes from the rainbow trout (Oncorhynchus mykiss) by flow cytometric analysis of intracellular calcium

Flow cytometric analysis of the forward/side light scatter (FSC/SSC) of density gradient-separated head kidney cells of the rainbow trout revealed three distinctly separated populations, which we defined as population 1, 2 and 3. In spleen cells, populations 1 and 2 were also found, whereas population 3 was not detected. Further characterization regarding the surface Ig (sIg) revealed that population 2 of the head kidney and spleen contained 37.4 and 34.4% sIg⁺-cells, respectively. Incubation of the head kidney and spleen cells with different concentrations of concanavalin A (ConA), phytohemagglutinin (PHA) and [PWM] induced a pronounced intracellular calcium increase only in cells of population 2. This reaction was concentration dependent and caused by a release of intracellular Ca²⁺-stores. FMLP, a chemotactic peptide, had no effect on intracellular calcium response in all three populations. Similarly, the stimulation with PMA had no effect. This indicates that population 2 of the head kidney as well as the spleen is characterized by a high forward and low side light scatter and contains both subpopulation of lymphocytes, B- and T-cells. We demonstrated that the analysis of intracellular calcium increase due to mitogens is a suitable approach to identify lymphocytes in fish and enables further functional studies in these cells.     

Migration of peripheral T and B cells into the thymus of aging (NZB × SJL)F1 female mice

Aging NZB × SJL (NS) female mice provide a unique model of thymopathology characterized by the intrathymic accumulation of large numbers of mature T and B cells. The purpose of the present work was to examine the possibility that this phenomenon results from the invasion of the thymus by cells from the periphery. Lymphoid cells labeled with chromium-51 or indium-111 were injected into syngeneic recipients to study their patterns of in vivo migration. Lymph node (LN) or spleen cells were found to localize significantly (1–2% of injected radioactivity) into the thymus of 12-month-old NS females but not into that of young recipients or of old NS males. However, intrathymic localization of injected LN cells was observed in castrated NS males which exhibit the same thymopathology as NS females. Both radiolabeled T and B cells were found to enter the thymus of aged NS females but the latter cells about three times less efficiently than the former. Moreover, while thymocytes from young NS females were unable to recirculate to LN, those of old NS females showed increased LN-seeking capacity and part (1%) of them did migrate back into the thymus of old but not young NS females. In additional cell transfer experiments, the intrathymic migration of B cells into old NS females was further documented by using the antibody response to sheep erythrocytes as a tracer. Taken together, these observations indicate that the thymus of aging NS female mice is permeable to recirculating lymphocytes, suggesting that at least part of the mature T and B cells detected in this thymus, are migrants from the periphery.