The goldfish immune response. I. Characterization of the humoral response to particulate antigens.

Anti-red blood cells (RBC) and anti-hapten antibody synthesis were studied in the goldfish, Carassius auratus. Spontaneous haemagglutination titres were found against all the antigens tested. A weak secondary response was observed in RBC-primed fish boosted during the end-phase of the primary antibody production. However, when the second antigenic challenge was performed during the early exponential phase of a primary stimulation, an important amplified response was obtained. The antibody production and immunological memory can be dissociated: no antibody synthesis occurred in glutaraldehyde-fixed RBC (F-RBC) primed was obtained when untreated or F-RBC were given to F-RBC primed animals. The amplified response to sheep red blood cells (SRBC) was significantly inhibited when fish were primed with a mixture of SRBC and Xenopus red blood cells (XRBC), demonstrating an antigenic competition phenomenon. Studies on anti-trinitrobenzene responses confirm the efficiency of E. coli lipopolysaccharide as a carrier for fish anti-hapten immunization. The kinetics and regulation of antibody synthesis in fish are discussed in relation to the described results.     

The ovine immune response to Chlamydia psittaci; histopathology of the lymph node

The histopathological response of the ovine popliteal lymph node to infection by an ovine abortion strain of Chlamydia psittaci was studied. After infection of 10 seronegative sheep by the subcutaneous route, the draining popliteal lymph nodes enlarged considerably. By day 6, expansion was more marked in the medulla than in the cortex but, by day 18, cortical follicles were prominent. Immunoglobulin-containing cells increased in number both in the medulla and cortex between days 6 and 18. C. psittaci was re-isolated from three nodes on day 6 and one on day 12, but at no stage was it demonstrated in tissue sections by an immunoperoxidase method. Thus it was shown that while C. psittaci could apparently become "latent" in lymphoid tissue, it could also stimulate a profound response at the same site.     

The requirement of macrophages in the secondary immune response to antigens of small and large size in vitro

Tissue culture techniques were combined with cell separation procedures, antimacrophage serum and soluble and particulate forms of sheep red cell antigen to investigate the cellular requirements for a secondary antibody response. By using the highly efficient active adherence column separation method of Shortman which almost completely removes phagocytic cells, it was found that the secondary response to SRC was macrophage dependent. This conclusion was verified by the use of specific antimacrophage serum and by a combination of both methods. Critical tests were used to verify that these separation methods acted on phagocytic and not on other cells, for example, thymus or bone marrow derived lymphocytes. In contrast, the secondary immune response to POL and solubilized SRC antigen was not dependent on the presence of phagocytic cells, as highly purified lymphocytes responded normally to these antigens. Antimacrophage serum did not depress the immune response to these soluble antigens. These results indicate that the requirement for macrophage activity depends on the physical size of the antigen. These findings, obtained in the secondary response in vitro, were closely analogous to previous findings from this laboratory on the cellular basis of the primary response to SRC and POL. The reasons for the different cellular bases of the secondary immune response to various molecular forms of antigen are discussed. The similar cellular basis of the primary and secondary response in vitro suggests that the basic mechanisms of immunization in these responses are the same.     

Phagocytosis and lipofuscin accumulation in lymph node macrophages

Lipofuscin is considered to be undigestible material present in secondary lysosomes. During histological and electron microscopical studies lipofuscin was observed in macrophages in popliteal lymph nodes in 3-6 month-old-male Wistar rats after occlusion of the afferent lymph flow to the lymph node. No pathological alterations were found in the lymph node after the operation. However, the number of macrophages was severely reduced. Simultaneously the number of secondary lysosomes increased in the remaining macrophages reaching a plateau 4 weeks after operation. At this time three quarters of the residual bodies already contained lipofuscin granules. In the following weeks almost all macrophages showed lipofuscin in increasing amounts. Macrophages in normal contralateral lymph nodes of the same rats rarely contained lipofuscin. The increased phagocytosis of the remaining macrophages thus preceded the appearance of lipofuscin. We suggest that lipofuscin results from an inadequate intralysosomal digestion.     

Targeting the tumor-draining lymph node with adjuvanted nanoparticles reshapes the anti-tumor immune response

Accumulating evidence implicates the tumor-draining lymph node (TDLN) in tumor-induced immune escape, as it drains regulatory molecules and leukocytes from the tumor microenvironment. We asked whether targeted delivery of adjuvant to the TDLN, presumably already bathed in tumor antigens, could promote anti-tumor immunity and hinder tumor growth. To this end, we used 30 nm polymeric nanoparticles (NPs) that effectively target dendritic cells (DCs, CD11c⁺) within the lymph node (LN) after intradermal administration. These NPs accumulated within the TDLN when administered in the limb ipsilateral (i.l.) to the tumor or in the non-TDLN when administered in the contralateral (c.l.) limb. Incorporating the adjuvants CpG or paclitaxel into the NPs (CpG-NP and PXL-NP) induced DC maturation in vitro. When administered daily i.l. and thus targeting the TDLN of a B16–F10 melanoma, adjuvanted NPs induced DC maturation within the TDLN and reshaped the CD4⁺ T cell distribution within the tumor towards a Th1 (CXCR3⁺) phenotype. Importantly, this also led to an increase in the frequency of antigen-specific CD8⁺ T cells within the tumor. This correlated with slowed tumor growth, in contrast to unhindered tumor growth after c.l. delivery of adjuvanted NPs (targeting a non-TDLN) or i.l. delivery of free adjuvant. CpG-NP treatment in the i.l. limb also was associated with an increase in CD8⁺/CD4⁺ T cell ratios and frequencies of activated (CD25⁺) CD8⁺ T cells within the TDLN whereas PXL-NP treatment reduced the frequency of regulatory T (FoxP3⁺ CD4⁺) cells in the TDLN. Together, these data implicate the TDLN as a delivery target for adjuvant therapy of solid tumors.     

Pathology of the lymph node and spleen in systemic mast cell disease

The spleen and lymph node are two of the most common organs involved in systemic mast cell disease (SMCD). However, SMCD infiltrates in the spleen and lymph node have a broad spectrum of morphological patterns which can make it difficult to recognize the diagnosis, especially when specimens are examined from patients in whom SMCD is not suspected. We reviewed the pathological features of 16 spleen and 23 lymph node specimens from 19 patients which represented all available material from a series of 58 Mayo Clinic patients with SMCD. The purpose of this study was to investigate the pathological manifestations of SMCD involvement in the spleen and lymph node and to address difficulties in differential diagnosis. All compartments of the spleen and lymph node were found to be affected by SMCD. SMCD lesions in the spleen were found in a paratrabecular (92%), parafollicular (69%), follicular (15%), and a diffuse red pulp (8%) distribution. In the lymph node, mast cell infiltrates affected the paracortex (88%), the parafollicular region (50%), the follicles (25%), the medullary cords (13%), and the sinuses (6%). Mast cells were frequently found in a perivascular location, and associated eosinophilia was common. Because of the broad spectrum of histological manifestations of SMCD in the spleen and lymph node, a wide range of differential diagnoses is discussed including follicular lymphoma, T-cell lymphoma, monocytoid B-cell hyperplasia and lymphoma, Kaposi's sarcoma, and Langerhans' cell granulomatosis.     

Limitation of nitric oxide production: cells from lymph node and spleen exhibit distinct difference in nitric oxide production

Many types of cells in the immune system have been found to produce nitric oxide (NO), which performs multiplex functions. However, in myelin basic protein peptide 68-86 (MBP 68-86)-induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats, we found that elevated NO production was generated from spleen cells (SC), not from lymph node cells (LNC). LNC expressed lower NO synthase 2 (NOS2) and produced lower levels of NO than SC upon MBP 68-86 stimulation. Expression of B7-1(CD80) and B7-2(CD86) molecules was much lower on LNC than on SC. Blocking of B7-1 or B7-2 ligation resulted in reduced NO production by SC. Unlike SC, LNC were resistant to interferon-gamma- or lipopolysaccharide-induced NO production. NO derived from SC suppressed cell proliferation and induced apoptosis in vitro. Addition of N(omega)-nitrol-L-arginine methylester (L-NAME) into cell cultures promoted cell expansion and reduced apoptosis. These results indicate that NO production originates from SC, but not from LNC. Low expression of co-stimulatory molecules and NOS2 of LNC limits NO induction. The high levels of NO derived from SC are involved in the self-limiting mechanisms of autoimmune responses by inhibiting cell expansion and promoting cell apoptosis.     

Response of the immune system of mammary tumor-bearing rats to cyclophosphamide and soluble low-molecular mass tumor-associated antigens: the spleen and lymph nodes

In a previous study, we showed that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this report, we analyzed the underlaying mechanisms. Studies were performed on the spleen and lymph nodes from the following groups of mammary tumor-bearing rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, i.v.) rats treated with CPA and sTAA. Different zones of the spleen and lymph nodes were measured and their T cell content (CD4(+) and CD8(+) cells) was analyzed immunohistochemically. CPA decreased the size and cell content of follicles, splenic areas related to the production of B cells, of the marginal zone and to a lesser extent of the periarterial lymph sheath, and decreased the number of CD4(+) and, at a lower rate, of CD8(+ )T cells in the spleen. Addition of sTAA restored activity in the splenic zones producing these cells. Similar effects of CPA and sTAA were found in lymph nodes with accumulation of B lymphocytes in the primary and secondary follicles and of T lymphocytes, including both CD4(+) and CD8(+) cells, in the paracortical zone. We suggest that inhibition of the functional activity of the immune system is one of the main reasons for the toxic effects of chemotherapeutic drugs such as CPA and that the tumor-suppressive antitoxic effects of sTAA result from their activation of B- and T-lymphocyte production in this system, particularly in the spleen and lymph nodes.     

Peritoneal macrophages in the immune response to SRBC in vitro

The role of macrophages in the immune response against SRBC in spleen cell suspension cultures obtained from mice was studied.

1. The presence of macrophages increases the number of PFC against SRBC in spleen cell cultures.

2. The ratio peritoneal exudate cells to spleen cells is very critical. Two × 10⁵ peritoneal exudate cells preincubated with sheep red blood cells stimulate 1 × 10⁷ mouse spleen cells to an optimal response. This reaction is suppressed when 2 × 10⁶ peritoneal cells are used.

3. Spleen cell populations which had lost the capability of producing antibodies by elimination of cells adhering to glass yielded a strong immune response upon addition of 2 × 10⁵ peritoneal exudate cells. The significance of macrophages for the induction of the immune response is discussed.

     

Control of the immune response. I. Depression of DNA synthesis by immune lymph node cells.

The DNA response in the regional lymph nodes draining the site of immunization with contact sensitizing agents was assessed by measuring the uptake of radioactive iododeoxyuridine. The DNA response in the regional lymph nodes reached a peak on day 3 after immunization and fell to pre-immunization levels by day 6. The hypothesis was tested that lymph node cells from mice immunized with picryl chloride might depress the DNA response to the same antigen. Immune lymph node cells were injected intravenously and the recipient mice were immunized with picryl chloride on the same day. The immune cells depressed the DNA response on day 4 by an average of about 60 per cent. Smaller but significant depression also occurred on day 3. The cells responsible for the depression appeared in the regional lymph nodes 3-4 days after immunization and disappeared by day 21. The transfer of small numbers of immune cells (less than 2-5 X 10(6)) increased the DNA response in recipients 4 days after immunization with picryl chloride. The depression of the DNA response was largely specific. Pooled data from ten experiments showed that cells immunized with 4-ethoxymethylene-phenyl oxazolone ('oxazolone') caused no depression of the DNA response to picryl chloride, although in two of these experiments significant depression of about 21 per cent was seen. Similar results were obtained when immune cells were injected into mice immunized with 'oxazolone'.     

The immune response of mice to antigen in macrophages

Peritoneal macrophages obtained following an injection of proteose peptone, and after uptake of Maia squinado haemocyanin were transferred to syngeneic hosts. Immunogenicity was tested by the capacity of macrophages containing the antigen to prime normal or irradiated (660–700 r) recipients for a secondary immune challenge. The immunogenicity of macrophages containing antigen depended on interaction with immunocompetent lymphoid cells since irradiated hosts were unresponsive unless normal lymphoid cells were also supplied. For optimal immune response the live macrophages had to gain access to lymphoid organs. Depending on the amount of antigen transferred with the macrophages, the recipient mice synthesized on secondary challenge 7S and/or 19S antibody. The kinetics of response to the antigen in macrophages were similar to those seen when using free soluble material except for some quantitative differences. Although the immune response was dependent on the total dose of antigen transferred with the macrophages, somewhat higher antibody titres were obtained with macrophages having a high antigen—cell ratio. Antigen in macrophages could elicit a secondary response in primed mice. The immunogenicity of macrophage-held haemocyanin was not impaired by X-irradiation of macrophage donors.     

Immunohistochemical study of S-100 protein in the bovine lymph node and spleen

Immunohistochemistry using anti-bovine S-100 protein serum was examined in the bovine lymph node and spleen. In the lymph node, immunoreactivity was found in endothelial cells of lymph vessels and in endothelial and reticular cells of the sinuses. In the spleen, immunoreactivity was observed in endothelial cells of the trabecular artery, central artery, penicillar artery, sheathed artery, terminal capillary, trabecular vein and lymph vessel. In addition, the follicular dendritic cells in germinal centers both of the lymph node and spleen were stained with S-100 protein. These findings suggest that S-100 protein of the vascular systems may be related to the flow of lymph and blood.