THE INTERACTION OF PARTICULATE HORSERADISH PEROXIDASE (HRP)-ANTI HRP IMMUNE COMPLEXES WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The uptake, distribution, and fate of particulate horseradish peroxidase (HRP)-anti HRP aggregates has been studied in homogeneous monolayers of mouse macrophages in vitro. Macrophages rapidly interiorize the immune complexes after binding to the cell surface. The rate of interiorization is maximal for complexes formed in a broad zone of 4-fold antibody excess to equivalence and corresponds to a rate of 10% of the administered load/10⁶ cells per hour. This rate is 4000-fold greater than the uptake of soluble HRP. The binding and endocytosis of HRP-anti HRP by macrophages is mediated by the trypsin insensitive F_c receptor. Cytochemically, intracellular HRP is localized within membrane bound vacuoles. After uptake of HRP, the enzymatic activity is degraded exponentially with a half-life of 14–18 hr until enzyme is no longer detectable. This half-life is twice as long as that previously observed for soluble uncomplexed HRP and is related to the combination of HRP with anti-HRP rather than the absolute amounts of enzyme or antibody ingested. The half-life of HRP-¹²⁵I was 30 hr. Exocytosis of cell associated enzyme or TCA precipitable counts was not detected, nor were persistent surface complexes demonstrable. The extensive capacity of macrophages to interiorize and destroy large amounts of antigen after the formation of antibody illustrates a role of this cell in the efferent limb of the immune response.     

Non-lymphoid cells of bronchus-associated lymphoid tissue of the rat in situ and in suspension

Immunohistochemical, enzyme-histochemical and electron-microscopical methods were used to study non-lymphoid cells of control and stimulated rat bronchus associated lymphoid tissue (BALT) in situ and in suspensions. Particular attention was paid to the so-called antigen-handling cells, i.e., the interdigitating cells (IDC), which are situated in the T-cell areas, the follicular dendritic cells (FDC), which appear to be restricted to germinal centers, and macrophages, present both in T-cell and B-cell areas. The interdigitating cells were distinguished by being Ia-positive and by the presence of acid phosphatase and non-specific esterase activity in an area near the nucleus. Follicular dendritic cells could be observed in situ by using a monoclonal antibody and by the in vitro trapping of HRP-anti-HRP complexes. Several types of macrophages were found. At the electron-microscopical level no well-developed IDC and FDC could be detected in control BALT. However, in BALT of lipopolysaccharide-stimulated and mycoplasma-infected rats, well-developed IDC and FDC were found. It can be concluded that IDC's and FDC's can be found in BALT.     

A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigen-processing cells

Recent scanning electron microscopic studies on isolated follicular dendritic cells (FDC) showed that dendrites of certain FDC were "beaded," i.e., consisting of a series of interconnected immune complex coated bodies (termed "iccosomes," measuring 0.3 to 0.7 micron diameter). In vitro these iccosomes detach from one another with ease. The major objectives herein were to establish whether these structures can be detected in sections and whether iccosomes serve to disseminate antigen in vivo. Beginning at day 1, the time point used for isolating beaded FDC, the popliteal lymph nodes of immune C3H mice were studied with light and transmission electron microscopy for 2 wk (i.e., at days 1, 3, 5, 8, and 14) after hind footpad injection of the histochemically detectable antigen, horseradish peroxidase (HRP). Iccosomes (0.25 to 0.38 micron diameter), contoured by a peroxidase (PO)-positive coat of HRP-anti-HRP complexes, were first detected by transmission electron microscopy at day 1 adjacent to cell bodies of certain FDC. Within their limiting membrane they contained flocculent material that was PO positive. At day 3 by light microscopy, germinal centers were seen enlarged and the antigen-retaining reticulum, composed of antigen-bearing FDC, appeared diffuse. This coincided with the transmission electron microscopic visualization of a dispersed state of iccosomes among the follicular lymphocytes. At that time iccosomes were seen attached to the surface of lymphocytes via PO-positive immune complexes and were surrounded by microvillous processes of these cells. Germinal center lymphocytes and tingible body macrophages both responded to contact with iccosomes by endocytosis. Antigen-containing tingible body macrophage were most conspicuous by light microscopy at day 5, when transmission electron microscopy showed that the majority of germinal center lymphocytes contained endocytosed HRP in secondary lysosome-like granules associated with the Golgi apparatus. The number of dispersed iccosomes was markedly reduced by day 5. In controls injected with HSA, a PO-negative antigen, lymphocytes and tingible body macrophages were PO-negative. The presence of antigen in both cell types was confirmed through the use of a gold-conjugated antigen (goat IgG). Simultaneous immunoperoxidase labeling of the same tissues with anti-Ia showed the gold conjugate containing B cells to be Ia+. Antigen-positive B cells and tingible body macrophages were greatly reduced in numbers by day 14, suggesting the intracellular fragmentation of the antigen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron microscopic investigation of cell surface antigens using a multivalent hybrid antibody with double specificity

Multivalent hybrid antibody (MHA) complexes with double specificity were prepared by combining two different antibodies, one specific against ferritin (Fer), the other against horseradish peroxidase using protein A from Staphylococcus aureus (SpA). Electron microscopy of mouse spleen lymphocytes and thymocytes (the latter coated with mouse anti-Thy-1 antibody) reacted with anti-HRP/SpA/anti-mIg complex and HRP showed the pattern of surface Ig receptors visualized by the specific peroxidase labelling. When IgG anti-Fer/SpA/IgG-anti-mIg complex was applied to the same cellular stuff, better resolution and higher accuracy were obtained although the distribution was similar. Surface Thy-1 alloantigen and Fe receptors (charged with hIgG) were simultaneously detected on the thymocyte surface by using in the same way a mixture of anti-Fer/SpA/anti-Thy-1 and anti-HRP/SpA/anti-Fab. The concomitant ultrastructural visualization of Thy-1 (with Fer) and Fc (with HRP) on mouse thymocytes clearly showed that their distribution was mainly independent and that the amount of Thy-1 antigen prevailed. These data show that electron microscopy with a mixture of MHA may be a useful technique for the concomitant location of two antigenic determinants on the cell surface.     

Analysis of bispecific monoclonal antibody binding to immobilized antigens using an optical biosensor

The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (K _ass) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The K _ass of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (k _diss) for anti-HRP shoulder of Babs was 21 times higher than k _diss for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody–enzyme conjugates in the case of binding of bivalent Mabs.     

Ultrastructural evidence for lack of tissue damage in a local immune complex reaction: a study of a mild passive Arthus reaction.

An electron microscopic study of a mild reversed passive Arthus reaction (RPAR) was performed using a horeradish peroxidase (HRP)-anti-HRP system to demonstrate the biological usefulness of the process which exists to clear tissue of immune complexes. To disclose the antigen, HRP, the biopsy material was subjected to a peroxidase reaction. HRP was mainly detected within irregular electron-dense precipitates which were considered insoluable HRP-anti-HRP immune complexes. Neutrophils were found to phagocytose and digest the deposited immune complexes in a similar way as described previously. But there was no vascular and other tissue damage. This study provides morphological evidence that the process of clearing tissues of immune complexes need not be harmful to the host.     

THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 10⁶ cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-¹²⁵I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-¹²⁵I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.     

Anti-horseradish peroxidase associated with tubular structures in the endoplasmic reticulum of plasma cells in stimulated lymph nodes of mice.

Tubular structures are described in dilated cisternae of the endoplasmic reticulum in plasma cells of lymph nodes stimulated with horseradish peroxidase (HRP). A positive reaction for anti-HRP antibody suggests that they are formed of immunoglobulins or are at least closely associated with them.     

The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum.

Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure.     

Classically activated macrophages use stable microtubules for matrix metalloproteinase-9 (MMP-9) secretion

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.     

Localisation of proteins in coated micropinocytotic vesicles during transport across rabbit yolk sac endoderm

Summary Rabbit yolk sac splanchnopleur exposed in utero to IgG-HRP and IgG-ferritin conjugates, rabbit and bovine anti-HRP antibodies, free HRP, ferritin and human IgG, was examined ultrastructurally in an attempt to determine whether or not coated micropinocytotic vesicles are involved in selectively transporting immunoglobulins across yolk sac endodermal cells. Human, rabbit and bovine IgG-HRP conjugates, rabbit anti-HRP antibodies, free HRP and human IgG, all become localised in coated micropinocytotic vesicles. Differences were observed in that only human IgG and rabbit anti-HRP antibodies could be located in the intercellular space and bovine IgG-HRP conjugate could not be detected in coated micropinocytotic vesicles in confluence with the lateral and basal plasmalemma. Bovine anti-HRP antibodies, IgG-ferritin conjugates, and free ferritin, could not be observed in coated micropinocytotic vesicles. All proteins were detected in macropinocytotic vesicles, and dense bodies resembling phagolysosomes. Results are discussed in the light of a proposal that selection occurs at the cell surface during formation of coated micropinocytotic vesicles and is not linked to intracellular proteolysis.Supported by an award from the Medical Research Council, to whom grateful acknowledgement is made     

Emergence of a surface immunoglobulin recycling process during B lymphocyte differentiation

Surface immunoglobulin (Ig)-mediated endocytosis has been investigated in rat B lymphocytes and plasma cells, using horseradish peroxidase (HRP)-labeled sheep anti-rat Ig Fab' fragment of antibody and HRP as monomeric ligands, respectively. Quantitative estimates of HRP activity associated either with plasma membrane or with endomembrane compartments were made in several experimental conditions. Binding of HRP-conjugate on B lymphocytes was followed by its endocytosis in combination with surface Ig, as shown by the progressive disappearance of plasma membrane-associated HRP activity. Between 1 and 6 h at 37 degrees C in presence of conjugate the total amount of cell-associated activity was constant. These results indicate that during this time no reappearance of surface Ig occurred by neosynthesis, by the expression of an intracellular pool or by the recycling in a free form of the previously internalized molecules. On the contrary, at saturating doses, internalization of HRP by anti-HRP plasma cells increased linearly with time at 37 degrees C in presence of antigen, when, during the same time, the plasma membrane HRP-binding capacity remained constant. Cycloheximide did not affect continuous HRP uptake. The existence of a large intracellular pool of receptors has been ruled out by experiments of removal of binding sites with pronase. In addition, monensin caused a progressive decrease in the number of surface receptors on plasma cells but not on B lymphocytes. Our data then indicate that, unlike B lymphocytes, plasma cells were able to recycle their surface Ig.     

Macrophage-associated erythropoiesis and lymphocytopoiesis in mouse fetal liver: ultrastructural and ISH analysis

To elucidate the process of fetal liver hematopoiesis, the relationships between stroma and hematopoietic cells involved in maturation were investigated. Cultured mouse fetal liver explants were established for morphological analysis of the interactions between fetal liver stroma and hematopoietic cells ex vivo. Fetal liver stroma comprised epithelial cells and macrophages, which occupied most of the culture surface. Macrophages proliferated extensively in primary culture, but almost disappeared after 3 passages. Close morphological and functional relationships were established between macrophages and hemopoietic cells, whereas epithelial cells did not interact with blood cells. Scanning electron microscopy revealed that macrophages were in close contact with erythroblasts and formed a three-dimensional network. In each erythroblastic island, 2-3 lymphocytes were also in contact with the macrophages; erythroblasts, lymphocytes and macrophages formed close, firm associations through their cytoplasmic membranes. This cell orientation was confirmed by transmission electron microscopy of fetal liver in vivo. In situ hybridization revealed that the macrophages expressed jagged-1, an important ligand of the Notch signaling system in hematopoiesis.     

Precise localization of antigens on follicular dendritic cells

Summary Horse-spleen ferritin or bovine serum albumin conjugated to colloidal gold (BSA-gold) were injected subcutaneously in preimmunized mice. In draining lymph nodes both antigens were located in macrophages or between the cytoplasmic processes of follicular dendritic cells (FDC). Some of the antigens remained trapped on FDC until day 31 after injection. Simultaneous injection of both antigens showed that they were located between the infoldings of the same FDC. These cells are thus able to retain at least two different antigens on their surface. The peculiar arrangement of ferritin between the cytoplasmic infoldings suggests that this antigen is fixed on both cell membranes by specific antibodies. The trapped immune complexes could thus stabilize the FDC membrane system.The antigen retention requires the presence of specific antibodies since BSA-gold or ferritin injected without preimmunization were not found between FDC processes. Nonantigenic materials, such as colloidal gold or carbon particles, are not trapped by FDC, except when injected in large amounts.The antigens were trapped on the surface of FDC, however unfrequently in close contact with lymphocytes. FDC might protect lymphocytes against an excess of immune complexes and act as regulators of contacts between lymphocytes and immune complexes.Abbreviations BSA bovine serum albumin - BSA-gold BSA conjugated to colloidal gold particles - FDC follicular dendritic cells     

Oxidized LDL immune complexes induce release of sphingosine kinase in human U937 monocytic cells

The transformation of macrophages into foam cells is a critical event in the development of atherosclerosis. The most studied aspect of this process is the uptake of modified LDL through the scavenger receptors. Another salient aspect is the effect of modified LDL immune complexes on macrophages activation and foam cell formation. Macrophages internalize oxidized LDL immune complexes (oxLDL-IC) via the Fc-gamma receptor and transform into activated foam cells. In this study we examined the effect of oxLDL-IC on sphingosine kinase 1 (SK1), an enzyme implicated in mediating pro-survival and inflammatory responses through the generation of the signaling molecule sphingosine-1-phosphate (S1P). Intriguingly, oxLDL-IC, but not oxLDL alone, induced an immediate translocation and release of SK1 into the conditioned medium as evidenced by fluorescence confocal microscopy. Immunoblot analysis of cell lysates and conditioned medium revealed a decrease in intracellular SK1 protein levels accompanied by a concomitant increase in extracellular SK1 levels. Furthermore, measurement of S1P formation showed that the activity of cell-associated SK decreased in response to oxLDL-IC compared to oxLDL alone, whereas the activity of SK increased extracellularly. Blocking oxLDL-IC binding to Fc-gamma receptors resulted in decreased levels of extracellular S1P. The data also show that cell survival of human U937 cells exposed to oxLDL-IC increased compared to oxLDL alone. Exogenously added S1P further increased cell survival induced by oxLDL-IC. Taken together, these findings indicate that S1P may be generated extracellularly in response to modified LDL immune complexes and may therefore promote cell survival and prolong cytokine release by activated macrophages.     

Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology

The objective of the present study was to investigate the mechanism of antigen migration from the site of initial localization in the lymph node subcapsular sinus (SS) to regions of follicular retention in the cortex. The migration of horseradish peroxidase (HRP), used as a histochemically identifiable antigen, was followed by light and electron microscopy in C3H mouse popliteal lymph nodes obtained 1, 5, 15, and 30 min, and 5 and 24 hr after hindfoot pad injection of HRP. The observations showed that as early as 1 min after HRP injection, localization of antigen occurred at distinct sites in the SS and subjacent areas of the cortex on the afferent side. At these sites, between 1 min and 24 hr, the antigen formed light microscopically identifiable trails, which reached progressively deeper into the cortex with time toward individual follicular regions. By 24 hr this apparent migration of antigen was complete, and HRP was localized in follicles. This migration pattern did not occur on the efferent sides of lymph nodes, and it was dependent on the systemic presence of specific antibodies since it was observable only in passively immunized but not in nonimmune mice. Temporary retention of antigen by typical macrophages was also observed in the SS on the efferent side. This was minimal in nonimmune mice and was significantly enhanced in passively immunized mice. Electron microscopy indicated that the apparent migration of immune complexes was mediated by a group of cells observed in the migration path that had immune complexes sequestered on their surface or in plasma membrane infoldings. These antigen transporting cells (ATC) were relatively large nonphagocytic cells, with lobated or irregular euchromatic nuclei and cell processes of various complexity. ATC observed in or near the SS appeared to be less differentiated, were monocyte-like, and resembled non-Birbeck granule-containing Langerhans cell precursors or veiled cells. Others, located deeper in the cortex, appeared more differentiated, interdigitated with antigen-retaining dendritic cells, and shared morphologic characteristics with follicular dendritic cells (FDC). The results support the concepts that immune complexes are trapped in the SS and are transported by a group of non-phagocytic cells, other than lymphocytes, to follicular regions. The mechanism of transport may involve the migration of ATC with a concomitant maturation into FDC, or by a mechanism of ATC to FDC transport utilizing dendritic cell processes and membrane fluidity, or by a combination of the two mechanisms.     

Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents

Cultured rat embryo fibroblasts were first allowed to store for 24 h fluorescein-labeled goat immunoglobulins directed against rabbit immunoglobulins (F anti-R IgG), and were subsequently exposed for 24 h to [(3)H]acetylated rabbit immunoglobulins known to bind to the cell membrane either specifically (anti-plasma membrane IgG: A anti-PM IgG) or unspecifically (contol IgG: AC IgG). As a result of immunological interaction between the two antibodies (no effect was found if the cells had been preloaded with control goat FC IgG), a substantial portion of the stored F anti-R IgG was unloaded from its intracellular storage site, appearing in the medium in the form of soluble immune complexes with rabbit A IgG. Part of the unloaded F anti-R IgG also was recovered in association with the plasma membrane, but only when A anti-PM IgG was used. In addition, significant reverse translocation of AC IgG from plasma membrane to lysosomes or some related intracellular storage compartment was also observed. With A anti-PM IgG, this translocation was less marked and affecte at the same time the plasma membrane marker 5’- nucleotidase. Cells that had stored horseradish peroxidase (HRP) simultaneously with F anti-R IgG did not unload HRP when exposed to A anti-PM IgG. These results support strongly, though not unequivocally, the concept that plasma membrane patches interiorized by endocytosis are recycled, or shuttled, back to the cell surface. In the framework of this concept, recycling antibody-coated membrane is taken to serve as vehicle for the selective intracellular capture and extracellular discharge of immunologically bound F anti-R IgG. The alternative explanation of regurgitation triggered off by immune complexes is considered less likely in view of the lack of HRP unloading.     

Mannose-specific binding sites for horseradish peroxidase in various cells of the rat

Mannose-specific binding sites for horseradish peroxidase (HRP) were studied in fixed sections of various tissues by a method reported previously. Liver sinusoidal cells, mast cells of lymph nodes, and alveolar macrophages of the lung and skin fibroblasts were main cell types showing mannose-specific binding of HRP. Macrophages, fibroblasts, and mast cells in the connective tissue of other organs also showed the reaction. However, macrophages of the spleen, and cultured 3T3 cells and L-cells did not give the reaction. The specificities of the binding reaction were studied by determining the approximate concentrations of competing sugars that suppressed the specific binding of HRP. It was found that the endogenous lectins in macrophages, fibroblasts, mast cells, and liver sinusoidal cells showed similar specificities toward various carbohydrates. D-Mannose and L-fucose had the highest affinity toward the lectins (competing ability for the binding of HRP). D-Mannose-6-phosphate, N-acetyl-D-glucosamine, D-glucose, D-ribose, and D-arabinose showed intermediate affinity, whereas D-xylose and D-galactose showed low affinity. Polymerized mannose in mannan and glycoproteins rich in mannose groups (invertase and ribonuclease B) showed much higher affinity to the binding sites than free mannose.     

Histology of the bone marrow antibody response

The histology of the specific and non-specific antibody response in mouse and rat bone marrow was studied after subcutaneous priming and intravenous boosting with horseradish peroxidase (HRP). Cells producing specific antibody against HRP were found only occasionally in the bone marrow after subcutaneous priming. After the intravenous boost injection their number gradually increased. These anti-HRP forming cells were found as single cells, randomly dispersed throughout the bone marrow. Such a random distribution was also found for cytoplasmic (non-specific) immunoglobulin containing cells. At no time point after immunization could lymphoid aggregates or trapping of immune complexes be observed in the bone marrow of either species. On the basis of these observations it is concluded that the bone marrow forms a suitable microenvironment for immigrating antibody-forming cells but does not contribute actively to the induction of the immune response.