Human Spleen Tissue Research Articles

Deletion of mesencephalic astrocyte-derived neurotrophic factor delays and damages the development of white pulp in spleen

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-induced protein, and it has been reported that ER stress and unfolded protein response (UPR) are closely related to the immune system. The spleen is an important immune organ and we have shown in our previous research that MANF is expressed in human spleen tissues. However, there have been limited studies about the effect of MANF on spleen development. In this study, we detected MANF expression in spleen tissues and found that MANF was expressed in the red pulp and marginal zone. Additionally, MANF was localized in the CD68+ and CD138+ cells of adult rat spleen tissues, but not in the CD3+ cells. We performed immunohistochemical staining to detect MANF expression in the spleen tissues of rats that were different ages, and we found that MANF+ cells were localized together in the spleen tissues of rats that were 1–4 weeks old. MANF was also expressed in CD68+ cells in the spleen tissues of rats and mice. Furthermore, we found that MANF deficiency inhibited white pulp development in MANF knockout mice, thus indicating that MANF played an important role in the white pulp development of rodent spleen tissues.

Genomic annotation for vaccine target identification and immunoinformatics-guided multi-epitope-based vaccine design against Songling virus through screening its whole genome encoded proteins.

Songling virus (SGLV), a newly discovered tick-borne orthonairovirus, was recently identified in human spleen tissue. It exhibits cytopathic effects in human hepatoma cells and is associated with clinical symptoms including headache, fever, depression, fatigue, and dizziness, but no treatments or vaccines exist for this pathogenic virus. In the current study, immunoinformatics techniques were employed to identify potential vaccine targets within SGLV by comprehensively analyzing SGLV proteins. Four proteins were chosen based on specific thresholds to identify B-cell and T-cell epitopes, validated through IFN-γ epitopes. Six overlap MHC-I, MHC-II, and B cell epitopes were chosen to design a comprehensive vaccine candidate, ensuring 100% global coverage. These structures were paired with different adjuvants for broader protection against international strains. Vaccine constructions' 3D models were high-quality and validated by structural analysis. After molecular docking, SGLV-V4 was selected for further research due to its lowest binding energy (-66.26 kcal/mol) and its suitable immunological and physiochemical properties. The vaccine gene is expressed significantly in E. coli bacteria through in silico cloning. Immunological research and MD simulations supported its molecular stability and robust immune response within the host cell. These findings can potentially be used in designing safer and more effective experimental SGLV-V4 vaccines.

Analysis of Butyrophilin-Mediated Activation of γδ T Cells from Human Spleen.

There is considerable interest in therapeutically engaging human γδ T cells. However, due to the unique TCRs of human γδ T cells, studies from animal models have provided limited directly applicable insights, and human γδ T cells from key immunological tissues remain poorly characterized. In this study, we investigated γδ T cells from human spleen tissue. Compared to blood, where Vδ2+Vγ9+ T cells are the dominant subset, splenic γδ T cells included a variety of TCR types, with Vδ1+ T cells typically being the most frequent. Intracellular cytokine staining revealed that IFN-γ was produced by a substantial fraction of splenic γδ T cells, IL-17A by a small fraction, and IL-4 was minimal. Primary splenic γδ T cells frequently expressed NKG2D (NK group 2 member D) and CD16, whereas expression of DNAM-1 (DNAX accessory molecule 1), CD28, PD-1, TIGIT, and CD94 varied according to subset, and there was generally little expression of natural cytotoxicity receptors, TIM-3, LAG-3, or killer Ig-like receptors. In vitro expansion was associated with marked changes in expression of these activating and inhibitory receptors. Analysis of functional responses of spleen-derived Vδ2+Vγ9+, Vδ1+Vγ9+, and Vδ1+Vγ9- T cell lines to recombinant butyrophilin BTN2A1 and BTN3A1 demonstrated that both Vδ2+Vγ9+ and Vδ1+Vγ9+ T cells were capable of responding to the extracellular domain of BTN2A1, whereas the addition of BTN3A1 only markedly enhanced the responses of Vδ2+Vγ9+ T cells. Conversely, Vδ1+Vγ9+ T cells appeared more responsive than Vδ2+Vγ9+ T cells to TCR-independent NKG2D stimulation. Thus, despite shared recognition of BTN2A1, differential effects of BTN3A1 and coreceptors may segregate target cell responses of Vδ2+Vγ9+ and Vδ1+Vγ9+ T cells.

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We develop a tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3000 (>9500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. It also enables precise quantification of ~600 phosphopeptides from 100 sorted cells (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 µm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.

Hemolysis in the spleen drives erythrocyte turnover

AbstractRed pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule–driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

Protective immunity elicited by the nematode-conserved As37 recombinant protein against Ascaris suum infection.

BackgroundAscaris lumbricoides is one of the three major soil-transmitted gastrointestinal helminths (STHs) that infect more than 440 million people in the world, ranking this neglected tropical disease among the most common afflictions of people living in poverty. Children infected with this roundworm suffer from malnutrition, growth stunting as well as cognitive and intellectual deficits. An effective vaccine is urgently needed to complement anthelmintic deworming as a better approach to control helminth infections. As37 is an immunodominant antigen of Ascaris suum, a pig roundworm closely related to the human A. lumbricoides parasite, recognized by protective immune sera from A. suum infected mice. In this study, the immunogenicity and vaccine efficacy of recombinant As37 were evaluated in a mouse model.Methodology/Principal findingsAs37 was cloned and expressed as a soluble recombinant protein (rAs37) in Escherichia coli. The expressed rAs37 was highly recognized by protective immune sera from A. suum egg-infected mice. Balb/c mice immunized with 25 μg rAs37 formulated with AddaVax™ adjuvant showed significant larval worm reduction after challenge with A. suum infective eggs when compared with a PBS (49.7%) or adjuvant control (48.7%). Protection was associated with mixed Th1/2-type immune responses characterized by high titers of serological IgG1 and IgG2a and stimulation of the production of cytokines IL-4, IL-5, IL-10 and IL-13. In this experiment, the AddaVax™ adjuvant induced better protection than the Th1-type adjuvant MPLA (38.9%) and the Th2-type adjuvant Alhydrogel (40.7%). Sequence analysis revealed that As37 is a member of the immunoglobulin superfamily (IgSF) and highly conserved in other human STHs. Anti-As37 antibodies strongly recognized homologs in hookworms (Necator americanus, Ancylostoma ceylanicum, A. caninum) and in the whipworm Trichuris muris, but there was no cross-reaction with human spleen tissue extracts. These results suggest that the nematode-conserved As37 could serve as a pan-helminth vaccine antigen to prevent all STH infections without cross-reaction with human IgSF molecules.Conclusions/SignificanceAs37 is an A. suum expressed immunodominant antigen that elicited significant protective immunity in mice when formulated with AddaVax™. As37 is highly conserved in other STHs, but not in humans, suggesting it could be further developed as a pan-helminth vaccine against STH co-infections.

Hemolysis in the Spleen Drives Erythrocyte Turnover

Erythrocytes circulate for an average of 120 days before they are removed from the circulation. Various processes and factors have been identified that may contribute to degradation of senescent erythrocytes, but this complex process is still not completely understood. Accumulation of removal signals such as phosphatidylserine exposure, changes in CD47 expression and oxidation of proteins and lipids that render them susceptible to complement deposition, may contribute to recognition and degradation by red pulp macrophages (RPM) of the spleen. However, many questions remain on the exact mechanisms that determine the fate of aged erythrocytes. This is well exemplified in a mouse study in which physiologically aged erythrocytes were found to undergo phagocytosis by RPM in vivo but not in vitro. This finding suggested that the splenic architecture may play an important role in facilitating erythrocyte turnover. Loss of membrane deformability may lead to the initial trapping of aged or damaged erythrocytes in the spleen, an event that precedes their degradation by macrophages. Loss of deformability can explain why certain genetic diseases that affect erythrocyte membrane deformability, such as is the case in sickle cell disease and spherocytosis, result in trapping in the spleen, giving rise to anaemia. Next to loss of deformability, activation of adhesion molecules, such as Lu/BCAM and CD44, specifically on aged erythrocytes has been proposed to contribute to retention of erythrocytes within the spleen, leading to their turnover. In this study we provide evidence that the splenic environment is of key importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPM we noted that only a small proportion of the macrophages were in the process of phagocytosing intact erythrocytes. Based on a range of variables, including the number of erythrocytes that are cleared daily, the number of RPM present in the spleen, the degradation rate of erythrocytes as well as differential contribution of spleen and liver to erythrocyte turnover, conservative estimates approximate that at least a 30-fold fewer erythrophagocytic events are observed in RPM than anticipated. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of a large population of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By in vivo imaging of the spleen and transfusion experiments we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis, thereby forming erythrocyte ghosts. Of note, we found that the levels of haptoglobin and hemopexin, two plasma proteins that are involved in scavenging of haemoglobin and heme, respectively, correlate well with the rate of hemolysis that was observed in the spleen. Additionally, we show that the erythrocyte adhesion molecules which are specifically activated on aged erythrocytes, Lu/BCAM and CD44, cause senescent erythrocytes to interact with the extracellular matrix of the spleen. This adhesion molecule-driven retention of senescent erythrocytes, under low shear conditions, was found to result in steady shrinkage of the erythrocytes and ultimately resulted in hemolysis and ghost formation. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghosts were found to be immediately recognized and rapidly degraded (1-3 hours) by RPM, thereby explaining the lack of phagocytosis of intact erythrocytes in the spleen. Together, these data identify hemolysis and ghost formation as key events in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated. Disclosures No relevant conflicts of interest to declare.

Spleen tissues from patients with lymphoma: magnetization measurements and Mössbauer spectroscopy

Spleen tissues from five patients with two types of non-Hodgkin B-cells lymphomas, namely, mantle cell lymphoma and marginal zone B-cell lymphoma, and one sample of healthy human spleen tissue were studied using Mossbauer spectroscopy and magnetization measurements. Magnetization measurements demonstrated small differences in the saturation magnetic moments and the presence of paramagnetic components. Mossbauer spectra of spleen tissues demonstrated some variations in the relative content of ferritin-like iron in tissues as well as small variation in the hyperfine parameters for normal and patients’ spleen tissues. Some changes in the ferritin iron core structure in patients’ spleen were suggested in comparison with normal subject.

Immunoglobulin Light Chains Form an Extensive and Highly Ordered Fibril Involving the N- and C-Termini.

Light-chain (AL)-associated amyloidosis is a systemic disorder involving the formation and deposition of immunoglobulin AL fibrils in various bodily organs. One severe instance of AL disease is exhibited by the patient-derived variable domain (VL) of the light chain AL-09, a 108 amino acid residue protein containing seven mutations relative to the corresponding germline protein, κI O18/O8 VL. Previous work has demonstrated that the thermodynamic stability of native AL-09 VL is greatly lowered by two of these mutations, Y87H and N34I, whereas a third mutation, K42Q, further increases the kinetics of fibril formation. However, detailed knowledge regarding the residues that are responsible for stabilizing the misfolded fibril structure is lacking. In this study, using solid-state NMR spectroscopy, we show that the majority of the AL-09 VL sequence is immobilized in the fibrils and that the N- and C-terminal portions of the sequence are particularly well-structured. Thus, AL-09 VL forms an extensively ordered and β-strand-rich fibril structure. Furthermore, we demonstrate that the predominant β-sheet secondary structure and rigidity observed for in vitro prepared AL-09 VL fibrils are qualitatively similar to those observed for AL fibrils extracted from postmortem human spleen tissue, suggesting that this conformation may be representative of a common feature of AL fibrils.

Transplantation of human spleen into immunodeficient NOD/SCID IL2Rγnull mice generates humanized mice that improve functional B cell development

We previously generated humanized TB34N mice that received human fetal thymus (T), bone tissue (B) and fetal liver-derived (FL)-CD34+ cells (34) in immunodeficient, NOD/SCID IL2Rγnull (N) mice. Although humanized TB34N mice had excellent hematopoiesis, here, we sought to further improve this model by additional transplantation of human spleen tissue (S) as a secondary hematopoietic tissue (TBS34N). The human spleen grafts were enlarged and differentiated into a similar morphology of adult humans, including follicular lymphoid structures with T- and B-cells. The TBS34N mice mimicked mature human immune system (HIS): mature T- and B-cells and follicular dendritic cells; activated germinal center B-cells expressing CD71, BR3+ cells, memory B-cells and activation-induced cytidine deaminase+ B-cells; CD138+ plasma cells were enriched in the mouse spleen. HBsAg-specific hIgG antibodies were secreted into the sera of all TBS34N mice upon immunization with HBsAg. Taken together, the humanized TBS34N mice improved mature HIS and achieved adaptive antibody responses.

Iron-rich complexes in human spleen in hereditary spherocytosis

Tissue iron plays an important role in the development of certain diseases. Although it is one of biogenic elements, its excess induces the reactive oxygen species (ROS) formation. The aim of the present work is to examine the protection against free or loosely bound iron from the view of morphology and chemical composition of iron-rich complexes in human spleen tissues with hereditary spherocytosis (HS) by scanning and transmission electron microscope with energy-dispersive microanalysis (EDX). The examination of human spleen tissues by scanning and transmission electron microscope showed covering of iron-rich particles. EDX revealed many iron-rich complexes of multi-element composition in HS samples with sulphur and phosphorus as the major elements. Detection was negative in the reference samples. The covering of iron-rich particles can be explained by elimination and isolation of ferritin/iron complexes from surrounding environment to prevent the ROS formation. Sulphur, phosphorus and their compounds are probably the most significant elements that influence the ROS formation (Fig. 5, Ref. 16). Full Text in PDF www.elis.sk.

Biomechanical response of human spleen in tensile loading

Blunt splenic injuries are most frequently caused as a result of motor vehicle collisions and are associated with high mortality rates. In order to accurately assess the risk of automotive related spleen injuries using tools such as finite element models, tissue level tolerance values and suitable material models must be developed and validated based on appropriate biomechanical data. This study presents a total of 41 tension tests performed on spleen parenchyma coupons and 29 tension tests performed on spleen capsule/parenchyma coupons. Standard dog-bone coupons were obtained from fresh human spleen and tested within 48h of death. Each coupon was tested once to failure at one of the four loading rates to investigate the effects of rate dependence. Load and acceleration data were obtained at each of the specimen grips. High-speed video and optical markers placed on the specimens were used to measure local displacement. Failure stress and strain were calculated at the location of failure in the gage length of the coupon. The results of the study showed that both the spleen parenchyma and the capsule are rate dependent, with higher loading rates yielding higher failure stresses and lower failure strains. The results also show that the failure stress of the splenic capsule is significantly greater than that of the underlying parenchyma. Overall, this study provides novel biomechanical data that demonstrate the rate dependent tissue level tolerance values of human spleen tissue in tensile loading, which can aid in the improvement of finite element models used to assess injury risk in blunt trauma.

Ex vivo perfusion of human spleens maintains clearing and processing functions

The spleen plays a central role in the pathophysiology of several potentially severe diseases such as inherited red cell membrane disorders, hemolytic anemias, and malaria. Research on these diseases is hampered by ethical constraints that limit human spleen tissue explorations. We identified a surgical situation--left splenopancreatectomy for benign pancreas tumors--allowing spleen retrieval at no risk for patients. Ex vivo perfusion of retrieved intact spleens for 4 to 6 hours maintained a preserved parenchymal structure, vascular flow, and metabolic activity. Function preservation was assessed by testing the ability of isolated-perfused spleens to retain Plasmodium falciparum-infected erythrocytes preexposed to the antimalarial drug artesunate (Art-iRBCs). More than 95% of Art-iRBCs were cleared from the perfusate in 2 hours. At each transit through isolated-perfused spleens, parasite remnants were removed from 0.2% to 0.23% of Art-iRBCs, a proportion consistent with the 0.02% to 1% pitting rate previously established in artesunate-treated patients. Histologic analysis showed that more than 90% of Art-iRBCs were retained and processed in the red pulp, providing the first direct evidence of a zone-dependent parasite clearance by the human spleen. Human-specific physiologic or pathophysiologic mechanisms involving clearing or processing functions of the spleen can now be experimentally explored in a human tissue context.

Molecular cloning of human IL-7cDNA and construction of eukaryotic vector expressing hIL-7

OBJECTIVE: To construct a vector expressing eukaryotic human interluken-7(hIL-7). METHODS: hIL-7 DNA was identified and cloned (cDNA) from human spleen tissue using reverse transcription polymerase chain reaction (RT-PCR). We incorporated the cDNA into the pMD18-T plasmid. The pMD18-T plasmid was then inserted into a dual expression vector (prokaryotic and eukaryotic) pBK-CMV and called pBK-CMV-hIL-7. We used pBK-CMV-hIL-7 vector to infect E.coli DH5alpha. The expression of the recombinant hIL-7 protein (rhIL-7) by E.coli DH5alpha was analyzed using SDS-PAGE and western blot testing. RESULTS: The genetically engineered E.coli DH5alpha did express rhIL-7 confirmed by western blot. CONCLUSION: The successful construction of genetically engineered eukaryotic gene for hIL-7 was done, This will enable further research into therapeutic uses for hIL-7.

Bacterial peptidoglycan polysaccharides in sterile human spleen induce proinflammatory cytokine production by human blood cells.

Peptidoglycan (PG) is the major component of the cell wall of gram-positive bacteria. In vitro, PG isolated from conventional bacterial cultures can induce secretion of proinflammatory cytokines by human monocytes, indicating that PG may be involved in immune responses against infections by gram-positive bacteria. To investigate the biologic activity of PG in human tissues, an improved method was developed to isolate significant amounts of PG from sterile human spleen tissue. Biochemical analysis demonstrated that PG isolated from human spleen is largely intact. Human whole blood cell cultures were able to produce the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 and -6 after stimulation with PG isolated from human spleen. Cytokine induction was not sensitive to inhibition by polymyxin B, in contrast to lipopolysaccharide. Collectively, the data show that intact PG in sterile human tissue is biologically active and may induce local proinflammatory cytokine production.

Reductive changes to polynuclear iron(III) clusters in iron-loaded human spleen tissue

Mössbauer spectroscopy was used to monitor, over a period of three days, the form of iron in iron-loaded spleen tissue following splenectomy from a patient with β-thalassemia. The tissue was stored at room temperature for a three day period in order to allow degradation and autolytic processes to take place. The majority of the iron in the fresh spleen tissue was found to be in the form of inorganic polynuclear iron(III) oxyhydroxide clusters associated with ferritin and hemosiderin. After three days, some of the iron had been reduced to high-spin iron(II).

The effect of histological processing on the form of iron in iron-loaded human tissues

Iron-loaded human spleen tissue was immersed in neutral buffered formalin over a period of 200 days. Over the first 60 days, iron leached steadily from the tissue until 3% had been lost. Thereafter, no further iron leaching was detected. Comparisons of Mössbauer spectra of freeze-dried tissue and tissue freeze-dried after immersion in formalin for 200 days showed no evidence of chemical transformation of the iron remaining in the tissue. The spectra indicated a difference in the heme-iron to non-heme iron ratio between the two samples probably reflecting inhomogeneity of the ratio throughout the spleen as measured on the centimetre scale. Mössbauer spectra of freeze-dried samples of iron-loaded human liver and pancreas tissue were compared with those for samples from the same patient that had been processed by routine hospital procedures for histology and archival. These spectra showed no evidence for chemical transformation of the iron present in the tissues. These results demonstrate that it is feasible to use archived fixed and embedded human tissue samples for studies aimed at gauging the relative fraction of goethite-like hemosiderin present in the tissue.

Tartrate-resistant acid phosphatase from human bone: purification and development of an immunoassay.

Tartrate-resistant acid phosphatase (TRAP) was purified 20,000-fold to apparent homogeneity from human bone. The purified enzyme consisted of one 32 kd subunit, which was cleaved by beta-mercaptoethanol into two subunits of 15 kd and 20 kd, as shown by sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The purified enzyme was identified by N-terminal amino acid sequencing, and it was shown to be homologous with previously purified TRAPs from other sources. We developed a polyclonal antiserum against the purified enzyme in mice. In immunohistochemistry, the antiserum recognized osteoclasts from human bone and alveolar macrophages from human lung tissue, but no cells from human spleen tissue. It also stained osteoclasts from rat bone cells cultured on bovine bone slices. Purified TRAP could be inhibited by vanadate and molybdate, but not by tartrate, and it was activated 2-fold by beta-mercaptoethanol. The glycoprotein structure of human bone TRAP was analyzed, and it was shown to contain only high-mannose type carbohydrates. We used the polyclonal antibody to develop a competitive fluorescence immunoassay for measuring serum TRAP concentrations. According to the assay, children have higher serum TRAP concentrations than adults, and postmenopausal women have higher concentrations than premenopausal women. Postmenopausal women also have higher serum TRAP concentrations than postmenopausal women on estrogen replacement therapy.

Human immune response to pneumococcal polysaccharides: complement-mediated localization preferentially on CD21-positive splenic marginal zone B cells and follicular dendritic cells

A functionally intact spleen with a marginal zone, containing B cells with high density of surface C3d-receptors (CD21), is essential for the ability to induce a primary immune response to thymus-independent type 2 (TI-2) antigens. Main representatives of natural TI-2 antigens are capsular pneumococcal polysaccharides (PPSs). In this study the localization of different types of PPS antigen is determined in human spleen tissue. Our findings indicate that a main type of TI-2 antigen, PPS, localizes preferentially in the marginal zone. PPSs show co-localization with C3, presumably C3d, at the surface of strongly CD21+ B cells equipped for rapid activation. This enables a rapid primary humoral response. The other main PPS localization at follicular dendritic cells in germinal centers, relevant for isotype switching of anti-PPS antibodies, does not seem to be dependent on the presence of specific immunoglobulin. This may explain the finding of specific IgG in an early stage after antigenic challenge. It seems likely that complement C3 fragments (likely C3d), bound to PPSs, enable PPS localization at B-cell and follicular dendritic cell surfaces by binding to CD21, the C3d receptor.

Differential processing of proenkephalin-A by human peripheral blood monocytes and T lymphocytes.

Human peripheral blood mononuclear cells are analyzed for preproenkephalin gene expression and peptide processing. Met-enkephalin immunoreactivity as detected with a specific antiserum is found in the cytoplasm of monocytes but not in T lymphocytes. Secretion of met-enkephalin was analyzed with an RIA that is specific for the met-enkephalin pentapeptide. Unfractionated PBMC spontaneously released 40 pg/ml met-enkephalin and this increased two- to fourfold after stimulation with PHA. Lower levels (less than 100 pg/ml) of met-enkephalin were detected in supernatants from purified T cells that were activated with PHA and IL-2. In contrast, stimulation of purified monocytes with LPS or PMA resulted in the release of up to 600 pg/ml of the processed peptide. To examine whether T cells can produce met-enkephalin precursor peptides, T cell conditioned media were treated with trypsin and carboxypeptidase-B, which is known to release met-enkephalin from the propeptide. This increased levels of met-enkephalin to 400 pg/ml, indicating that lymphocytes secrete the propeptide but do not process it to met-enkephalin. The 1.4-kb preproenkephalin mRNA is detected in activated blood mononuclear cells and in purified monocytes and T cells. To determine whether monocytes or lymphocytes express met-enkephalin in vivo, lymphoid tissues were analyzed by immunohistochemistry. In human spleen tissue, positive cells were found in the red pulp but not in the follicles, which is also consistent with met-enkephalin expression in monocytes. In summary, these results show that human peripheral blood mononuclear cells express preproenkephalin mRNA and that monocytes, but not T cells, process the propeptide to metenkephalin.